Molecular tumor board: molecularly adjusted therapy upon identification and functional validation of a novel ALK resistance mutation in a case of lung adenocarcinoma

Abstract

We report a case of a long-term surviving patient with EML4/ALK translocated non-small cell adenocarcinoma of the lung in UICC8 stage IVA. During recurrence under continuous crizotinib therapy, a hitherto insufficiently characterized missense mutation in the ALK gene (Arg1181His) was identified through targeted sequencing. The aforementioned EML4/ALK translocation could still be detected in this situation. Employing a 3D reconstruction of the ALK tertiary structure, considering its interaction with various ALK inhibitors at the molecular binding site, our analysis indicated the presence of a mutation associated with crizotinib resistance. To validate the biological relevance of this previously unknown mutation, we carried out an in vitro validation approach in cell culture in addition to the molecular diagnostics accompanied by the molecular tumor board. The tumor scenario was mimicked through retroviral transfection. Our comparative in vitro treatment regimen paired with the clinical trajectory of the patient, corroborated our initial clinical and biochemical suspicions. Our approach demonstrates preclinical, in silico, and clinical evidence of a novel crizotinib resistance mutation in ALK as well as sensitivity toward brigatinib and potentially lorlatinib. In future cases, this procedure represents an important contribution to functional diagnostics in the context of molecular tumor boards.

Keywords: ALK inhibitors; ALK translocations; NSCLC; functional validation; molecular profiling.

Graphical Abstract

Figure 1.

Clinical, therapeutic, CT graphic, and histopathologic sequence of the disease. (a) Swimmer plot, which illustrates the clinical course and the sequence of therapies administered. (b) 47-year-old man with NSCLC cTxpN3 pM1a (ple). CT scans in initial staging and follow-up. (I) Initial CT and PET imaging (not shown) after malignancy confirmation in pleural biopsy revealed advanced-stage disease with primary tumor localization in the right lower lobe and adjacent atelectasis. (II) After 3 chemotherapy cycles of cisplatin/pemetrexed and pemetrexed maintenance treatment, a partial response (PR) was achieved. (III) CT scan after 22 months showed progressive disease (PD) with a significant increase in size; second-line crizotinib was initiated. (IV) Progression 120 months after initial diagnosis and 96 months after crizotinib initiation. (V) First CT scan evaluation 3 months after TKI switch to brigatinib revealed partial remission and current CT shows persistent stable disease up to 11 months after change in therapy (not shown). (c) Representative microphotographs of tumor samples from 2012 (I), 2014 (II), and 2022 (III) including immunohistochemical stainings for TTF-1/Napsin (IV; double-staining) and ALK (V; clone D5F3). Abbreviations: TTF-1, thyroid transcription factor 1; ALK, anaplastic lymphoma kinase. Scale bar = 100 µm. (d) The detection of the EML4/ALK fusion in FISH diagnostics.

Figure 2.

Overview of the results from molecular diagnostics. (a) Comparative ultra-deep panel sequencing of tissue material from initial diagnosis (2012) and recurrence after 8 years of crizotinib therapy (2022). Clonal evolution of the CDKN2A-bearing clone can be deduced. The loss of the TP53 alteration in the more recent tissue sample is worth mentioning. A missense mutation in the ALK gene leading to a p.Arg1181His replacement was newly detected in 2022. (b) The Lollipop plot illustrates all resistance mutations in the ALK gene segments after extensive literature research. The p.Arg1181His mutation detected in the patient’s tissue sample is reported for the first time in NSCLC. (c, d) The B-factor of ALK-ligand complexes. (c) Putty representation of the ALK-ligand complex highlighting high to low flexible regions of the molecule color-coded in rainbow (low b-factor in blue, high b-factor in red). The ligands are also color-coded based on the rigid and flexible parts within the molecule (gray circle). (d) The ligand molecule from the trajectories highlighted with flexible groups (gray circle) over 100 ns of MD simulations.

Figure 3.

EML4/ALK^R1181H variant confers partial resistance to ALK-ATP competitive inhibitors compared to non-mutated EML4/ALK based on functional validation studies in vitro. (a) Absolute cell numbers over time were measured in the absence of IL-3 by trypan blue exclusion (n = 3). Data are shown as mean ±  SD. (b) Immunoblot analysis of serum-starved Ba/F3 cells expressing EML4/ALK or EML4/ALK^R1181H with indicated antibodies. (c) MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based cell proliferation analysis of Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated concentrations of crizotinib for 48 hours. Data are shown as mean ± SD (n = 3). (d) Immunoblot analysis of Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated concentrations of crizotinib for 4 hours. A representative image of 2 independent experiments is shown (n = 2). (e) MTS-based cell proliferation analysis of Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H with indicated concentrations of brigatinib for 48 hours. Data are shown as mean ± SD (n = 3). (f) Immunoblot analysis of Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated concentrations of brigatinib for 4 hours. A representative image of 2 independent experiments is shown (n = 2). (g) MTS-based cell proliferation analysis of Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H with indicated concentrations of lorlatinib for 48 hours. Data are shown as mean ± SD (n = 3). (h) Immunoblot analysis of Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated concentrations of lorlatinib for 4 hours. (i) Annexin-V staining was performed on Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated concentrations of crizotinib for 48 hours. Data are shown as mean ± SD (n = 3). (j) Annexin-V staining was performed on Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated concentrations of brigatinib for 48 hours. Data are shown as mean ± SD (n = 3). (k) Annexin-V staining was performed on Ba/F3 cells expressing EML4/ALK and EML4/ALK^R1181H cultured with indicated s of lorlatinib for 48hrs. Data are shown as mean ± SD (n = 3). Abbreviation: OD, optical density.