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10.1371/journal.pone.0310079
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Repeated rebiopsy for detection of EGFR T790M mutation in patients with advanced-stage lung adenocarcinoma: Associated factors and treatment outcomes of Osimertinib
Repeated rebiopsy for EGFR T790M
Kim Taeyun Data curation Formal analysis Writing – original draft 1 2
Choe Junsu Data curation Validation 1
Shin Sun Hye Validation 1
Jeong Byeong-Ho Validation 1
Lee Kyungjong Validation 1
Kim Hojoong Validation 1
Lee Se-Hoon Validation 3
https://orcid.org/0000-0002-9765-9068
Um Sang-Won Conceptualization Formal analysis Funding acquisition Validation Writing – review & editing 1 4 *
1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
2 Department of Medicine, Kosin University Gospel Hospital, Kosin University College of Medicine, Busan, Republic of Korea
3 Department of Medicine, Division of Hematology and Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
4 Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea
Montazeri Aliabadi Hamidreza Editor
Chapman University, UNITED STATES OF AMERICA
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: sangwonum@skku.edu
19 9 2024
2024
19 9 e03100798 4 2024
23 8 2024
© 2024 Kim et al
2024
Kim et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Objectives

This study was performed to investigate the detection rate of EGFR T790M mutation by repeated rebiopsy, to identify the clinical factors related to repeated rebiopsy, and to assess survival outcomes according to the methods and numbers of repeated rebiopsies in patients with lung adenocarcinoma who received sequential osimertinib after failure of previous 1st or 2nd generation EGFR-tyrosine kinase inhibitors.

Methods

This retrospective study included patients with advanced-stage lung adenocarcinoma who were confirmed to have EGFR T790M mutation and to have received osimertinib from January 2020 to February 2021 at Samsung Medical Center. The presence of T790M mutation was assessed based on either plasma circulating tumor DNA (ctDNA) or tissue specimens.

Results

A total of 443 patients underwent rebiopsy, with 186 (42.0%) testing positive for the T790M mutation by the sixth rebiopsy. The final analysis included 143 eligible patients. Progression-free survival was not significantly different in terms of the methods (tissue: 13.3 months, 95% confidence interval [CI]: [9.4, 23.5] vs plasma: 11.1 months, 95% CI: [8.1, 19.4], p = 0.33) and numbers (one: 13.4 months, 95% CI: [9.4, 23.5] vs two or more: 11.0 months, 95% CI: [8.1, 14.8], p = 0.51) of repeated rebiopsies. Longer overall survival (OS) was found in patients in whom T790M was detected by tissue specimens rather than by plasma ctDNA (2-year OS rate: 81.7% for tissue vs 63.9% for plasma, p = 0.0038). Factors related to the lower numbers of rebiopsies included age and bone metastasis. Factor associated with T790M detection in tissue rather than in plasma was pleural metastasis, while advanced tumor stage was related to T790M confirmation in plasma rather than in tissue.

Conclusions

Repeated rebiopsy for T790M detection in patients with NSCLC can increase the detection rate of the mutation. Detection of T790M by plasma ctDNA might be related to poor survival outcomes.

National Research Foundation of Korea 2020R1A2C2006282 https://orcid.org/0000-0002-9765-9068
Um Sang-Won This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1A2C2006282). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the article and its Supporting Information files.
Data Availability

All relevant data are within the article and its Supporting Information files.
==== Body
pmcIntroduction

In patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation, EGFR tyrosine kinase inhibitors (TKIs) yield significant treatment responses. However, most cases progress after initial tumor shrinkage, and secondary resistance to EGFR-TKIs unavoidably leads to treatment failure [1]. T790M mutation is detected in approximately 50% of patients with NSCLC who develop resistance to EGFR-TKIs [2]. Osimertinib is effective against the T790M mutation and markedly improves survival outcomes for patients with advanced-stage EGFR-mutated NSCLC, especially for lung adenocarcinoma (LUAD), in whom previous regimens have failed [3].

Identification of the T790M mutation can guide physicians to properly initiate osimertinib or next-generation EGFR-TKIs for patients whose disease has progressed. At first rebiopsy, however, only 44% of patients were found to have the T790M mutation [4]. This may lead to subsequent rebiopsy attempts, which may in turn increase the detection rate of T790M for the prescription of osimertinib [5,6]. Therefore, it is necessary to attempt repeated rebiopsy to confirm the T790M mutation. However, few data exist regarding whether the T790M detection rate can be enhanced by repeated rebiopsy, which factors are associated with repeated rebiopsy, or survival outcomes in relation to these attempts. A recent meta-analysis of eight studies involving 1,013 patients with NSCLC revealed an increased T790M detection rate by repeated rebiopsy and no difference in progression-free survival (PFS) between first and repeated rebiopsy [6]. However, considerable heterogeneity was present among the studies because of the small numbers of participants and non-uniform measurement of EGFR mutation across different clinics [6].

In this context, the present study was performed to investigate the detection rate of T790M by repeated rebiopsy, identify the clinical factors related to repeated rebiopsy, and assess survival outcomes according to the methods and number of repeated rebiopsies in patients with lung adenocarcinoma who received sequential osimertinib after failure of previous 1st or 2nd generation EGFR-TKIs.

Materials and methods

Datasets and patient selection

This retrospective single-center observational study was performed at Samsung Medical Center. The electronic medical records of patients who met the following selection criteria from January 2020 to February 2021 were reviewed: aged ≥19 years; pathologically confirmed LUAD; advanced stage of disease, i.e., American Joint Committee on Cancer (AJCC) stage 3, 4A, or 4B; and the patients who received rebiopsy for the detection of T790M mutation.

The study protocol was approved by the Institutional Review Board of Samsung Medical Center (no. 2023-05-057). The study was conducted in accordance with the Declaration of Helsinki. All procedures were performed in accordance with relevant guidelines and regulations.

Measurements

Information was collected on the patients’ baseline characteristics, including age, sex, body mass index, smoking status (never or ever), tumor stage based on the 8th edition of the AJCC staging manual, and the Charlson comorbidity index. We also collected data on the number of metastatic organs, presence of metastasis to specific organs (brain, liver, bone, lung, or pleura), previous curative surgical treatment or radiotherapy, and the number of regimens before osimertinib. We collected data from the point at which treatment commenced in the palliative setting.

Tissue, plasma, and body fluid specimens were obtained for EGFR mutation analysis. For tissue specimens, EGFR mutations were confirmed using either a peptide nucleic acid clamp kit and real-time polymerase chain reaction (Roche Cobas EGFR mutation test; Roche Molecular Systems, Pleasanton, CA, USA) or next-generation sequencing (NGS) using a customized targeted panel (TruSight Oncology 500 Assay; Illumina, San Diego, CA, USA). Plasma circulating tumor DNA (ctDNA) was analyzed using either real-time polymerase chain reaction (Roche Cobas EGFR mutation test) or NGS (TruSight Oncology 500 Assay; Illumina, San Diego, CA, USA). Meanwhile, the next target for rebiopsy was based on the clinician’s decision.

Outcomes

PFS with osimertinib and overall survival (OS) were measured. PFS was defined as the time from initiation of osimertinib to tumor progression or death during osimertinib therapy. OS was defined as the time from the initiation of osimertinib to death of any cause. Data from patients who were still receiving treatment or were alive at the time of data collection were censored (31 May 2023).

Statistical analysis

Data are presented as mean ± standard deviation or median (interquartile range: IQR) for continuous variables and n (%) for categorical variables. Exploratory data analyses were performed to identify the impact of patients’ baseline characteristics. Student’s t-test and the chi-square test or Fisher’s exact test were used to compare the methods and number of repeated rebiopsies according to the patients’ characteristics for continuous and categorical variables, respectively. The Kaplan–Meier method and log-rank test were performed to visualize and compare PFS and OS according to the methods and number of rebiopsies for T790M confirmation. Subgroup analyses were performed by sex, age, smoking status, and AJCC stage. A multivariable logistic regression model with a stepwise selection process was used to identify factors related to repeated rebiopsy. P-value below 0.05 was considered statistically significant. All statistical analyses were performed using R software (version 4.3.3 for Windows; R Development Core Team).

Results

Baseline characteristics

Fig 1 shows the patient selection process. A total of 443 patients with LUAD received rebiopsies, and 186 were positive for T790M after sequential rebiopsies (Table 1), with the cumulative rate of T790M detection increasing as follows: 14.2% after the first rebiopsy, 31.8% after the second, 38.8% after the third, 41.1% after the fourth, 41.5% after the fifth, and 42.0% after the sixth. Because this study was designed to analyze patients with LUAD who were confirmed to have T790M mutation and to have received sequential osimertinib in a palliative setting, we excluded 43 patients for the following reasons: underwent rebiopsy on the same day as positive confirmation of T790M (n = 6), treatment with osimertinib after concurrent radiotherapy (n = 8), treatment with osimertinib as first-line therapy (n = 12), positivity for T790M before first-line therapy (e.g., de novo T790M mutation) (n = 4), no available data (n = 7), death within a few days of osimertinib initiation (n = 1), and received other 3rd generation TKIs (e.g., lazertinib or olmutinib). For the final analysis, 143 patients were included.

10.1371/journal.pone.0310079.g001 Fig 1 Patient selection process.

LUAD, lung adenocarcinoma; AJCC, American Joint Committee on Cancer; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor.

10.1371/journal.pone.0310079.t001 Table 1 Detection rate of T790M in all patients who received a rebiopsy during the study period.

	First	Second	Third	Fourth	Fifth	Sixth	
T790M, n (%)							
    Positive	63 (14.2)	78 (21.5)	31 (16.1)	10 (10.1)	2 (4.8)	2 (12.5)	
    Negative	380 (85.8)	285 (78.5)	162 (83.9)	89 (89.9)	40 (95.2)	14 (87.5)	
Total	443	363	193	99	42	16	
    Liquid, n (%)	330 (74.5)	148 (40.8)	80 (41.5)	43 (43.4)	18 (42.9)	5 (31.3)	
    Tissue, n (%)	113 (25.5)	215 (59.2)	113 (58.5)	56 (56.6)	24 (57.1)	11 (68.8)	
Cumulative % for T790M	14.2	31.8	38.8	41.1	41.5	42.0	

Clinical characteristics of 143 study subjects are displayed in Tables 2 and S1. The patients’ mean age was 66 years, and 55.9% were female (Table 2). Stage 4B cancer was present in 52.4% of the patients. Of all 143 patients, the most prevalent site of metastasis was the lung (n = 71, 49.7%), followed by the pleura (n = 67, 46.9%), bone (n = 55, 38.5%), brain (n = 50, 35.0%), and liver (n = 18, 12.6%). Curative chemoradiotherapy had been performed previously in 20 (14.0%) patients, and a curative operation had been previously performed in 35 (24.5%) patients. Exon 19 deletion was found in 88 (61.5%) patients, which was higher than the rate of L858R (52, 36.4%). Most of patients received gefitinib (68, 47.6%) or afatinib (62, 43.4%) before the initiation of osimertinib.

10.1371/journal.pone.0310079.t002 Table 2 Patients’ baseline characteristics (N = 143).

Age, years	66.0 ± 10.2	
Female sex	80 (55.9)	
Body mass index, kg/m 2	23.3 ± 3.5	
Smoking status		
    Never	91 (63.6)	
    Ever	52 (36.4)	
Charlson comorbidity index		
    6	119 (83.2)	
    ≥ 7	24 (16.8)	
AJCC tumor stage		
    3–4A	68 (47.5)	
    4B	75 (52.4)	
Number of metastatic organs		
    0–1	56 (39.2)	
    2–3	68 (47.6)	
    ≥ 4	19 (13.3)	
EGFR mutation		
    Exon 19 Deletion	88 (61.5)	
    L858R	52 (36.4)	
    Others	3 (2.1)	
Brain metastasis	50 (35.0)	
Liver metastasis	18 (12.6)	
Bone metastasis	55 (38.5)	
Lung-to-lung metastasis	71 (49.7)	
Pleural metastasis	67 (46.9)	
Previous curative chemoradiotherapy	20 (14.0)	
Previous curative surgery	35 (24.5)	
Previous EGFR-TKI treatment
Gefitinib
Afatinib	
68 (47.6)
62 (43.4)	
    Erlotinib	13 (9.1)	
Number of previous regimens		
    1	108 (75.5)	
    2	21 (14.7)	
    3	14 (9.8)	
Data are presented as mean ± standard deviation for continuous variables and n (%) for categorical variables.

AJCC, American Joint Committee on Cancer.

Clinical outcomes and survival

The median interval between the first and second rebiopsy was 12.5 days (IQR: 8.5–21.0 days). PFS was not significantly different in terms of the methods (tissue: 13.3 months, 95% confidence interval [CI]: [9.4, 23.5] vs plasma: 11.1 months, 95% CI: [8.1, 19.4], p = 0.33) and numbers (one: 13.4 months, 95% CI: [9.4, 23.5] vs two or more: 11.0 months, 95% CI: [8.1, 14.8], p = 0.51) of repeated rebiopsies (Table 3). When confining the analysis to patients whose tumor stage was > 3A at the initial diagnosis, the result was not different (S2 Table).

10.1371/journal.pone.0310079.t003 Table 3 Progression-free survival (months) according to the number of rebiopsies and methods for confirmation of T790M mutation.

	1-year PFS rate	Median	95% CI	p	
Number of rebiopsies				0.51	
One	54.7%	13.4	10.3–20.5		
Two or more	40.9%	11.0	8.1–14.8		
Confirmation of T790M				0.33	
Tissue	55.2%	13.3	9.4–23.5		
Plasma	46.9%	11.1	8.1–19.4		
Confirmation of T790M				0.49	
Tissue, first	62.2%	16.6	12.0–28.8		
Plasma, first	50.3%	12.2	8.3–19.6		
Tissue, second or more	47.6%	11.2	8.1–26.4		
Plasma, second or more	16.7%	9.0	5.6–NA		
PFS, progression free survival; CI, confidence interval; NA, not available.

In terms of OS, no significant difference according to the number of rebiopsies was found (Table 4). However, when patients were stratified by the method used to confirm T790M, a significant difference in OS was observed (Fig 2): patients whose mutation was confirmed through a tissue specimen survived for a longer period than patients whose mutation was detected in a plasma sample (i.e., ctDNA) (2-year OS rate: 81.7% for tissue vs 63.9% for plasma, p = 0.0038). A similar trend was found when further stratifying patients by both the method and number of repeated rebiopsies. When confining the analysis to patients with an advanced tumor stage at the initial diagnosis, it was also found that the OS was different between the methods of T790M confirmation (tissue vs. plasma) (S1 Fig and S3 Table).

10.1371/journal.pone.0310079.g002 Fig 2 Difference in OS between the methods of T790M confirmation.

OS, overall survival.

10.1371/journal.pone.0310079.t004 Table 4 Overall survival (months) according to the number of rebiopsies and methods for confirmation of T790M mutation.

	2-year OS rate	Median	95% CI	p	
Number of rebiopsies				0.13	
One	70.6%	NR	32.2–NA		
Two or more	78.3%	NR	NA–NA		
Confirmation of T790M				0.0038	
Tissue	81.7%	NR	NA–NA		
Plasma	63.9%	NR	26.3–NA		
Confirmation of T790M				0.031	
Tissue, first	79.9%	NR	NA–NA		
Plasma, first	64.7%	NR	27.8–NA		
Tissue, second or more	84.1%	NR	NA–NA		
Plasma, second or more	53.3%	NR	14.2–NA		
OS, overall survival; CI, confidence interval; NA, not available; NR, not reached.

Clinical characteristics associated with the number of rebiopsies

The multivariable-adjusted odds ratios of factors influencing the number of rebiopsies are shown in Table 5. Older age and bone metastasis were significantly associated with a decreased likelihood of undergoing a second or subsequent rebiopsies.

10.1371/journal.pone.0310079.t005 Table 5 Factors associating influencing the number of rebiopsies and the confirmation of T790M by plasma ctDNA analysis.

	OR*	95% CI	p	
2 or more rebiopsies				
Age	0.94	0.90–0.98	0.006	
Bone metastasis	0.19	0.06–0.46	<0.001	
Number of previous regimens				
≥ 2 (reference: 1)	0.39	0.13–1.09	0.087	
T790M confirmation by plasma ctDNA analysis				
Age	1.03	0.99–1.08	0.124	
Stage 4B (reference: 3–4A)	3.81	1.70–8.87	0.001	
Pleural metastasis	0.44	0.19–0.99	0.048	
Liver metastasis	2.68	0.76–11.26	0.144	
Number of previous treatment regimens	3.91	1.00–19.34	0.064	
≥ 2 (reference: 1)	1.96	0.77–5.12	0.16	
* OR was calculated using a stepwise logistic regression model considering age, sex, body mass index, smoking status, EGFR mutation status, Charlson’s comorbidity index, tumor stage, numbers of organ metastasis, presence of brain, liver, bone, and pleural metastasis, and the numbers of previous regimens.

OR of > 1 indicates a higher probability of undergoing multiple rebiopsies or detecting T790M by plasma.

OR, odds ratio; ctDNA, circulating tumor DNA.

Clinical characteristics associated with detection of T790M in plasma ctDNA

The characteristics of patients according to the sites of T790M detection are summarized in S4 Table. Stage 4B, brain metastasis, bone metastasis and pleural metastasis were more common in the group with T7900M confirmation using plasma ctDNA compared to those with T790M confirmation using tissue biopsy. The multivariable-adjusted odds ratios of T790M confirmation through plasma ctDNA are presented in Table 5. Pleural metastasis was related to mutation detection in tissue specimens. Conversely, more advanced tumor stage (4B) was related to the detection in plasma samples.

Discussion

In this single-institution retrospective study, we analyzed patients with advanced-stage LUAD who underwent failed EGFR-TKI therapy and other therapies and were subsequently treated with osimertinib in the presence of T790M mutation. We comprehensively followed the trajectories of T790M confirmation according to the number of rebiopsies and the site of mutation detection. The detection rate of T790M detection rate in all patients who received sequential rebiopsies was 42.0%. OS were longer in patients whose mutation was detected in tissue than in plasma: it was similar in the analysis that confined to the patients who were initially diagnosed at an advanced stage and had received no previous curative surgery or chemoradiotherapy. Specifically, patients whose mutation was initially found in tissue specimens had the longest survival, followed by those whose mutation was found in tissue specimens at the second or more rebiopsy, plasma samples at the first rebiopsy, and plasma samples at the second or more rebiopsy. Factors associated with the number of rebiopsies included older age and bone metastasis. Factor associated with T790M confirmation in tissue rather than in plasma was the presence of pleural metastasis, while advanced tumor stage was related to T790M confirmation in plasma rather than in tissue.

OS was significantly longer when the T790M mutation was found in tissue than in plasma. This difference in survival might be attributed to the fact that patients whose mutation was detected in plasma samples had more advanced disease and a higher rate of metastasis to specific organs at the initiation of osimertinib (S4 Table). Although several prior studies have examined differences in survival outcomes (e.g., PFS or OS) based on the sites at which T790M mutation was confirmed, the results are inconsistent [5,7–12]. For example, some studies showed differences in PFS according to the rebiopsy site [7,9,10], whereas others showed no differences [8,12]. Relatively few studies have reported relevant data on OS, but they consistently showed poorer OS when the mutation was found in plasma rather than in tissue [10,11]. Our findings are in line with these previous results regarding OS.

The overall detection rate of T790M was comparable but slightly lower to previous reports [6,7,13–15]. In our study, the detection rate after first rebiopsy was 14.2%, 31.8% after the second, 38.8% after the third, 41.1% after the fourth, 41.5% after the fifth, and 42.0% after the sixth. A retrospective study of 80 patients with NSCLC showed that, in 53.7% of patients who progressed after first-line therapy with first- or second-generation T790M mutation in EGFR-TKIs was found at the first rebiopsy, and subsequent rebiopsies increased the detection rate by 12.5%, 2.5%, 1.2%, and 1.2% [14]. Another meta-analysis showed that the detection rate after the first rebiopsy was 44.2%, and the rate after repeated rebiopsy was 46.5%; the pooled detection rate of the first and repeated rebiopsies was 54.5% [6]. This discrepancy likely originated from the differences in the method by which the repeated rebiopsy performed: In our institution, liquid biopsy was used as the primary method for T790M detection (74.5%, 330/443), likely due to the simplicity of blood sampling compared to tissue acquisition. Meanwhile, tissue biopsy was the predominant method for the second rebiopsy (59.2%, 215/363). Interindividual spatiotemporal heterogeneity of EGFR mutations, especially T790M, has been demonstrated in patients with NSCLC for whom EGFR-TKI therapy failed [16,17]. This could result in an unsuccessful single attempt at the detection of T790M mutation, necessitating additional attempts at different sites. Many advances in techniques have led to the use of liquid biopsy as a preferred method for T790M detection [18]. However, there still exists a group of patients in whom T790M is more likely to be confirmed within tissue specimens [19]. An individualized approach to detect T790M mutation is necessary.

With respect to establishing individualized strategies for T790M detection, our findings regarding the factors related to the number of rebiopsies and the sites of mutation detection add valuable information to the literature. Some studies have suggested potential factors related to positive T790M detection. A multicenter retrospective study in Italy showed that bone metastasis and high numbers of metastatic organs were associated with T790M detection [20]. Our study similarly indicated that patients with advanced disease at the time of rebiopsy and patients with long-standing disease may benefit from the less invasive method of ctDNA sampling to detect T790M. Meanwhile, a negative correlation was found between pleural metastasis and the possibility of detection by a plasma sample. This difference from other organs might be related to the tumor microenvironment, molecular pathways, or genetic traits that affect tumor affinity [21]. Data on the negative correlation of pleural metastasis with other sites of metastasis have accumulated from observational studies [22,23].

Our study has several limitations. First, it was a retrospective study based on electronic medical records of a single institution, and the results are not generalizable to all hospitals or clinical settings. For example, multiple rebiopsies might not be feasible in many institutions. Second, given the nature of the study design, causality in the relationships of associated factors with the number and methods of repeated rebiopsies should be inferred with caution. Third, because our institution initiated liquid biopsy in 2018, the follow-up period was relatively short. Further data after the survival period are necessary to elucidate the longitudinal impact of repeated rebiopsy.

Conclusion

Repeated rebiopsy for the confirmation of T790M mutation in patients with LUAD who underwent treatment failure with previous regimens, including EGFR-TKI therapy, may increase the detection rate of T790M mutation. This could in turn guide physicians in terms of starting their patients on osimertinib. In particular, active rebiopsy of tissue could be considered in younger patients or those with a lower disease stage because there is a higher likelihood that mutations will not be detected in plasma samples. Confirmation of T790M in plasma rather than tissue may be related to poor survival outcomes, which might reflect advanced disease. Given the inter- and intraindividual spatiotemporal heterogeneity of EGFR mutations, it is important to personalize the strategy for repeated rebiopsy.

Supporting information

S1 Fig Difference in OS between the methods of T790M confirmation in patients with advanced-stage disease at diagnosis.

OS, overall survival. *Patients who were diagnosed with stage 3, 4A, or 4B at their first diagnosis were analyzed.

(TIF)

S1 Table Clinical data set of 143 study subjects.

(XLSX)

S2 Table Progression-free survival (months) according to the number of rebiopsies and methods for confirmation of T790M mutation in patients initially diagnosed with advanced disease.

(DOCX)

S3 Table Overall survival (months) according to the number of rebiopsies and methods for confirmation of T790M mutation in patients initially diagnosed with advanced disease.

(DOCX)

S4 Table Patient characteristics according to sites of T790M confirmation.

(DOCX)

10.1371/journal.pone.0310079.r001
Decision Letter 0
Montazeri Aliabadi Hamidreza Academic Editor
© 2024 Hamidreza Montazeri Aliabadi
2024
Hamidreza Montazeri Aliabadi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
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PONE-D-24-12639Repeated rebiopsy for detection of EGFR T790M mutation in patients with advanced-stage lung adenocarcinoma: associated factors and treatment outcomes of OsimertinibPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting study about the T790M yield of repeated biopsies under EGFR TKI progression and their association with patient outcomes.

I have the following comments:

1. It would have been helpful to give some information about the entire patient population: 100% had T790M after max 3 biopsies, but how many received biopsies (i.e. how many other patients had all biopsies negative) in the same time period?

2. It would be important to add the type of EGFR mutation in Table 1 (del19 vs. L858R vs. other rare mutations)

3. Regarding the analysis of survival: it would be important to also check other prognostic parameters, like the type of EGFR mutation. It is known that the type of EGFR mutation and presence of brain metastases are independent predictors of OS for these patients, which should be taken into account in the analysis, see for example https://pubmed.ncbi.nlm.nih.gov/32871455/, which could also be added in the Discussion.

Reviewer #2: Um et al reported implications of repeated rebiopsy for T790M detection in patients with EGFR-mutant NSCLC and associated factors and treatment outcomes of osimertinib.

Their manuscript includes several interesting points for readers. However, there is a critical methodologic problem in their study, and major modification is necessary.

Major points:

1. Their study population focused on only osimertinib-administered patients. They insisted T790M detection rate on first rebiopsy of 69.9%. This is absolutely incorrect. As they mentioned, many studies reported clinical T790M detection rate on first rebiopsy of 30-50%. These previous studies consecutively examined all patients who received rebiopsy to detect T790M in their hospitals. The authors should also consecutively examine all rebiopsied patients in their institute. This is a critical problem in their study.

2. Similarly, T790M detection rates on second and third rebiopsies were incorrect. They excluded cases without T790M, since their study only examined T790M-positive cases.

3. They described “The median interval between the first and second rebiopsy was 12.5 days (IQR: 8.5-21.0 days).” This is astonished. After failure of first rebiopsy, was second rebiopsy performed on the same target? or another target? Their manuscript did not show detailed information on rebiopsy targets.

Minor point:

1. EGFR should be italicized when they express a gene. (e.g., EGFR mutation)

**********

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Reviewer #1: Yes: Petros Christopoulos

Reviewer #2: No

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10.1371/journal.pone.0310079.r002
Author response to Decision Letter 0
Submission Version1
30 Jul 2024

Reviewer 1

Comment 1: It would have been helpful to give some information about the entire patient population: 100% had T790M after max 3 biopsies, but how many received biopsies (i.e. how many other patients had all biopsies negative) in the same time period?

Response 1: Thank you for the valuable comment.

In response to the comments from both reviewers, we re-checked the entire patient population who received rebiopsies during the study period. Therefore, we have also revised the patient selection process. In brief, a total of 443 patients received rebiopsies, with the cumulative rate of T790M detection increasing as follows: 14.2% after the first rebiopsy, 31.8% after the second, 38.8% after the third, 41.1% after the fourth, 41.5% after the fifth, and 42.0% after the sixth.

We summarized these results as Fig 1 and Table 1. Also, we have revised the related sections: Abstract (Results), Materials and Methods, 1st paragraph of Results, and 1st and 3rd paragraph of Discussion.

Comment 2 & 3: It would be important to add the type of EGFR mutation in Table 1 (del19 vs. L858R vs. other rare mutations). Regarding the analysis of survival: it would be important to also check other prognostic parameters, like the type of EGFR mutation. It is known that the type of EGFR mutation and presence of brain metastases are independent predictors of OS for these patients, which should be taken into account in the analysis, see for example https://pubmed.ncbi.nlm.nih.gov/32871455/, which could also be added in the Discussion.

Response 2 & 3: Thank you for the comment and sharing a valuable reference.

First, we have added numbers and percentages of EGFR mutation in Table 2. Second, we have re-estimated odds ratio from multivariable analyses additionally considering EGFR mutation status. Results are summarized in Table 5: however, in this analysis we could not find EGFR mutation as a distinguishing parameter in terms of number of rebiopsy and method for rebiopsy. Please refer to the Results section.

Reviewer 2

Major points:

Comment 1 & 2: Their study population focused on only osimertinib-administered patients. They insisted T790M detection rate on first rebiopsy of 69.9%. This is absolutely incorrect. As they mentioned, many studies reported clinical T790M detection rate on first rebiopsy of 30-50%. These previous studies consecutively examined all patients who received rebiopsy to detect T790M in their hospitals. The authors should also consecutively examine all rebiopsied patients in their institute. This is a critical problem in their study.

Similarly, T790M detection rates on second and third rebiopsies were incorrect. They excluded cases without T790M, since their study only examined T790M-positive cases.

Response 1 & 2: Thank you for the valuable comment.

In response to the comments from both reviewers, we have re-checked the entire patient population who received rebiopsies during the study period. Therefore, we have also revised the patient selection process. In brief, a total of 443 patients received rebiopsies, with the cumulative rate of T790M detection increasing as follows: 14.2% after the first rebiopsy, 31.8% after the second, 38.8% after the third, 41.1% after the fourth, 41.5% after the fifth, and 42.0% after the sixth.

We have summarized these results as Fig 1 and Table 1. Also, we have revised the related sections: Abstract (Results), Materials and Methods, 1st paragraph of Results, and 1st and 3rd paragraph of Discussion.

Comment 3: They described “The median interval between the first and second rebiopsy was 12.5 days (IQR: 8.5-21.0 days).” This is astonished. After failure of first rebiopsy, was second rebiopsy performed on the same target? or another target? Their manuscript did not show detailed information on rebiopsy targets.

Response 3: Thank you for the comment. The next target for rebiopsy was based on the clinician’s decision rather than on a protocolized approach. Therefore, we clarified this point in the method section.

Minor point:

Comment 1: EGFR should be italicized when they express a gene. (e.g., EGFR mutation)

Response 1: We have italicized EGFR when it expresses the gene.

Attachment Submitted filename: Response to Reviewers_revised_240727.docx

10.1371/journal.pone.0310079.r003
Decision Letter 1
Montazeri Aliabadi Hamidreza Academic Editor
© 2024 Hamidreza Montazeri Aliabadi
2024
Hamidreza Montazeri Aliabadi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
16 Aug 2024

PONE-D-24-12639R1Repeated rebiopsy for detection of EGFR T790M mutation in patients with advanced-stage lung adenocarcinoma: associated factors and treatment outcomes of OsimertinibPLOS ONE

Dear Dr. Um,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please make sure to specifically address the concerns raised by Reviewer 2.

Please submit your revised manuscript by Sep 30 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Hamidreza Montazeri Aliabadi

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all reviewers' comments and improved the manuscript accordingly.

There are no further comments

Reviewer #2: Um et al revised and resubmitted their manuscript with major modifications.

Although their manuscript has several interesting and clinically meaningful points, there are still some unresolved critical problems in the manuscript.

I feel Figure 1 was well modified and easy to understand for readers and their conclusions are excellent.

Major points:

1. Results of abstract is inappropriate for a scientific paper. They exhibited little numeric data. Guide for authors of PLOS ONE describes, “Results – What did you find? Briefly give the key findings of your study. Include key numeric data (including confidence intervals or p values), where possible.”. They should thoroughly modify it. Table 1 is impressive and easy to understand for readers, and the findings should be included in the Results section.

2. S3 Table appears first in the Discussion. Also, sentences on lines 256-259: “In our institution, liquid biopsy was used as the primary method for T790M detection (73.7%, 330/448), likely due to the simplicity of blood sampling compared to tissue acquisition. Meanwhile, tissue biopsy was the predominant method for the second rebiopsy (59.2%, 215/363).” appear first in the Discussion. Such expressions are really inappropriate for scientific papers. What the authors did not mention in the Result should not be used in the Discussion. They must mention these results in the Result section, previously.

3. T790M-positive rate at first rebiopsy was only 14%. This is lower than former pivotal reports on T790M-positive rate: 30-50%. They considered high rate of liquid biopsy caused this lower T790M-positive rate. So, they should demonstrate rates of liquid and histologic biopsies from first to sixth rebiopsy in the Result section.

4. On lines 146-147, they described, “For the final analysis, 143 patients were included and they received a maximum of three repeated rebiopsies.”. This is confusing. Table 1 shows some patients received sixth rebiopsy. What?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Petros Christopoulos

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0310079.r004
Author response to Decision Letter 1
Submission Version2
19 Aug 2024

Response to Reviewer’s comments

Um et al revised and resubmitted their manuscript with major modifications.

Although their manuscript has several interesting and clinically meaningful points, there are still some unresolved critical problems in the manuscript.

I feel Figure 1 was well modified and easy to understand for readers and their conclusions are excellent.

Major points:

Comment 1: Results of abstract is inappropriate for a scientific paper. They exhibited little numeric data. Guide for authors of PLOS ONE describes, “Results – What did you find? Briefly give the key findings of your study. Include key numeric data (including confidence intervals or p values), where possible.”. They should thoroughly modify it. Table 1 is impressive and easy to understand for readers, and the findings should be included in the Results section.

Response 1: Thank you for the comment. We have added key numerical data with p values to the Results section of Abstract as follows.

“Progression-free survival was not significantly different in terms of the methods (tissue: 13.3 months, 95% confidence interval [CI]: [9.4, 23.5] vs plasma: 11.1 months, 95% CI: [8.1, 19.4], p=0.33) and numbers (one: 13.4 months, 95% CI: [9.4, 23.5] vs two or more: 11.0 months, 95% CI: [8.1, 14.8], p=0.51) of repeated rebiopsies. Longer overall survival (OS) was found in patients in whom T790M was detected by tissue specimens rather than by plasma ctDNA (2-year OS rate: 81.7% for tissue vs 63.9% for plasma, p = 0.0038).”

We have also added the key numerical data in the Results section.

Comment 2: S3 Table appears first in the Discussion. Also, sentences on lines 256-259: “In our institution, liquid biopsy was used as the primary method for T790M detection (73.7%, 330/448), likely due to the simplicity of blood sampling compared to tissue acquisition. Meanwhile, tissue biopsy was the predominant method for the second rebiopsy (59.2%, 215/363).” appear first in the Discussion. Such expressions are really inappropriate for scientific papers. What the authors did not mention in the Result should not be used in the Discussion. They must mention these results in the Result section, previously.

Response 2: Thank you for your comments.

We have added following sentences in the Results section (Clinical characteristics associated with detection of T790M in plasma ctDNA).

“The characteristics of patients according to the sites of T790M detection are summarized in S3 Table. Stage 4B, brain metastasis, bone metastasis and pleural metastasis were more common in the group with T7900M confirmation using plasma ctDNA compared to those with T790M confirmation using tissue biopsy.”

We have also added following sentences in the Results section (Baseline Characteristics).

“The majority of patients (74.5%) received their first rebiopsy using a liquid biopsy, while most patients at the second rebiopsy received a tissue biopsy (59.2%).”

Comment 3: T790M-positive rate at first rebiopsy was only 14%. This is lower than former pivotal reports on T790M-positive rate: 30-50%. They considered high rate of liquid biopsy caused this lower T790M-positive rate. So, they should demonstrate rates of liquid and histologic biopsies from first to sixth rebiopsy in the Result section.

Response 3: Thank you for your insightful comment. We have added the data on the rates of liquid and tissue biopsies from first to sixth rebiopsy in Table 1 and have described the content in the Results section

Comment 4: On lines 146-147, they described, “For the final analysis, 143 patients were included and they received a maximum of three repeated rebiopsies.”. This is confusing. Table 1 shows some patients received sixth rebiopsy. What?

Response 4: We apologize for the confusion. To be clear, we have revised the sentence as follows: “For the final analysis, 143 patients were included”

10.1371/journal.pone.0310079.r005
Decision Letter 2
Montazeri Aliabadi Hamidreza Academic Editor
© 2024 Hamidreza Montazeri Aliabadi
2024
Hamidreza Montazeri Aliabadi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
26 Aug 2024

Repeated rebiopsy for detection of EGFR T790M mutation in patients with advanced-stage lung adenocarcinoma: associated factors and treatment outcomes of Osimertinib

PONE-D-24-12639R2

Dear Dr. Um,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hamidreza Montazeri Aliabadi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

10.1371/journal.pone.0310079.r006
Acceptance letter
Montazeri Aliabadi Hamidreza Academic Editor
© 2024 Hamidreza Montazeri Aliabadi
2024
Hamidreza Montazeri Aliabadi
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
10 Sep 2024

PONE-D-24-12639R2

PLOS ONE

Dear Dr. Um,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Hamidreza Montazeri Aliabadi

Academic Editor

PLOS ONE
==== Refs
References

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2 Westover D , Zugazagoitia J , Cho BC , Lovly CM , Paz-Ares L . Mechanisms of acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors. Ann Oncol. 2018;29 (suppl_1 ):i10–i9. doi: 10.1093/annonc/mdx703 ; PubMed Central PMCID: PMC6454547.29462254
3 Mok TS , Wu YL , Ahn MJ , Garassino MC , Kim HR , Ramalingam SS , et al . Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer. N Engl J Med. 2017;376 (7 ):629–40. Epub 20161206. doi: 10.1056/NEJMoa1612674 ; PubMed Central PMCID: PMC6762027.27959700
4 Kawamura T , Kenmotsu H , Omori S , Nakashima K , Wakuda K , Ono A , et al . Clinical Factors Predicting Detection of T790M Mutation in Rebiopsy for EGFR-Mutant Non-small-cell Lung Cancer. Clin Lung Cancer. 2018;19 (2 ):e247–e52. Epub 20170810. doi: 10.1016/j.cllc.2017.07.002 .28866043
5 Ninomaru T , Hata A , Kokan C , Okada H , Tomimatsu H , Ishida J . Higher osimertinib introduction rate achieved by multiple repeated rebiopsy after acquired resistance to first/second generation EGFR-TKIs. Thorac Cancer. 2021;12 (6 ):746–51. Epub 20210121. doi: 10.1111/1759-7714.13822 ; PubMed Central PMCID: PMC7952804.33475261
6 Kim S , Kim SH , Kim J , Kim MH , Lee MK , Eom JS . Should We Perform Repeated Re-biopsy for the Detection of T790M Mutation? Cancer Res Treat. 2023;55 (4 ):1190–7. Epub 20230417. doi: 10.4143/crt.2023.320 ; PubMed Central PMCID: PMC10582528.37080607
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