Concurrent EGFR L858R and K860I mutations: PCR-negative but NGS-positive alterations that respond to EGFR inhibitors in non-small cell lung cancer

doi:10.21203/rs.3.rs-5234168/v1

Introduction: Clinical routine amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and next generation sequencing (NGS) are able to identify sensitizing EGFR mutations of non-small cell lung cancer (NSCLC) patients. Nevertheless, the detection and treatment response of NSCLC patients with concurrent L858R and uncommon EGFRexon 21 mutations remain unclear.

Methods: Concurrent NGS and ARMS-PCR were performed to identify EGFR mutations in 5033 NSCLC cases.

Results: Totally, EGFRL858R mutations were identified in 1024 (20.3%) cases, among which 54 concurrent L858R and uncommon EGFR exon 21 mutations were identified by NGS but not ARMS-PCR. The most common concurrent pathogenic mutations in EGFR exon 21 were V834L, K860I and L833V. Of those, 23 patients received EGFR-TKIs as the first-line treatment with median progression-free survival (PFS) of 16.7 months (95% CI: 12.8–20.7 months) and objective response rate (ORR) of 38.9% (95% CI: 9.1% – 46.9%). Interestingly, concurrent L858R and K860I mutations were observed in 10 cases by NGS, while ARMS-PCR showed completely negative results in EGFR mutations. Of those, three patients received EGFR-TKIs as the first-line treatment, with median PFS of 11.0 months and ORR of 100.0%. Furthermore, three patients with concurrent L858R and K860I mutations identified by NGS received adjuvant targeted therapy after surgery with median disease free survival of 10.0 months.

Conclusion: NSCLC patients with concurrentEGFR L858R and K860I mutations are able to benefit from EGFR-TKIs, which can be detected by NGS but not by ARMS-PCR.

non-small cell lung cancer

concurrent EGFR L858R and K860I mutations

next generation sequencing

amplification refractory mutation system-polymerase chain reaction

targeted therapy

Lung cancer is the leading cause of cancer related death around the world(Collins et al. 2007), among which non-small cell lung cancer (NSCLC) is the most common histological type(Sung et al. 2021). EGFR mutations are one of the most common oncogenic driver alterations in NSCLC (Marinaş et al. 2012; Harrison et al. 2020a; Levantini et al. 2022). Accounting for almost 80-85% EGFR mutations(Ladanyi and Pao 2008; Shi et al. 2014; Lee et al. 2015), common EGFR mutations (L858R and exon 19 deletion) are widely acknowledged as strong predictors for good clinical response to EGFR tyrosine kinase inhibitors (EGFR-TKIs)(da Cunha Santos et al. 2011; Harrison et al. 2020b). In recent years, EGFR-TKIs have become the standard of care among many advanced NSCLC patients with common EGFR mutations, among which L858R is the most common subtype in China(Maemondo et al. 2010; Han et al. 2012; Soria et al. 2018). However, the treatment response for NSCLC patients with complex EGFR mutations such as concurrent L858R and uncommon EGFR exon 21 mutations remains largely unexplored(Saxon et al. 2017; Levantini et al. 2022). Especially, effective clinical evidence about treatment strategies for patients with these uncommon EGFR mutations is lacking due to challenges in trial recruitment(Harrison et al. 2020b).

Nowadays, both next generation sequencing (NGS) and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) are widely recommended and applied to identify targetable EGFR alterations in NSCLC(Hanna et al. 2017; Lindeman et al. 2018; Ettinger et al. 2018). As a classical approach to rapidly amplify targeted DNA sequences in small amount of samples with low price, ARM-PCR is widely applied in multiple malignant tumors(Rr 2022), while our previous study reported that around 5% of NSCLC patients carried uncommon EGFR mutations that could not be identified by ARMS-PCR(Li et al. 2018). With the ability to detect small mutations across all regions of desired genes with high throughput(2021), NGS have revolutionized genome sequencing in precision medicine these years(Aggarwal et al. 2019, 2021; Rr 2022), while the requirement of specific equipment and expertise as well as the demanding and time-consuming processing limited its popularization(Kockum et al. 2023). Most studies comparing different detecting strategies focused on common EGFR mutations while little is known about the difference in detecting complex EGFR mutations between ARMS-PCR and NGS, such as concurrent L858R and uncommon EGFR exon 21 mutations .

In this study, both NGS and ARMS-PCR were performed in a large cohort to identify and characterize concurrent L858R and uncommon EGFR exon 21 mutations. Besides, the association of L858R and concurrent uncommon EGFR exon 21 mutations (especially K860I) as well as the treatment response to EGFR-TKIs were further explored to uncover promising detection and treatment strategy of NSCLC patients with concurrent L858R and uncommon EGFR exon 21 mutations.

In total, formalin-fixed, paraffin-embedded (FFPE) tumor tissues were collected from 5033 NSCLC patients and detected by DNA NGS and PCR concurrently, identifying 2414 EGFR mutations and 1024 L858R mutations. This study was approved by the Institutional Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College. All the experimental and analytical methods were carried out in accord with the acknowledged guidelines. Informed consent was obtained from all patients involved.

ARMS- PCR

The detection of EGFR mutations was accomplished using ARMS-PCR with the Human EGFR Gene Mutation Detection Kits (ACCB, Beijing, China)(Li et al. 2018). Briefly, 5 ng of genomic DNA and PCR master mix were added into the PCR tubes and ARMS-PCR was performed for 5 min at 95 C, followed by 40 cycles of 95 C for 15 s, and 60 C for 1 min(Li et al. 2018).

NGS

We performed targeted-capture NGS with genomic DNA using a 56-gene panel(Li et al. 2018). In brief, 100 to 200 ng of genomic DNA was fragmented and barcoded to construct libraries, which were hybridized with the DNA panel. The libraries were sequenced and data were analyzed to identify mutations, copy number variants and rearrangements(Li et al. 2018).

Patients and treatment responses

The clinical and pathological information were collected from electronic clinical records between October 2017 and December 2023 in Cancer Hospital, Chinese Academy of Medical Sciences. Treatment responses were evaluated by computed tomography (CT) or magnetic resonance imaging (MRI) every 2 to 3 months. The objective response rate (ORR) was calculated as the total percentage of patients with complete response (CR) and partial response (PR); the disease control rate (DCR) was calculated as the percentage of patients with CR, PR and stable disease (SD), according to Response Evaluation Criteria in Solid Tumors version1.1 (RECIST v1.1)(Eisenhauer et al. 2009). The PFS was measured from the initiation date of EGFR-TKIs treatment to the date of disease progression or death of any cause.

Statistical analysis

All the statistics were performed by R v 4.2.1 or SPSS 25.0 Software. A two-sided probability less than 0.05 was considered statistically significant. Spearman correlation analysis was performed and the one-variable linear regression model was accomplished using the least square method, tested by F-test. The survival analysis was conducted using the Kaplan-Meier method and Log-rank tests. All the graphs and were produced by R v 4.2.1 or Adobe Illustrator 2022.

Concurrent ARMS-PCR and NGS in 5033 cases

Using ARMS-PCR and NGS concurrently, EGFR mutations were identified in 2414 of 5033 (48.0%) FFPE NSCLC cases. Of those, 1024 (20.3%) cases harbored EGFR L858R mutations, among which concurrent L858R and uncommon EGFR exon 21 mutations were identified in 54 cases (Figure 1A). The distribution of these concurrent uncommon EGFR exon 21 mutations are illustrated in Figure 1B. The most common concurrent EGFR exon 21 mutations were V834L (24.07%, 13/54), K860I (18.52%, 10/54) and L833V (14.81%, 8/54) in this study.

Concurrent L858R and K860I mutations in NSCLC patients

To identify driver alterations in NSCLC, both NGS and ARMS-PCR were performed for each case, NGS can detect concurrent L858R and uncommon EGFR exon 21 mutations while ARMS-PCR can only identify EGFR L858R mutation. Interestingly, concurrent L858R and K860I mutations were identified by NGS in 10 cases, meanwhile ARMS-PCR showed completely negative results (Figure 2).

Furthermore, high concordance of mutant allele frequencies (MAFs) was identified between L858R and concurrent uncommon EGFR exon 21 mutations using Spearman correlation analysis (n=54, p<0.001, Figure 3A). Also, among patients with concurrent L858R and K860I mutations, the MAFs of L858R are significantly correlated with those of K860I (n=10, P<0.0001, Figure 3B), indicating that K860I and L858R might be on the same allele from the same mutant clone.

Targeted therapy and clinical outcome

Among the 54 NSCLC patients with concurrent L858R and uncommon EGFR exon 21 mutations, 23 received EGFR-TKIs as the first-line treatment. The optimal efficacy was PR, SD and PD in 7 (31.8%), 10 (45.4%), and 1 (4.5%) cases, respectively. The ORR was 38.9% (95% confidence interval [CI]: 9.1% – 46.9%) and the median PFS was 16.7 months (95% CI: 12.8–20.7 months) (Table 1 and Supplementary Table 3). No statistically significant difference was identified between patients with concurrent L858R and uncommon EGFR exon 21 mutations and those with L858R mutation when treated with EGFR-TKIs at first line setting. (Supplementary Figure 1). It’s noteworthy that one patient with concurrent L833V and L858R mutations received Icotinib treatment with PFS of 18.4 months. Repeated DNA-NGS of lymph node biopsy after disease progression revealed that acquired K852T might be its unique resistant mechanism.

Among 10 patients with concurrent L858R and K860I mutations, three received EGFR-TKIs as the first-line treatment, with a median PFS of 11.0 months and ORR of 100.0% (Table 2). Besides, three patients with concurrent L858R and K860I mutations received adjuvant targeted therapy after surgery with a median disease free survival (DFS) of 10.0 months. Remarkably, a 56-year-old female with a tumor measuring 6.1cm*6cm in the superior lobe of right lung and a brain metastatic lesion measuring 2.3cm*1.8cm was diagnosed as stageⅣ(T4N1M1b) lung adenocarcinoma. Concurrent L858R (21.1%) and K860I (22.8%) mutations were identified by NGS but not ARMS-PCR. She received Dacomitinib as the first-line treatment and achieved a partial response for 11 months (Figure 4). After treatment failure, NGS of repeated biopsy from the superior lobe of the right lung identified a newly emerged T790M mutation (34.0%) as the resistant mechanism. She then took Osimertinib orally and gained a stable disease for 5 months (Figure 4).

Concurrent L858R and uncommon EGFR exon 21 mutations can be observed in NSCLC. Nevertheless, the detection characteristics and treatment response of these patients remain elusive. In this study, the most common EGFR exon 21 mutations that coexisted with L858R mutations were V834L (24.07%, 13/54), K860I (18.52%, 10/54) and L833V (14.81%, 8/54), all of which could not be detected by ARMS-PCR. Especially, PCR negative but NGS positive alterations were observed for concurrent EGFR L858R and K860I mutations, and NSCLC patients with these mutations identified by NGS are able to benefit from EGFR-TKIs.

ARMS-PCR is a classical approach to selectively amplify targeted DNA sequences and is widely applied to identify EGFR mutations. However, it is not able to identify uncommon or unknown mutations(Cai et al. 2019; Sousa et al. 2020). It was reported that more than 40% of NSCLC patients harboring EGFR ex20ins mutations may be undetected by ARMS-PCR assays(He et al. 2022; Ou et al. 2023). Our previous study found that around 5% NSCLC patients carried uncommon EGFR mutations that could not be identified by ARMS-PCR(Li et al. 2018). Nevertheless, NGS enables ambitious large-scale genomic sequencing efforts with its massively parallel sequencing capacity and high sensitivity(Koboldt 2020, 2021). In this study, we observed that NSCLC patients with concurrent uncommon EGFR exon 21 mutations identified by NGS could not be identified by ARMS-PCR. Interestingly, completely negative results were observed for patients with concurrent L858R and K860I mutations identified by NGS. Moreover, high concordance was observed between the MAFs of L858R and K860I, indicating that L858R and K860I mutations might be on the same allele from the same mutant clone. Thus, the base substitution sites of c.2573 T > G and c.2579 A > T are too close for the primers of ARMS-PCR to bind, leading to a completely false negative result. However, The hybrid capture-based NGS analyzes gene mutations with probes can detect both L858R and K860I.

Some previous studies explored the association of complex EGFR mutations with the efficacy of EGFR-TKI treatment. For example, Zhang’s study analyzed the treatment response of EGFR-TKIs at first-line setting for advanced adenocarcinoma patients with concurrent EGFR Del-19/21 L858R and uncommon EGFR mutations, the median PFS was 9.7 months and ORR was 60.0%(Zhang et al. 2018). However, the treatment efficacy of EGFR-TKIs among patients with concurrent L858R and uncommon EGFR exon 21 mutations is largely unknown. Here, our study revealed that NSCLC patients with concurrent L858R and uncommon EGFR exon 21 mutations are able to benefit from EGFR-TKIs. Importantly, patients with concurrent L858R and K860I mutations, which may be detected by NGS but not by ARMS-PCR, are associated with good response to EGFR-TKIs with a median PFS of 11.0 months and a median DFS of 10.0 months. Furthermore, we noticed that one patient with concurrent L858R and K860I mutations had a 11-month partial response to Dacomitinib before acquiring T790M, which is the most common resistant mechanism among patients with common EGFR mutations.

Our study has some limitations: On one hand, the number of NSCLC patients with concurrent L858R and uncommon EGFR exon 21 mutations in our study is relatively small, and further studies in a larger cohort are warranted to confirm the conclusions. On the other hand, the time of follow-up of these patients treated with second or third generation EGFR-TKIs is relatively short.

In summary, our study found that ARMS-PCR failed to identify concurrent L858R and K860I mutations in NSCLC, while NGS provided comprehensive information to optimize clinical outcomes. In clinical practice, if we merely rely on the results of ARMS-PCR, patients with concurrent L858R and K860I mutations may miss the chance to benefit from EGFR-TKIs, confirmed to be highly effective in this study. Thus, NGS should be recommended for NSCLC patients to identify driver alterations such as EGFR mutations, especially when negative results are obtained by ARMS-PCR.

Grant Support

This work was supported by grants from the National Natural Science Foundation of China (81702436, 81972808).

Disclosure of Potential Conflicts of Interest

All the authors do not have conflicts of interest to declare.

Miss Shi^1,# and Dr Li¹^,# contributed equally to this work.

Author contributions: All authors contributed to the study conception and design. Software, investigation, formal analysis, writing original draft, visualization, validation were performed by Huiyang Shi; conceptualization, methodology, writing-reviewing and editing, software, project administration were performed by Weihua Li; data curation, supervision, resources and funding acquisition were provided from Lei Guo, Jianming Ying and Xingsheng Hu. All the authors read and approved the final manuscript.

Data availability：The authors declare that all data supporting the conclusions can be found in the main text and the supplementary files. The raw data produced in this study are not publicly available, but are available for non-commercial purposes from the corresponding authors on reasonable request.

Ethics approval: This study was approved by the Institutional Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College.

Consent to participate: Informed consent was obtained from all patients involved.

Acknowledgments: We would like to thank all the participants and their families.

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Table 1 Optimal response of EGFR-TKIs among patients with concurrent L858R and uncommon EGFR exon 21 mutations (n=23).

Table 1 Optimal response of EGFR-TKIs among patients with concurrent L858R and uncommon EGFR exon 21 mutations (n=23).

	No (%)
	Concurrent L858R and uncommon EGFR exon 21 mutations (N=23)
Optimal Response
CR	0 (0.0)
PR	7 (31.8)
SD	10 (45.4)
PD	1 (4.5)
NA	4 (18.1)
ORR	7 (38.9)
DCR	17 (94.4)
Treatment response was evaluated according to RECIST v1.1.

Table 2 Inconsistent results of NGS and ARMS-PCR among NSCLC patients with concurrent L858R and K860I mutations.

Table 2. Inconsistent results of NGS and ARMS-PCR among NSCLC patients with concurrent L858R and K860I mutations.
Case	Gender	Age	Hystological type	Stage	DNA NGS	Frequency (%)	PCR	EGFR-TKI treatment	Optimal response	PFS (months)
P01	Female	54	adenocarcinoma	2	p.L858R, p.K860I	21.66; 22.81	—	Gefitinib	PR	18
P02	Male	65	adenocarcinoma	2	p.L858R, p.K860I	35.01; 34.64	—	Osimertinib	NA	5
P03	Male	75	adenocarcinoma	2	p.L858R, p.K860I	30.60; 31.71	—	Icotinib	NA	4
P04	Female	74	adenocarcinoma	2	p.L858R, p.K860I	16.81; 18.33	—	Osimertinib	NA	NR
P05	Female	56	adenocarcinoma	2	p.L858R, p.K860I	21.07; 22.80	—	Dacomitinib	PR	11
P06	Male	48	adenocarcinoma	2	p.L858R, p.K860I	17.5; 17.4	—	Osimertinib	PR	4
Abbreviations: PR: partial response; NA: not available; PFS: progression-free survival; NR: not reached.

No competing interests reported.

SupplementaryFigure1.pdf
Supplementary Figure 1 Survival analysis of patients with L858R single mutation (Blue) and those with concurrent L858R and EGFR exon 21 mutations (Red) using the K-M method and log-rank test. All patients were treated EGFR-TKIs as the first-line treatment.
supplementarytable1.docx
Supplementary Table 1 Clinicopathological feature of patients with concurrent L858R and uncommon EGFR exon 21 mutations.
supplementarytable2.docx
Supplementary Table 2 Concurrent L858R and uncommon EGFR exon 21 mutations in the study population.
Supplementarytable3.docx
Supplementary Table 3 EGFR-TKIs treatment response among patients with concurrent L858R and uncommon EGFRexon 21 mutations.

Concurrent EGFR L858R and K860I mutations: PCR-negative but NGS-positive alterations that respond to EGFR inhibitors in non-small cell lung cancer

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Abstract

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Introduction

Patients and specimens

Results

Discussion

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References

Tables

Additional Declarations

Supplementary Files

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