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Molecular follow-up of first-line treatment by osimertinib in lung cancer: Importance of using appropriate tools for detecting EGFR resistance mutation C797S

Open AccessPublished:June 09, 2021DOI:https://doi.org/10.1016/j.cancergen.2021.06.001

      Highlights

      • First-line osimertinib improved the response rate of patients with EGFR-mutated lung cancer.
      • EGFR-C797S is a resistance mutation to osimertinib, following the primary activating EGFR and the T790M resistance mutation.
      • We report the case of a patient primarily treated by osimertinib showing a EGFR-C797S mutation without EGFR-T790M.
      • Some targeted methods used in routine practice may overlook C797S.
      • Challenges of the identification of an isolated C797S mutation, without any association with T790M in routine molecular oncology practice.

      Abstract

      The C797S mutation encoded by EGFR exon 20 is classically observed as a tertiary event in EGFR-mutant non-small-cell lung carcinoma (NSCLC) primarily treated by first generation tyrosine kinase inhibitors (TKI) and secondarily treated by third-generation TKI, such as osimertinib, if the EGFR-T790M resistance mutation is detected. Recently, significant prolonged progression free survival has been observed following first-line osimertinib, in EGFR-mutant NSLC. While mechanisms of molecular resistance to first-generation TKI have been well studied, little is known about resistance induced by primary third-generation TKI treatments. We report the case of a 65 year-old female treated by first-line osimertinib for a multimetastatic exon 19-EGFR-mutant NSCLC. EGFR-C797S resistance mutation and PIK3CA mutation were detected together with the remaining EGFR-exon 19 deletion. This observation provides insights of acquired resistance to first line-osimertinib. It also highlights the importance of making molecular platforms which perform routine EGFR testing in lung cancer aware of the kind of therapeutic protocols given to the patient. Indeed, for rapid results or low-costs procedures, some targeted methods specifically targeting T790M may be used at relapse and may overlook alterations such as C797S or PIK3CA mutations. Targeted next generation sequencing is therefore a recommended option.

      Keywords

      Introduction

      Tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) have dramatically improved the response rate and progression free survival (PFS) of patients with EGFR-mutated non-small cell lung carcinoma (NSCLC) [
      • Lee CK
      • Brown C
      • Gralla RJ
      • et al.
      Impact of EGFR Inhibitor in Non–Small Cell Lung Cancer on Progression-Free and Overall Survival: A Meta-Analysis..
      ]. Detection of alterations in the DNA sequence of exons 18-21 of EGFR are currently done by molecular biology platforms worldwide in order to identify patients who will strongly benefit from such a treatment. Notably, small in-frame deletions in exon 19 and point mutations in exon 21, such as L858R, are the most frequent predictive biomarkers of response. However, following initial efficacy, disease progression due to acquired resistance is unavoidable after a median time of 12 months [
      • Maemondo M
      • Inoue A
      • Kobayashi K
      • et al.
      Gefitinib or Chemotherapy for Non–Small-Cell Lung Cancer with Mutated EGFR.
      ,
      • Mitsudomi T
      • Morita S
      • Yatabe Y
      • et al.
      Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial.
      ,
      • Rosell R
      • Carcereny E
      • Gervais R
      • et al.
      Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
      ]. The T790M mutation encoded by exon 20 of EGFR is the most frequent acquired resistance mechanism to first-generation EGFR TKI such as gefitinib or erlotinib [
      • Nagano T
      • Tachihara M
      • Nishimura Y.
      Mechanism of Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors and a Potential Treatment Strategy.
      ]. Considerable advances have been done in conception of third generation TKI able to target resistance mutations. Osimertinib is a TKI that acts in EGFR-TKI sensitive and T790M mutant tumors by selectively and irreversibly targeting the cysteine-797-residue in the ATP-binding site via covalent bond formation [
      • Cross DA
      • Ashton SE
      • Ghiorghiu S
      • et al.
      AZD9291, an Irreversible EGFR TKI, Overcomes T790M-Mediated Resistance to EGFR Inhibitors in Lung Cancer.
      ]. It was initially approved in 2015 in the USA and in 2016 in Europe as a second line therapy in patients treated by first generation TKI who underwent disease progression due to occurrence of T790M mutation [
      • Yu HA
      • Arcila ME
      • Rekhtman N
      • et al.
      Analysis of Tumor Specimens at the Time of Acquired Resistance to EGFR-TKI Therapy in 155 Patients with EGFR-Mutant Lung Cancers.
      ,
      • Sacher AG
      • Paweletz C
      • Dahlberg SE
      • et al.
      Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer.
      ]. Therefore molecular testing of T790M on tumor tissue or liquid biopsy became mandatory before a switch to osimertinib. While protocols based on first-generation TKI followed by osimertinib at occurrence of T790M mutation definitively improved the treatment of NSCLC [
      • Mok TS
      • Wu Y-L
      • Ahn M-J
      • et al.
      Osimertinib or Platinum–Pemetrexed in EGFR T790M–Positive Lung Cancer.
      ], they could not escape to the rule of acquired resistance, often within the first year of osimertinib treatment [
      • Nagano T
      • Tachihara M
      • Nishimura Y.
      Mechanism of Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors and a Potential Treatment Strategy.
      ]. Approximately 16% of patients [
      • Nagano T
      • Tachihara M
      • Nishimura Y.
      Mechanism of Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors and a Potential Treatment Strategy.
      ] harbor a C797S mutation as a tertiary event, following the primary and secondary events: primary activating EGFR mutation and T790M resistance mutation, respectively [
      • Thress KS
      • Paweletz CP
      • Felip E
      • et al.
      Acquired EGFR C797S mutation mediates resistance to AZD9291 in non–small cell lung cancer harboring EGFR T790M.
      ]. Osimertinib became later a valuable option for first-line treatment of EGFR-mutated lung cancer (NSCLC) [
      • Soria JC
      • Ohe Y
      • Vansteenkiste J
      • Reungwetwattana T
      • et al.
      Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer.
      ,
      • Ramalingam SS
      • Vansteenkiste J
      • Planchard D
      • et al.
      Overall Survival with Osimertinib in Untreated, EGFR -Mutated Advanced NSCLC.
      ]. The phase 3 FLAURA trial demonstrated a consistent improvement of median PFS (18.9 versus 10.2 months) as well as a good tolerance for patients affected by advanced NSCLC primarily treated by osimertinib in comparison to patients treated by standard first-generation TKI [
      • Ramalingam SS
      • Vansteenkiste J
      • Planchard D
      • et al.
      Overall Survival with Osimertinib in Untreated, EGFR -Mutated Advanced NSCLC.
      ]. First-line osimertinib was approved in 2018 and 2020 in the USA and Europe, respectively. So far, little is known about resistance markers specific to first-line osimertinib protocols [
      • Starrett JH
      • Guernet AA
      • Cuomo ME
      • et al.
      Drug Sensitivity and Allele Specificity of First-Line Osimertinib Resistance EGFR Mutations.
      ]. Here, we report the first detailed molecular analysis of EGFR-C797S and PIK3CA mutations without T790M or amplification of MET or any other amplification following primary osimertinib treatment in a multimetastatic NSCLC. We discuss the challenging issues of the appropriate identification of resistance markers induced by emerging therapeutic approaches in routine molecular oncology practice.

      Materials and methods

      Tumor DNA extraction

      Genomic DNA was extracted from formalin-fixed-paraffin embedded (FFPE) tumor samples using Promega Maxwell 16 FFPE Plus LEV DNA kit (Promega, Madison, WI).

      Targeted DNA next generation sequencing

      Targeted next generation sequencing (NGS) of tumor DNA was performed using Ion AmpliSeq Cancer Colon-Lung Panel v2 and Ampliseq OST Plus Panel (design updated in 2018), followed by Ion Torrent semiconductor-based sequencing (Thermo Fisher Scientific, Waltham, MA). The Ion AmpliSeq Colon-Lung Panel v2 was designed to amplify and sequence 92 amplicons covering the 22 following genes: AKT, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, FBXW7, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11 and TP53. The library from each DNA sample was barcoded with a sample-specific 10 nucleotide barcode sequence (Ion Xpress barcode adapter kit) and processed on a 318 v2 chip. Base calling, barcode sorting and trimming, alignment to the human reference genome (hg19), and variant calling were achieved using the Torrent Suite v3.6.2 and the Torrent Variant Caller plugin with the “Somatic-Low stringency” default parameters. Mutations were annotated using Ion Reporter v5.0. Common variants (Global Minor Allele Frequency, (GMAF) <0.01) were filtered out and only those affecting the amino acid sequence were retained.

      Comparative genomic hybridization and single nucleotide polymorphism on arrays (aCGH/aSNP)

      aCGH/aSNP analyses were done retrospectively on both pre- and post-osimertinib biopsy samples. We used 80ng input of genomic DNA (Affymetrix, Santa Clara, CA). Copy number alterations and loss of heterozygosity (LOH) of genomic DNA were evaluated using Affymetrix OncoScan CNV FFPE Assay. Data were analyzed using the Chromosome Analysis Suite (ChAS 3.3) software (ThermoFisher Scientific, Waltham, MA). Annotations were based on the human reference hg19 (Genome Reference Consortium Human Build 37 (GRCh37)).

      Results

      Case study

      In October 2018, a 63-year-old woman who had quitted smoking in 2004 presented at the emergency unit with cough, dyspnea and arterial hypertension. There was no evidence of peculiar medical or familial history. Computed tomography (CT) scan showed a 23 mm opacity of the right upper lobe (Fig. 1A) and multiple metastases of a deleted exon 19 EGFR-mutant (Del19) well differentiated NSCLC. The patient received first-line osimertinib (80 mg/day) treatment in November 2018. A partial response was reached in February 2019 (Fig. 1B). The disease started to rapidly progress in March 2020: bone and cerebral metastases resulted in significant dizziness, visual impairment and nausea (Fig. 1C, D). The patient died in palliative care unit in May 2020, 18 months after initial diagnosis, before receiving a second line of treatment.
      Fig. 1
      Fig. 1Computed-Tomography scan before (A) and after (B,C,D) first-line osimertinib treatment. Lesions are indicated by red arrows. (A) November 2018: primary right upper lobe nodule and mediastinal lymphadenopathies. (B) February 2019: partial response in all lesions, according to Response Criteria in Solid Tumors 1.1 (RECIST). March 2020: widespread thoracic progression (C), cerebral metastasis (D).

      Molecular analyses

      NGS of a pleural metastasis sample done by video-assisted thoracoscopic surgery before TKI treatment showed a deletion of the EGFR Del19 (c.2240_2254del15; p. L747_T751del; allele frequency (AF): 29%) and a point mutation in exon 3 of CTNNB1 (c.94G>C; p.D32H; AF: 6%). In March 2020, NGS analysis of a bone metastasis biopsy at progression post-osimertinib showed a c.2390G>C; p.C797S mutation in exon 20 of EGFR (AF: 20%) in addition to the remaining EGFR exon 19 deletion (AF: 42%) and CTNNB1 mutation (AF: 17%) (Fig. 2). Moreover, a mutation in exon 10 of PIK3CA (c.1633G>A; p.E545K; AF: 16%) was observed. aCGH analysis was retrospectively performed in those two tumor samples. Both tumors shared a few gains of chromosomal segments (1q21.1-q44; 8q11.1-q23.3; 17q21.32-q24.3) and of whole chromosomes 7 and 14 as well as segmental losses (6q11.1-q27; 9p21.3; 13q11-q21.32), including deletion of CDKN2A/CDKN2B (9p21) and RB1 (13q14) (data not shown). In addition to a few alterations that were identical to those observed in the pre-osimertinib sample, losses of several whole chromosomes were detected in the post-osimertinib biopsy. No amplification was detected. Notably, there was no detectable amplification of EGFR, MET, ERBB2, ALK and ROS1.
      Fig. 2
      Fig. 2Next generation sequencing (NGS) analysis of the bone metastasis biopsy sample in March 2020 after 16 months of first-line osimertinib. Binary Alignment Map visualization Images and allele frequency were retrieved from Alamut visual software (Interactive Biosoftware, Version 2.14). (A,B) 12 mutations observed both before and after osimertinib. (C,D) mutations observed only after osimertinib. (A) EGFR exon 19 deletion (c.2240_2254del15; p. L747_T751del; allele frequency (AF): 42% (AF before osimertinib: 29%). (B) CTNNB1 exon 3 mutation (c.94G>C; p.D32H; AF: 17% (AF before osimertinib: 6%). (C) EGFR exon 20 C797S (c.2390G>C; p.C797S; AF: 20%). (D) PIK3CA exon 10 mutation (c.1633G>A; p.E545K; AF: 16%).

      Discussion

      Among the cohort of the FLAURA trial, the most common resistance marker found in the samples of 91 patients which underwent molecular analysis when post-primary osimertinib progression occurred, was MET amplification (14/91; 15%) while EGFR-C797S mutation was detected in 6 patients (7%) [
      • Ramalingam SS
      • Cheng Y
      • Zhou C
      • et al.
      Mechanisms of acquired resistance to first-line osimertinib: Preliminary data from the phase III FLAURA study.
      ]. Here, we report the first detailed molecular analysis of a progressive multimetastatic Del19 NSCLC following first-line osimertinib treatment. We observed EGFR-C797S and PIK3CA-E545K mutations without EGFR-T790M. Amplification of MET or any other amplification was not detected. This observation strengthens the importance of using molecular methods able to detect C797S mutation in such a context. Indeed, because of rapid and low-cost results, methods such as pyrosequencing or real-time PCR only focused on investigating EFGR exon 19 and 21 main mutations or T790M are sometimes preferentially used instead of NGS at relapse. However, such methods may overlook emerging mutations related to first-line osimertinib, such as EGFR-C797S or L718V/Q. Although there is currently no treatment specifically targeting EGFR-C797S resistance mutation, this information is important both for advances in research of novel drugs active in resistant tumors and for care optimization of each patient. In preclinical models, EGFR-C797S mutant NSCL showed response to first-generation TKI [
      • Rangachari D
      • To C
      • Shpilsky JE
      • VanderLaan PA
      • Kobayashi SS
      • Mushajiang M
      • et al.
      EGFR Mutated Lung Cancers Resistant to Osimertinib through EGFR C797S Respond to First-Generation Reversible EGFR Inhibitors but Eventually Acquire EGFR T790M/C797S in Preclinical Models and Clinical Samples.
      ]. However this response was transitory and T790M eventually occurred. Interestingly, Starrett et al [
      • Starrett JH
      • Guernet AA
      • Cuomo ME
      • et al.
      Drug Sensitivity and Allele Specificity of First-Line Osimertinib Resistance EGFR Mutations.
      ] showed that in preclinical models, erlotinib was effective in cells with C797S but not with 718L/D mutations and that combination of first-line erlotinib and osimertinib prevents secondary mutations. Moreover, these authors, in a retrospective study of circulating free DNA of EGFR-mutant patient's blood samples showed that C797S mutation was more often associated to Del19 whereas L718V/Q mutations were correlated to L858R. Unfortunately, in this study data on treatment given to the patients were not available. Nevertheless, if confirmed, such data might be useful for orientating the search of resistance mutations. The PIK3CA mutation that was associated with C797S in the present case has also been described in a few cases following second-line osimertinib treatment [
      • Rosell R
      • Carcereny E
      • Gervais R
      • et al.
      Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
      ]. This mutation might also be considered for alternative therapeutic options though PIK3CA inhibitors demonstrated only a modest clinical efficacy as single-agent early clinical studies [
      • Rodon J
      • Dienstmann R
      • Serra V
      • et al.
      Development of PI3K inhibitors: lessons learned from early clinical trials.
      ]. The role of other somatic alterations observed in the present case is still unclear. CTNNB1 mutations, such as the one present in our case in pre- and post osimertinib samples has been described to limit EGFR-inhibitor response in EGFR-mutant lung cancers [
      • Blakely CM
      • Watkins TBK
      • Wu W
      • Gini B
      • Chabon JJ
      • McCoach CE
      • et al.
      Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers.
      ]. Targeting the Wnt-catenin pathway might therefore be used for counteracting TKI-resistance. In addition, we observed RB1 and CDKN2A homozygous deletions. Both alterations may have played a role in the tumor progression. RB1 deletion has been described to be a marker of transformation from NSCLC into small cell lung carcinoma [
      • Taniguchi H
      • Sen T
      • Rudin CM.
      Targeted Therapies and Biomarkers in Small Cell Lung Cancer.
      ] while association of CDKN2A deletion with an activating EGFR mutation has been correlated to a poor response to TKI [
      • Jiang J
      • Gu Y
      • Liu J
      • Wu R
      • Fu L
      • Zhao J
      • et al.
      Coexistence of p16/CDKN2A homozygous deletions and activating EGFR mutations in lung adenocarcinoma patients signifies a poor response to EGFR-TKIs.
      ].
      To the best of our knowledge, this is the first detailed case report with genomic data which shows EGFR-C797S mutation without EGFR T790M, after first-line osimertinib in a metastatic Del19 NSCLC. Our report underlines the importance for molecular platforms to be fully informed on therapeutic protocols prescribed to NSCLC patients, in order to make them able to use the optimal methods of molecular detection when they start the molecular screening of a tumor sample. Targeted NGS appears as a recommended option since it detects main resistance EGFR mutations and is also able to identify MET amplification. This is crucial both for precision medicine and for understanding resistance mechanisms to novel TKI protocols, such as first-line osimertinib.

      Declaration of Competing Interest

      FP and VDT report honoraria from AstraZeneca, outside the submitted work. HB reports consulting and personal fees from Bristol-Myers Squibb and personal fees from Novartis, MSD and Roche outside the submitted work. The others authors declare that they have no conflict of interest.

      Acknowledgments

      We thank R. Grattery, AC Peyron, T. Fabas, S. Gimet, M. Delhorbe and J. Sallenave-Petipas for skillful technical assistance and M. Grobon for editing the manuscript.

      Funding

      Prime d'intéressement à la recherche (Direction de la Recherche et de l'Innovation, Nice University Hospital).

      Sample credit author statement

      YF, IDM, FP, VDT: Conceptualization, Methodology, Data curation, Writing- Original draft preparation; Resources: LTB, HB, HB; Investigastion, Supervision: HB, LTM, HB; Formal analysis: SG. Project administration: IDM, FP; Funding acquisition: FP.

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