case_images: PMC11783293_01
case_images: 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 microm. (d) The detection of the EML4/ALK fusion in FISH diagnostics.
case_images: oyae143_fig1.jpg
case_images: PMC11783293_01_oyae143_fig1.jpg
case_images: F1
case_images: Figure 1c). Fluorescence in-situ hybridization (FisH) identified EML4/ALK rearrangement acting as the potential driver of the disease. Combination chemotherapy was stopped after 3 cycles due to severe ototoxicity followed by 22 months of pemetrexed maintenance until tumor progression (Figure 1a, 1b). Histopathological reevaluation confirmed the previously identified ALK translocation (Supplementary Figure S1). Ten years after initial diagnosis, a follow-up CT scan of the chest demonstrated progression of the primary site in the right lower lobe (measuring 45 x 39 mm in transversal extension), 2 new suspicious lung nodules as well as an enhancement in the right-sided pleural effusion (Figure 1b). More specifically, the EML4-ALK variant E6;A19 could be identified (Supplementary Figure S1).
case_images: 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.
case_images: oyae143_fig2.jpg
case_images: PMC11783293_01_oyae143_fig2.jpg
case_images: F2
case_images: ALK inhibitor resistance and, to potentially guide the further treatment strategy. Apart from the previously known ALK translocation, molecular diagnostics identified pathogenic variants in CDKN2A and FBXW2 as well as an acquired single nucleotide variant (SNV) in the ALK gene (c.3542G > A, p.Arg.1181His; variant allele frequency 37.55%; coverage 1337x; Figure 2a). TKI resistance (eg, p.Leu1196Met, p.Cys1156Tyr or p.Leu1152Arg; Figure 2b). To the best of our knowledge, this variant has so far not been reported in NSCLC (Supplementary Methods). Our in silico experiments (Supplementary Methods) suspected a potential responsiveness toward brigatinib and lorlatinib compared to crizotinib due to a more flexible beta-layer sheet which facilitates binding opportunities for the next generation ALK inhibitors (Figure 2c; Supplementary Table S1 and Figure S2). These calculations suggest an increase in binding energy for lorlatinib in interaction with the mutated ALK structure (Supplementary Table S2 and Figure S2).
case_images: EML4/ALKR1181H 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/ALKR1181H 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/ALKR1181H 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/ALKR1181H 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/ALKR1181H 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/ALKR1181H 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/ALKR1181H 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/ALKR1181H cultured with indicated concentrations of lorlatinib for 4 hours. (i) Annexin-V staining was performed on Ba/F3 cells expressing EML4/ALK and EML4/ALKR1181H 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/ALKR1181H 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/ALKR1181H cultured with indicated s of lorlatinib for 48hrs. Data are shown as mean +- SD (n = 3). Abbreviation: OD, optical density.
case_images: oyae143_fig3.jpg
case_images: PMC11783293_01_oyae143_fig3.jpg
case_images: F3
case_images: NSCLC cell line (BaF3) via the retroviral vector PIG (plasmid-IRES-EGFP-Puromycin). Next, we demonstrated that the EML4/ALKR1181H variant is equally oncogenic as the EML4/ALK variant (Figure 3a; Supplementary Figure S3). AKT are strongly activated in both EML4/ALK cells and the EML4/ALKR1181H variant expressing cells, suggesting the crucial role of STAT3 which is independent of JAK family kinase, MAP kinase and PI3K pathways activation in ALK-transfected cells (Figure 3b). Analysis of resistance toward different ALK inhibitors showed that the cellular IC50 value in the EML4/ALKR1181H variant was 3 times higher (~300 nM) compared to the wild-type EML4/ALK (~105 nM) variant in the setting of in vitro treatment with crizotinib (Figures 3c, 3d; Supplementary Table S3).