# Concomitant ALK translocation and EGFR C797S mutation as resistance mechanisms to osimertinib in an EGFR-mutant NSCLC patient

Elvire Pons-Tostivint, Pauline Hulo, Christine Sagan, Thomas Papazyan, Guillaume Herbreteau, Marc Denis

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Elvire Pons-Tostivint, Pauline Hulo, Christine Sagan, Thomas Papazyan, Guillaume Herbreteau, et al. Concomitant ALK translocation and EGFR C797S mutation as resistance mechanisms to osimertinib in an EGFR-mutant NSCLC patient. Respiratory Medicine and Research, 2024, 86, pp.101116. 10.1016/j.resmer.2024.101116. hal-05088665

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Short communication

# Concomitant ALK translocation and EGFR C797S mutation as resistance mechanisms to osimertinib in an EGFR-mutant NSCLC patient

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Elvire Pons-Tostivint<sup>a,b,*</sup>, Pauline Hulo<sup>a,b</sup>, Christine Sagan<sup>c</sup>, Thomas Papazyan<sup>a</sup>, Guillaume Herbreteau<sup>d</sup>, Marc Denis<sup>b,d</sup>

## ARTICLE INFO

**Article history:**

Received 8 January 2024

Accepted 10 May 2024

Available online 14 May 2024

**Keywords:**

EGFR-mutant NSCLC

Osimertinib

Resistance mechanisms

C797S

ALK translocation

Osimertinib, a third-line generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor (TKI), has emerged as the first-line standard of care for advanced EGFR-mutant NSCLC patients. However, acquired resistance develops in nearly all patients, encompassing both on-target (i.e. EGFR-dependent, involving EGFR secondary mutations or amplifications) and off-target mechanisms (i.e. EGFR-independent, including oncogenic fusions, activation of bypass pathway and histological transformation) [1]. We discuss a case of advanced EGFR-mutant lung adenocarcinoma patient, treated front-line with osimertinib, who presented at relapse apparition of concomitant secondary EGFR p.C797S mutation and ALK gene translocation.

A 75-year-old woman, previously treated for melanoma, was diagnosed with non-smoking lung cancer following haemoptysis. A chest CT scan revealed a 32 mm pulmonary tumor in the left inferior lobe with metastases to mediastinal lymph nodes, lungs, and pericardium. Abdominal and pelvic CT scans detected liver and bone metastases. Brain MRI showed multiple metastatic lesions, the largest in the cerebellum at 17 mm (Fig. 1). She had good overall health and no neurological symptoms. A liver tumor biopsy revealed the presence of a TTF1 + (90 % of tumour cells) adenocarcinoma with a PD-L1 expression in 60 % of tumor cells. Molecular analysis performed by

NGS (QIAseq Targeted DNA custom panel of 22 genes including BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PDGFRA, PIK3CA, RET and TP53 - data analysis with the Biomedical Genomics Workbench software, Qiagen, Hilden, Germany) identified an activating mutation in EGFR exon 19 and a TP53 nonsense mutation (Fig. 1). The EGFRdel19 mutation was also detected in circulating tumor DNA (ctDNA)) using the COBAS EGFR assay (Roche Diagnostic). The tumor biopsy did not express ALK or ROS1 as determined by immunohistochemistry (clones 5A4 and D4D6, respectively).

After 10 months of first-line therapy with Osimertinib 80 mg, stability and significant reduction in lung and hepatic lesions were achieved. Eleven months later, due to grade 2 cutaneous toxicity, the dosage was reduced to 40 mg. After 15 months, a CT scan revealed asymptomatic recurrence in hepatic site with new lesions emerging (Fig. 1). Additionally, brain MRI unveiled the emergence of a singular cerebellar lesion. Analysis of ctDNA by NGS revealed the persistence of the EGFRdel19 and TP53 mutations previously identified in the liver biopsy, as well as an additional EGFR p.C797S mutation (p. Cys797Ser; c.2389T>A). Simultaneously, a liver biopsy validated the existence of adenocarcinoma cells displaying TTF1 positivity, although with a PD-L1 expression of 0 %. Molecular analysis (NGS) showed the EGFRdel19 and TP53 mutations, but the p.C797S mutation was not detected. Digital PCR (kit IDEGFR-RESIST, ID solutions, France; Crystal Digital PCR performed on a Naica system, Stilla technologies, France) confirmed that this resistance mutation was not present in this new biopsy. By contrast, ALK immunohistochemistry

<footnote><sup>a</sup> Nantes University, Centre Hospitalier Universitaire Nantes, Medical oncology, F-44000 Nantes, France</footnote>

<footnote><sup>b</sup> Nantes Université, Inserm UMR 1307, CNRS UMR 6075, Université d'Angers, CRCI2NA, Nantes, France</footnote>

<footnote><sup>c</sup> Nantes University, Centre Hospitalier Universitaire Nantes, Pathology department, F-44000 Nantes, France</footnote>

<footnote><sup>d</sup> Nantes University, Centre Hospitalier Universitaire Nantes, Department of Bichemistry, F-44000 Nantes, France</footnote>

<footnote>* Corresponding author at: Hôpital Laennec, CHU de Nantes, Boulevard Professeur Jacques Monod, 44800 Saint-Herblain, France.
E-mail address: elvire.pons@chu-nantes.fr (E. Pons-Tostivint).</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Timeline of the patient's history with imaging, biopsy, and molecular biology results.</figcaption></figure>

was strongly positive (3+ in all tumor cells), and the rearrangement was confirmed by FISH. The existence of a gene fusion was confirmed at the RNA level by RNA sequencing (QIAseq RNA Fusion XP targeted panel, Qiagen), which revealed a fusion of exon 6 of the *EML4* gene to exon 20 of the *ALK* gene (usually described as *EML4-ALK* variant v3) (Fig. 1). The patient underwent stereotactic radiation therapy for the solitary brain lesion. Osimertinib was discontinued and a switch was made to an ALK inhibitor, alectinib. However, only a few weeks later, a CT scan revealed a multifocal progression involving the brain, lungs, and liver (Fig. 1). Plasma was collected and analysed by NGS. The variant allele fraction of the *EGFRdel19* was higher than at progression on osimertinib (18.5 % as compared to 3.8 %), whereas the variant allele fraction of the p.C797S mutation slightly decreased (1.1 % as compared to 2.3 %). The patient made the decision to discontinue all treatment, and she passed away a few weeks thereafter.

Managing resistance to osimertinib is a significant clinical challenge due to the diversity of resistance mechanisms. Common genomic alterations include *EGFR* p.C797X mutations, *MET* amplification, *ERBB2* (HER2 gene) amplification, and non-EGFR pathway changes [1]. The *EGFR* p.C797S mutation in exon 20 is a key resistance mechanism to osimertinib, impairing its binding capacity and leading to resistance [1,2]. It is more frequent in *EGFRdel19* patients, especially in those receiving second-line or later osimertinib, with rates of up to 24 % [2,3]. However, this mutation is observed in only 5–7 % of patients starting osimertinib as their initial therapy. A recent study on 327 patients highlighted resistance mechanisms to upfront osimertinib [4]. Among 95 patients with post-progression biopsies, resistance mechanisms were identified in half. Among these, off-target alterations, including gene amplifications, single-gene mutations or acquired fusions were found in 24 out of 95 patients. On-target *EGFR* alterations were detected in 12/95 patients (*EGFR* amplification, p.C797S or p.C724S mutations). No cases of *ALK* translocation were observed. Another publication assessed ctDNA from paired plasma samples (obtained at both baseline and disease progression) from patients treated with osimertinib as part of the

FLAURA study [2]. Among 109 patients, 35 % showed acquired resistance mechanisms. The most common were *MET* amplification (17 patients), followed by on-target *EGFR* mutations (11 patients) including p.C797S mutation (7 patients). Notably, one patient with baseline *EGFRdel19* had a simultaneous recurrence with p.C797S mutation, *BRAF* p.V600E mutation, and *ALK* fusion. In *MET*-amplified, EGFR-mutant NSCLC patients, the combination of tepotinib and osimertinib has demonstrated efficacy, whereas tepotinib monotherapy exhibited very low activity (Mazieres et al. ESMO 2022). This underscores the importance of sustained EGFR inhibition in the latter line. Recently, clinically meaningful improvements over osimertinib alone have been demonstrated in first-line, with the combination of osimertinib–chemotherapy, or the combination of the EGFR-MET bispecific antibody, amivantamab, with a brain-penetrant EGFR TKI, lazertinib (5; Cho et al. ESMO 2023). The advent of these novel first-line therapeutic strategies is likely to reshape the disease profile at relapse.

In our case, the patient underwent both liquid and tissue biopsies at progression. The objective to perform biopsy upon relapse is to identify and address potential mechanisms of resistance, as recommended in guidelines [5]. Biopsying a progressing tumor lesion is recommended to uncover subclonal resistance events. Liquid biopsy, on the other hand, offers several advantages over tumor biopsy, especially for detecting clonal evolution under the selective pressure of therapy. In our patient, we observed an interesting disparity between blood and tissue samples results. While the acquired p.C797S mutation appeared in the blood, it was absent in the liver (despite progressive lesions). This is probably related to a subclone not present in the liver that released p.C797S *EGFR*-mutated DNA in the blood. Conversely, an *ALK* translocation was detected in the liver metastasis. Thus, we decided to stop osimertinib and to treat patient with an ALK inhibitor. This choice was driven by the belief that the *ALK* translocation clone contributed to liver disease progression, alongside limited options against p.C797S mutations. Unfortunately, this strategy was ineffective, as the
patient experienced rapid progression in liver, brain, and lung
lesions. A few weeks before death, we observed that the variant
allele fraction of the *EGFRdel19* increased, whereas the variant
allele fraction of the p.C797S mutation slightly decreased. These
modifications were probably related to the absence of therapeutic
pressure on the *EGFR*-mutated tumor cells.

Our case highlights the complexity of osimertinib resistance
mechanisms, emphasizing the presence of subclonal alterations in
specific tumor sites. It underscores the complementary roles of tissue
and ctDNA testing at progression.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.

Funding

The authors declare that no funds, grants or other support were
received during the preparation of this manuscript.

References

[1] Passaro A, Jänne PA, Mok T, Peters S. Overcoming therapy resistance in EGFR-mutant lung cancer. Nat Cancer 2021;2(4):377-91.

[2] Chmielecki J, Gray JE, Cheng Y, Ohe Y, Imamura F, Cho BC, et al. Candidate mechanisms of acquired resistance to first-line osimertinib in EGFR-mutated advanced non-small cell lung cancer. Nat Commun 2023;14(1):1070.

[3] Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 2015;21(6):560-2.

[4] Choudhury NJ, Marra A, Sui JSY, Flynn J, Yang SR, Falcon CJ, et al. Molecular bio-markers of disease outcomes and mechanisms of acquired resistance to first-line osi-mentinib in advanced EGFR-mutant lung cancers. J Thorac Oncol 2023;18(4):463-75.

[5] Hendriks LE, Kerr KM, Menis J, Mok TS, Nestle U, Passaro A, et al. Oncogene-addicted metastatic non-small-cell lung cancer: ESMO clinical practice guideline for diagnosis, treatment and follow-up. Ann Oncol 2023;34(4):339-57.