EML4-ALK Variant Research Articles

Impact of EML4-ALK Variants and Co-Occurring TP53 Mutations on Duration of First-Line ALK Tyrosine Kinase Inhibitor Treatment and Overall Survival in ALK Fusion-Positive NSCLC: Real-World Outcomes From the GuardantINFORM database

IntroductionTyrosine kinase inhibitors (TKIs) are first-line treatment options for ALK-positive (ALK+) NSCLC. Factors such as variant allele frequencies (VAFs), EML4-ALK fusion variant, and concurrent TP53 mutations (TP53mt) in circulating tumor DNA (ctDNA) may affect treatment outcomes. We evaluated their effects on time to discontinuation (TTD) of first-line treatment with next-generation ALK TKIs in a real-world setting. MethodsAdults with advanced or metastatic NSCLC and ctDNA-detected ALK fusion who received first-line next-generation ALK TKI monotherapy were identified in GuardantINFORM. Effects of ALK fusion VAF, EML4-ALK variants, and TP53mt detection on TTD were evaluated. ResultsA total of 307 patients with ALK fusion in baseline ctDNA received first-line alectinib (n = 280), brigatinib (n = 15), lorlatinib (n = 9), or ceritinib (n = 3); 150 patients (49%) had ALK-fusion VAF greater than or equal to 1%. Among 232 patients with EML4-ALK fusions (v1, 50%; v3, 36%), TP53mt co-occurred with v1 in 42 (18%) and v3 in 32 (14%). Patients with VAF less than 1% versus greater than or equal to 1% had a median TTD of 32.2 (95% confidence interval [CI]: 20.7–not estimable [NE]) versus 14.7 months (10.4–19.9; hazard ratio [HR] = 1.57 [95% CI: 1.09–2.26]; p = 0.0146). Median TTD was 13.1 (9.5–19.9) versus 27.6 months (17.3–NE) in patients with versus without TP53mt detected (HR = 1.53 [1.07–2.19]; p = 0.0202) and 20.3 (14.4–NE) versus 11.5 months (7.4–31.1) in patients with v1 versus v3 (HR = 1.29 [0.83–2.01]; p = 0.2641). Patients with TP53mt and v3 had a median TTD of 7.4 months (95% CI: 4.2–31.1). ConclusionHigh ctDNA VAF, EML4-ALK v3, and TP53mt were associated with early discontinuation of first-line ALK TKIs.

Transforming tumoroids derived from ALK-positive pulmonary adenocarcinoma to squamous cell carcinoma in vivo

Approximately 3–5% of non-small cell lung cancers (NSCLC) harbor ALK fusion genes and may be responsive to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors. There are only a few reports on cell lines with EML4-ALK variant 3 (v3) and tumoroids that can be subject to long-term culture (> 3 months). In this study, we established tumoroids (PDT-LUAD#119) from a patient with lung cancer harboring EML4-ALK that could be cultured for 12 months. Whole-exome sequencing and RNA sequencing analyses revealed TP53 mutations and an EML4-ALK v3 mutation. PDT-LUAD#119 lung tumoroids were sensitive to the ALK tyrosine kinase inhibitors (ALK TKIs) crizotinib, alectinib, entrectinib, and lorlatinib, similar to NCI-H3122 cells harboring EML4-ALK variant 1 (v1). Unexpectedly, clear squamous cell carcinoma and solid adenocarcinoma were observed in xenografts from PDT-LUAD#119 lung tumoroids, indicating adenosquamous carcinoma. Immunostaining revealed that the squamous cell carcinoma was ALK positive, suggesting a squamous transformation of the adenocarcinoma. Besides providing a novel cancer model to support basic research on ALK-positive lung cancer, PDT-LUAD#119 lung tumoroids will help elucidate the pathogenesis of adenosquamous carcinoma.

Clinical difference on the variants and co-mutation in a Chinese cohort with ALK-positive advanced non-small cell lung cancer.

Despite the generally favourable prognoses observed in patients with ALK-positive non-small cell lung cancer (NSCLC), there remains significant variability in clinical outcomes. The objective of this study is to enhance patient stratification by examining both the specific sites of gene fusion and the presence of co-occurring mutations. We collected retrospective clinical and pathological data on ALK-positive patients with locally advanced or metastatic disease. ALK fusion variants and concomitant mutations were identified through next-generation sequencing technology. We then assessed treatment efficacy via tumor response and survival metrics. This study included a total of 59 patients, with 49 harboring echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions and 10 presenting with rare fusions. The median follow-up period was 33 months. Clinical outcomes between non-EML4-ALK and EML4-ALK patients were comparable. Among the EML4-ALK cohort, patients with longer variants (v1, v2, v8) demonstrated superior progression-free survival (PFS) (median PFS: 34 months vs. 11 months; hazard ratio [HR]: 2.28; P = 0.05) compared to those with shorter variants (v3, v5). Furthermore, patients treated with second-generation ALK inhibitors (ALKi) displayed a progression-free survival advantage (median PFS: not reached [NR] vs. 9 months; HR: 5.37; P = 0.013). Baseline TP53 co-mutation were linked with a substantially shorter OS (median OS,37 months vs. NR; HR 2.74; P = 0.047). In ALK+ NSCLC, longer EML4-ALK variants correlate with improved prognosis and enhanced response to second-generation ALKi, while TP53 co-mutations indicate a negative prognosis.

Concurrent inhibition of ALK and SRC kinases disrupts the ALK lung tumor cell proteome

Precision oncology has revolutionized the treatment of ALK-positive lung cancer with targeted therapies. However, an unmet clinical need still to address is the treatment of refractory tumors that contain drug-induced resistant mutations in the driver oncogene or exhibit resistance through the activation of diverse mechanisms. In this study, we established mouse tumor-derived cell models representing the two most prevalent EML4-ALK variants in human lung adenocarcinomas and characterized their proteomic profiles to gain insights into the underlying resistance mechanisms. We showed that Eml4-Alk variant 3 confers a worse response to ALK inhibitors, suggesting its role in promoting resistance to targeted therapy. In addition, proteomic analysis of brigatinib-treated cells revealed the upregulation of SRC kinase, a protein frequently activated in cancer. Co-targeting of ALK and SRC showed remarkable inhibitory effects in both ALK-driven murine and ALK-patient-derived lung tumor cells. This combination induced cell death through a multifaceted mechanism characterized by profound perturbation of the (phospho)proteomic landscape and a synergistic suppressive effect on the mTOR pathway. Our study demonstrates that the simultaneous inhibition of ALK and SRC can potentially overcome resistance mechanisms and enhance clinical outcomes in ALK-positive lung cancer patients. One Sentence SummaryCo-targeting ALK and SRC enhances ALK inhibitor response in lung cancer by affecting the proteomic profile, offering hope for overcoming resistance and improving clinical outcomes.

Overcoming Central β-Sheet #6 (Cβ6) ALK Mutation (L1256F), TP53 Mutations and Short Forms of EML4-ALK v3/b and v5a/b Splice Variants are the Unmet Need That a Re-Imagined 5th-Generation (5G) ALK TKI Must Deliver.

Despite the development and approval of seven anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) spanning over three "generations" since the discovery of ALK fusion positive (ALK+) non-small cell lung cancer (NSCLC), there remains intrinsic and acquired resistances to these approved TKIs. Currently, a fourth-generation (4G) ALK TKI, NVL-655, is being developed to attack some of the unmet needs such as compound resistance mutations in cis. However, EML4-ALK variant 3 and TP53 mutations are intrinsic genomic alterations that negatively modulate efficacy of ALK TKIs. Potentially, in the shifting landscape where lorlatinib should be the first-line ALK TKI of choice based on the CROWN trial, the central β-sheet #6 (Cβ6) mutation ALK L1256F will be the potential acquired resistance mutation to lorlatinib which may be resistant to current ALK TKIs. Here we opine on what additional capacities a putative fifth-generation (5G) ALK TKI will need to possess if it can be achieved in one single molecule. We propose randomized trial schemas targeting some of the intrinsic resistance mechanisms that will lead to approval of a prototypic fifth-generation (5G) ALK TKI and actually be beneficial to ALK+ NSCLC patients rather than just design a positive pivotal superiority trial for the sole purpose of drug approval.

HER3 overexpression: a predictive marker for poor prognosis in advanced ALK-positive non-small cell lung cancer treated with ALK inhibitors.

Anaplastic lymphoma kinase (ALK)-targeted tyrosine kinase inhibitors (TKIs) improve patient survival; however, some patients develop ALK-TKI resistance with unidentified mechanisms. We investigated ErbB family and c-MET expression in patients with ALK-positive non-small cell lung cancer (NSCLC) to understand their roles in the ALK-TKI response. We studied 72 patients with advanced ALK-positive NSCLC with EML4-ALK fusion variant subtyping and immunostaining for c-MET, EGFR, HER2, and HER3 on tissue specimens both pre- (primary) and post-treatment (secondary) with ALK-TKI. We investigated the association of their expression with survival outcomes and assessed the effectiveness of combining ALK and EGFR inhibitors in ALK-positive NSCLC cell lines stimulated with the HER3-specific ligand HRG1. High expression of c-MET, EGFR, HER2, and HER3 was observed in 4.9%, 18.0%, 1.6%, and 25.8% of primary tumors, respectively, and 18.5%, 37.0%, 10.7%, and 35.7% of secondary tumors, respectively. HER3 overexpression in primary tumors showed inferior survival (P=0.132). In the subgroup with EML4-ALK variant 1/2 (V1/V2), HER3 overexpression was significantly associated with inferior survival in both primary and secondary tumors (P=0.022 and P=0.004, respectively). Combination treatment with lorlatinib and erlotinib significantly reduced HRG1-induced activation of RTK signaling in ALK-positive NSCLC cells. HER3 overexpression has potential as a prognostic marker in ALK-positive NSCLCs, including ALK-TKI naïve and treated cases, especially those with EML4-ALK V1/V2. Assessing HER3 expression may be crucial for treatment planning and outcome prediction in these patients.

ALK F1174S mutation impairs ALK kinase activity in EML4-ALK variant 1 and sensitizes EML4-ALK variant 3 to crizotinib.

To assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3. We constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions. In addition, we characterized the effect of different F1174 kinase domain mutations in the context of EML4-ALK fusions. In contrast to the oncogenic ALK-F1174S mutation that has been reported to be activating in the context of full-length ALK in neuroblastoma, EML4-ALK (F1174S) variant 1 exhibits impaired kinase activity leading to loss of oncogenicity. Furthermore, unlike the previously reported F1174C/L/V mutations, mutation of F1174 to S sensitizes EML4-ALK variants 3a and 3b to crizotinib. These findings highlight the complexity of drug selection when treating patients harboring resistance mutations and suggest that the F1174S mutation in EML4-ALK variant 1 is likely not a potent oncogenic driver. Additional oncogenic driver or other resistance mechanisms should be considered in the case of EML4-ALK variant 1 with F1174S mutation.

Molecular heterogeneity and co-altered genes in MET-amplified ALK-positive lung cancer: Implications for MET targeted therapy

ObjectivesMET amplification is a common mechanism of resistance to second- and third-generation anaplastic lymphoma kinase (ALK) inhibitors. In case series of MET-amplified ALK-rearranged (ALK + ) lung cancer, durability of responses to combinations targeting ALK and MET is variable, suggesting heterogeneity across tumors. However, little is known about the molecular composition of this subset of ALK-rearranged (ALK + ) NSCLC. Materials and methodsWe queried tissue and plasma databases to compile a group of > 50 specimens with ALK rearrangements and concurrent MET amplification. Fluorescence in-situ hybridization (FISH) and next-generation sequencing (NGS) were utilized to quantify the range of MET copies and describe the global molecular landscape of co-altered genes. ResultsBy FISH, high-level amplification (overall MET/centromere 7 probe ratio ≥ 5) was detected in 75 % of MET-amplified ALK + NSCLC tissue specimens. Intralesional heterogeneity of MET copies was observed, with high-level amplification identified even in cells from tumors with overall low-level MET amplification. Analysis of 48 MET-amplified ALK + NSCLC plasma specimens suggested that high-level amplification is rarely (17 %) detected in plasma. In both tissue and plasma, EML4-ALK variant 1 was the predominant variant (51 %) identified in MET-amplified specimens. ALK kinase domain mutations were only present in a minority of MET-amplified ALK + NSCLCs. MET-amplified ALK + NSCLC plasma specimens were enriched for TP53 mutations (81 % vs 45 %, p = 0.002), EGFR amplification (17 % vs 4 %, p < 0.001), and MYC amplification (21 % vs 3 %, p < 0.001) compared to ALK + NSCLC specimens lacking MET amplification. ConclusionsMET-amplified, ALK + NSCLC often presents with high-level and heterogeneous amplification in tissue, seldom overlaps with ALK mutations, and frequently co-occurs with alterations associated with aggressive tumor biology.

Large-scale transcriptomic profiling of the tumor immune microenvironment in ALK+ lung cancer.

9020 Background: Patients (pts) with ALK+ Non-Small Cell Lung Cancer (NSCLC) do not derive significant clinical benefit from immune checkpoint inhibitors. To better understand this lack of immunotherapy sensitivity, we aimed to characterize major immune components of the tumor microenvironment (TME) by comprehensive transcriptomic and immunohistochemistry (IHC) analyses. Methods: We analyzed NGS data from 5490 NSCLC pts that underwent DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS) sequencing at Caris Life Sciences (Phoenix, AZ). 374 ALK-rearranged cases were evaluated, along with 3169 KRAS-mut ( STK11/ KEAP1-wt) and 1947 EGFR-mut cases serving as comparators with known heterogenous and inert immune TMEs, respectively. PD-L1 (22C3) was evaluated by IHC. Immune cell fractions were inferred using quanTIseq. Gene expression profiles were analyzed for a T cell-inflamed signature (TIS) predictive of response to immunotherapy and for other immune modulatory genes such as IFNG, GZMB, TGFB1, and those of the adenosine pathway ( CD73/NT5E, CD39/ENTPD1, ADORA1, ADORA2A/B). A significant difference between genomic subgroups was defined as fold-change &gt; 1.2. In an independent cohort of 14 ALK+ NSCLC pts, density and spatial organization of CD4+ and CD8+ T cells, Tregs, major myeloid lineage cells, PDL1, and CD73 were assessed by quantitative IHC. Results: ALK+ tumors were associated with high PD-L1 (≥50%) expression (40% vs 47% for KRAS-mut vs 18% for EGFR-mut, p &lt; 0.001) and low TMB (median 3 mut/MB vs 9 for KRAS-mut vs 4 for EGFR-mut, p &lt; 0.001). The abundance of CD8+ T cells (fold-change -1.3, p &lt; 0.001), Tregs (fold-change -1.2, p &lt; 0.001), M2 macrophages (fold-change 1.2, p &lt; 0.001), and CD4+ T cells (fold-change 1.9, p &lt; 0.001) differed from KRAS-mut; notably, similar to EGFR-mut. In ALK+ tumors, IFNG (fold-change -1.5, p &lt; 0.001), GZMB (fold-change -1.6, p &lt; 0.001), TGFB (fold-change -1.3, p &lt; 0.001), LAG-3 (fold-change -1.4 p &lt; 0.001), CD73/NT53 (fold-change -1.7 p &lt; 0.001) , and ADORA2A (fold-change -1.4, p &lt; 0.001) were decreased while ADORA1 (fold-change 1.3, p &lt; 0.001) was increased compared to KRAS-mut. EML4-ALK comprised 94.7% of the ALK+ tumors and distribution of EML4-ALK variants was consistent with prior reports (e.g. v1 35.6%, v3 35.1%). Immune cell fractions and immune-related gene expression did not vary significantly between major variant subgroups (v1 vs non-v1, and v3 vs non-v3, p &gt; 0.05). Conclusions: To our knowledge this is the largest transcriptomic analysis of the ALK+ NSCLC TME. Despite high levels of PD-L1, ALK+ tumors exhibit multiple features of an inert immune TME, primarily characterized by low TMB and decreased CD8+ T cells and immune activation markers. Our findings suggest that, while immunosuppressive factors such as M2 macrophages and adenosine signaling may also be targeted, strategies to enhance immunogenicity are critical for an effective immune response in ALK+ NSCLC.

A real-world study of ALK fusion detection, treatment patterns, and survival outcomes of patients with advanced non–small-cell lung cancer (aNSCLC) in Brazil.

e18655 Background: Clinical trials have firmly established that ALK tyrosine kinase inhibitors (TKIs) are safe and effective in aNSCLC, with substantial anti-tumor activity of multiple agents across different treatment lines. Real-world ALK fusion diagnosis and targeted therapy effectiveness have not been described in Brazilian private healthcare setting, with ALK TKIs approved since 2016. Methods: This study retrospectively explored the patterns of use and outcome after initial exposure to ALK TKIs in patients with ALK positive aNSCLC in a real-world clinical cohort from Oncoclínicas Group, Brazil's largest network of community oncology practices, between 2016 and 2022. For data collection we used a platform where structured EHR variables are integrated with unstructured elements from physician notes using technology-based abstraction and expert curation. We also describe molecular epidemiology and ALK fusion variant prevalence for a subset of patients tested with an in-house RNA sequencing assay (GS180, ArcherDx) at Oncoclínicas Precision Medicine Lab from 2020 to 2022. Results: Out of 100 ALK-positive aNSCLC patients treated with ALK TKIs, median age was 60 years and 62% were female. Overall, median number of treatment lines was 2, with 29% receiving 3 or more regimens. In first line, 64% of the patients received an ALK TKI (58% crizotinib, 33% alectinib, and 9% brigatinib, ceritinib or lorlatinib). The remaining 36% were treated with chemo +/- immunotherapy first, and 28% switched to an ALK TKI right after driver diagnosis. When comparing first line treatment practice in 2020-2022 versus 2016-2019 we detected major reduction in crizotinib as first ALK TKI (83% versus 35%). Overall, 14% received 2 or more sequential ALK TKIs. With a median follow-up of 12 months, median Overall Survival (mOS) from start of first ALK TKI was 28 months (CI95% 18-NR). In first line, exposure to alectinib was associated with numerically higher mOS (36 months) when compared to crizotinib (18 months, HR = 0.58, CI95% 0.2-1.6). From 592 cases of non-squamous NSCLC cases with in-house RNA sequencing, ALK fusions were detected in 5.2%. EML4-ALK fusion was found in 84%, with most common variants being E5:A20 (non-canonical) in 41% and E13:E20 (v1) in 38%. We also found novel fusions partners HIP1, KLC1, LTK and STRN in 16%. Conclusions: The largest clinico-genomics cohort in Latin America reveals that the impact of ALK TKIs in ALK fusion aNSCLC may not be as profound in the real world as in clinical trials. Fast access to molecular diagnosis and exposure to ALK TKI in first line are being incentivized. EML4-ALK variant prevalence in Brazilian patients with aNSCLC is different from published cohorts, and the effect that non-canonical variants may have on ALK TKI activity must be studied further.

EML4-ALK biology and drug resistance in non-small cell lung cancer: a new phase of discoveries.

Anaplastic lymphoma kinase (ALK) can be driven to oncogenic activity by different types of mutational events such as point-mutations, for example F1174L in neuroblastoma, and gene fusions, for example with echinoderm microtubule-associated protein-like 4 (EML4) in non-small cell lung cancer (NSCLC). EML4-ALK variants result from different breakpoints, generating fusions of different sizes and properties. The most common variants (Variant 1 and Variant 3) form cellular compartments with distinct physical properties. The presence of a partial, probably misfolded beta-propeller domain in variant 1 confers solid-like properties to the compartments it forms, greater dependence on Hsp90 for protein stability and higher cell sensitivity to ALK tyrosine kinase inhibitors (TKIs). These differences translate to the clinic because variant 3, on average, worsens patient prognosis and increases metastatic risk. Latest generation ALK-TKIs are beneficial for most patients with EML4-ALK fusions. However, resistance to ALK inhibitors can occur via point-mutations within the kinase domain of the EML4-ALK fusion, for example G1202R, reducing inhibitor effectiveness. Here, we discuss the biology of EML4-ALK variants, their impact on treatment response, ALK-TKI drug resistance mechanisms and potential combination therapies.

Efficacy of Brigatinib in Patients With Advanced ALK-Positive NSCLC Who Progressed on Alectinib or Ceritinib: ALK in Lung Cancer Trial of brigAtinib-2 (ALTA-2)

IntroductionBrigatinib is a potent next-generation ALK tyrosine kinase inhibitor approved for treatment-naive and crizotinib-refractory advanced ALK-positive (ALK+) NSCLC. We evaluated brigatinib after other next-generation ALK tyrosine kinase inhibitors. MethodsIn this single-arm, phase 2, ALK in Lung Cancer Trial of brigAtinib-2 (NCT03535740), patients with advanced ALK+ NSCLC whose disease progressed on alectinib or ceritinib received brigatinib 180 mg once daily (after 7-d 90-mg lead-in). Primary end point was independent review committee (IRC)-assessed overall response rate (ORR). Circulating tumor DNA (ctDNA) was analyzed. ResultsAmong 103 patients (data cutoff: September 30, 2020; median follow-up [range]: 10.8 [0.5–17.7] mo), confirmed IRC-ORR was 26.2% (95% confidence interval [CI]: 18.0–35.8), median duration of response, 6.3 months (95% CI: 5.6–not reached), and median progression-free survival (mPFS), 3.8 months (95% CI: 3.5–5.8). mPFS was 1.9 months (95% CI: 1.8–3.7) in patients with ctDNA-detectable baseline ALK fusion (n = 64). Among 86 patients who progressed on alectinib, IRC-ORR was 29.1% (95% CI: 19.8–39.9); mPFS was 3.8 months (95% CI: 1.9–5.4). Resistance mutations were present in 33.3% (26 of 78) of baseline ctDNA; 54% (14 of 26) of mutations were G1202R; 52% (33 of 64) of patients with detectable ALK fusion had EML4-ALK variant 3. Most common all-grade treatment-related adverse events were increased creatine phosphokinase (32%) and diarrhea (27%). The mean dose intensity of brigatinib (180 mg once daily) was 85.9%. ConclusionsIn ALK in Lung Cancer Trial of brigAtinib-2, brigatinib was found to have a limited activity in patients with ALK+ NSCLC post-ceritinib or post-alectinib therapy. mPFS was longer with brigatinib in patients without baseline detectable plasma ALK fusion.

Potential Therapeutic Strategy for EGFR-Mutant Lung Cancer With Concomitant EML4-ALK Rearrangement—Combination of EGFR Tyrosine Kinase Inhibitors and ALK Inhibitors

IntroductionAlthough driver gene mutations have been believed to be mutually exclusive, some patients with NSCLC and concomitant EGFR mutations and EML4-ALK rearrangements have been reported. In this study, we reported a case of a patient with lung cancer who harbored both EGFR mutation and the EML4-ALK rearrangement after acquiring resistance to the EGFR tyrosine kinase inhibitor treatment. EGFR-mutant and ALK fusion proteins were detected in the same tumor cells through immunohistochemical analysis. Investigation of the molecular mechanisms of concomitant EGFR mutation and the EML4-ALK rearrangement in the same tumor cell can help discover an appropriate treatment for these patients. MethodsPC-9 cells, expressing EGFR exon 19 deletion, were transfected with EML4-ALK variant 3a (v3a) and variant 3b (v3b) separately and selected, and the effect of EGFR and ALK inhibitors was evaluated in vitro and in vivo. ResultsPC-9_v3a-gef and PC-9_v3b-gef cells were resistant to gefitinib and ALK inhibitors alone, but ALK inhibitors enhanced gefitinib-induced cytotoxicity. In animal studies, gefitinib completely inhibited the tumor growth in PC-9_vector cells but not in PC-9_v3a-gef and PC-9_v3b-gef cells. A combination of ALK inhibitor and gefitinib was found to be more potent than gefitinib alone in PC-9_v3a-gef and PC-9_v3b-gef cells. Furthermore, combination treatment with osimertinib and ceritinib caused a decrease in liver tumor size of the patient with liver metastases. ConclusionsOur data suggest that combination treatment with EGFR and ALK inhibitors can be a therapeutic strategy for treating NSCLC with concomitant EGFR mutation and EML4-ALK rearrangement.

EP16.03-012 Novel Human-derived EML4-ALK Fusion cell lines identify ribonucleotide reductase RRM2 as a Target of Activated ALK in NSCLC

Echinoderm microtubule-associated protein-like 4 (EML4)-Anaplastic Lymphoma Kinase (ALK) rearrangements occur in 7% to 10% of lung adenocarcinomas and are targets for treatment with tyrosine kinase inhibitors. Here we have developed three novel EML4-ALK-positive patient-derived Non-Small-Cell-Lung-Cancer (NSCLC) cancer cell lines, CUTO8 (variant 1), 9 (variant 1), and 29 (variant 3), and included a fourth ALK-positive cell line YU1077 (EML4-ALK variant 3) to study ALK-positive signaling and responses.

Novel human-derived EML4-ALK fusion cell lines identify ribonucleotide reductase RRM2 as a target of activated ALK in NSCLC

IntroductionEchinoderm microtubule-associated protein-like 4 (EML4)-Anaplastic Lymphoma Kinase (ALK) rearrangements occur in 3% to 7% of lung adenocarcinomas and are targets for treatment with tyrosine kinase inhibitors (TKIs). Here we have developed three novel EML4-ALK-positive patient–derived Non-Small-Cell-Lung-Cancer (NSCLC) cancer cell lines, CUTO8 (variant 1), CUTO9 (variant 1) and CUTO29 (variant 3) and included a fourth ALK-positive cell line YU1077 (variant 3) to study ALK-positive signaling and responses. Variants 1 and 3 are the most common EML4-ALK variants expressed in ALK-positive NSCLC, and currently cell lines representing these EML4-ALK variants are limited. Materials and methodsResazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting. RNA sequencing was performed in all four cell lines to identify differentially expressed genes. Whole-genome sequencing was performed to determine the presence of EML4-ALK fusion and ALK tyrosine kinase inhibitor resistance mutations. ResultsIn this study, we have confirmed expression of the corresponding ALK fusion protein and assessed their sensitivity to a range of ALK tyrosine kinase inhibitors. These patient derived cell lines exhibit differential sensitivity to lorlatinib, brigatinib and alectinib, with EML4-ALK variant 3 containing cell lines exhibiting increased sensitivity to lorlatinib and brigatinib as compared to alectinib. These cell lines were further characterized by whole genome sequencing and RNA-seq analysis that identified the ribonucleotide reductase regulatory subunit 2 (RRM2) as a downstream and potential therapeutic target in ALK-positive NSCLC. ConclusionWe provide a characterization of four novel EML4-ALK-positive NSCLC cell lines, highlighting genomic heterogeneity and differential responses to ALK TKI treatment. The RNA-Seq characterization of ALK-positive NSCLC CUTO8, CUTO9, CUTO29 and YU1077 cell lines reported here, has been compiled in an interactive ShinyApp resource for public data exploration (https://ccgg.ugent.be/shiny/nsclc_rrm2_2022/).

Alternative Treatment Options to ALK Inhibitor Monotherapy for EML4-ALK-Driven Lung Cancer.

Simple SummaryLung cancer remains one of the most common and difficult cancers to treat. However, excellent progress in identifying the genetic causes of lung cancers has revealed many of the mutant proteins driving progression of this cancer. This in turn has led to development of new precision medicines that target these proteins providing significant improvement to patient outcomes. Unfortunately, this benefit tends to be relatively short-lived as the cancers develop resistance to these targeted agents. In this review, we look specifically at cancers driven by the EML4-ALK protein that is present in around 5% of lung cancer patients, occurring more frequently in young non-smokers. We consider the recent evidence for how resistance develops to current clinically approved targeted ALK inhibitors in these patients. Furthermore, we explore how additional research is revealing exciting alternative treatment options that may lead to a more sustained response and increased long-term survival for these patients.EML4-ALK is an oncogenic fusion protein that accounts for approximately 5% of NSCLC cases. Targeted inhibitors of ALK are the standard of care treatment, often leading to a good initial response. Sadly, some patients do not respond well, and most will develop resistance over time, emphasizing the need for alternative treatments. This review discusses recent advances in our understanding of the mechanisms behind EML4-ALK-driven NSCLC progression and the opportunities they present for alternative treatment options to ALK inhibitor monotherapy. Targeting ALK-dependent signalling pathways can overcome resistance that has developed due to mutations in the ALK catalytic domain, as well as through activation of bypass mechanisms that utilise the same pathways. We also consider evidence for polytherapy approaches that combine targeted inhibition of these pathways with ALK inhibitors. Lastly, we review combination approaches that use targeted inhibitors of ALK together with chemotherapy, radiotherapy or immunotherapy. Throughout this article, we highlight the importance of alternative breakpoints in the EML4 gene that result in the generation of distinct EML4-ALK variants with different biological and pathological properties and consider monotherapy and polytherapy approaches that may be selective to particular variants.

Abstract 4120: Acquired TPM3-NTRK1 fusion resistant to larotrectinib in a non-small cell lung cancer with EML4-ALK fusion progressed on lorlatinib

Abstract Background: As oncogenic driver genes have been detected, targeted therapy plays a crucial role in non-small cell lung cancer (NSCLC) with targeted mutation. Lorlatinib, a selective third-generation inhibitor of anaplastic lymphoma kinase (ALK), is a treatment option for ALK-positive NSCLC patients who previously failed on ALK tyrosine kinase inhibitors (TKIs). Larotrectinib, a highly selective tropomyosin receptor kinase (TRK) inhibitor, is a treatment option for NSCLC with neurotrophic tyrosine receptor kinase (NTRK) mutation. Herein, we present an acquired TPM3-NTRK1 fusion resistant to larotrectinib in lung adenocarcinoma with EML4-ALK fusion progressed on lorlatinib. Case Presentation: A 48-year-old male with stage IV adenocarcinoma of lung with metastatic disease of bone was initially treated with carboplatin, pemetrexed, and pembrolizumab. Follow up CT in 4 months showed numerous hepatic lesions and liver biopsy demonstrated large cell neuroendocrine carcinoma. Upon progression of disease (PD), systemic therapy regimen was switched to combination of carboplatin, paclitaxel, and bevacizumab. After two months since starting the new regimen, a second PD was observed. Follow-up MRI brain demonstrated multiple small metastatic lesions. Subsequently, he was started on alectinib 600 mg twice a day given EML 4-ALK fusion mutation [4.8% of variant allele frequency (VAF)] in circulating tumor DNA (ctDNA) NGS assay. Follow-up CT chest in 5 months showed 5 mm left lower lobe nodule which remained unchanged in size. He maintained SD for 9 months before repeat MRI brain demonstrated PD with new leptomeningeal carcinomatosis. Subsequently, he received whole brain radiotherapy and the treatment regimen was switched to lorlatinib 100 mg daily. In 7-week follow-up CT, the 5 mm nodule in the left lower lobe was stable. Follow-up MRI brain in 16 weeks showed decrease in size and number of numerous enhancing parenchymal and leptomeningeal lesions. Overall SD was maintained for 8 months before follow-up MRI brain showed new punctate lesions. Repeat ctDNA NGS assay revealed new TPM3-NTRK1 fusion (VAF 0.2%) and ALK L1196M (VAF 2.8%), and a decision was made to add larotrectinib 100 mg daily. The 6-week follow-up MRI brain showed new parenchymal brain metastases. Discussion: It has been reported that the development of ALK resistance mutations may be associated with specific EML4-ALK variants. However, the influence of the specific EML4-ALK variants in NSCLC was not fully investigated. We report the case of acquired TPM3-NTRK1 fusion resistant to larotrectinib and acquired ALK L1196M in lung adenocarcinoma with EML4-ALK fusion progressed on lorlatinib. Further investigations are warranted to explore the mechanism of resistance to ALK TKIs. Citation Format: Horyun Choi, Jinah Kim, Yeun Ho Lee, Leeseul Kim, Na Hyun Kim, Young Kwang Chae. Acquired TPM3-NTRK1 fusion resistant to larotrectinib in a non-small cell lung cancer with EML4-ALK fusion progressed on lorlatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4120.

Phase 3 trial of lorlatinib in treatment-naive patients (Pts) with ALK-positive advanced non–small cell lung cancer (NSCLC): Comprehensive plasma and tumor genomic analyses.

9070 Background: Lorlatinib, a third-generation ALK tyrosine kinase inhibitor, has shown overall and intracranial activity in ALK+ advanced NSCLC. In the randomized, multicenter, phase 3 study in pts with previously untreated ALK+ advanced NSCLC (CROWN; NCT03052608), lorlatinib showed a statistically significant and clinically meaningful improvement in progression-free survival (PFS) vs crizotinib (Shaw AT, et al. N Engl J Med. 2020;383:2018-2029). Comprehensive molecular profiling of circulating tumor DNA (ctDNA) and tumor tissue was performed to identify molecular correlates of response. Methods: At baseline (BL), plasma samples were available from 134 and 129 pts in the lorlatinib and crizotinib arms, respectively. Analyses returned results for tumor tissue (archived or new biopsy) from 147 pts across both arms. Plasma and tumor DNA were analyzed by next-generation sequencing (NGS; Guardant360 and TissueNext, respectively, Guardant Health, Inc.). Objective response rate (ORR), duration of response, and PFS based on the September 20, 2021, cutoff, all assessed by blinded independent central review, were summarized according to mutation and tumor mutation burden (TMB) status. Results: At BL, 22% of pts had no detectable ctDNA. ALK missense mutations (n=19) or deletion (n=1) were detected in plasma of 12 pts (n=5 and 7 in the lorlatinib and crizotinib arms, respectively). Most pts harbored 1 mutation, but 3 pts harbored ≥3 mutations. In tumor samples, no somatic ALK mutation was detected. ALK fusions were detected in plasma of 48% of pts and in tumor of 80%. EML4-ALK variant (v) subtypes were highly concordant between ctDNA and tumor tissue. Based on ctDNA, ORRs were generally higher in the lorlatinib vs crizotinib arm, reaching 80% and 72% for EML4-ALK v1 and v3, respectively, in the lorlatinib arm, and 50% and 74% in the crizotinib arm. Median PFS was not reached for v1 in the lorlatinib arm and was 7.4 mo in the crizotinib arm; for v3, mPFS was 33.3 and 5.5 mo, respectively. TP53 mutations were found in 42% of pts with detectable ctDNA, and their presence did not seem to influence lorlatinib activity. In the crizotinib arm, absence of TP53 mutations led to longer PFS. These findings are being verified in tumor tissue. A pt treated with lorlatinib with an ongoing partial response in tumor lesions at the data cutoff date was found to have a KRAS G12V mutation and the presence of ALK fusion in tumor tissue but had no ctDNA detected at BL. Conclusions: Pts with untreated ALK+ advanced NSCLC had higher ORRs and potentially longer PFS across predefined biomarker subgroups when treated with lorlatinib compared with crizotinib in the phase 3 CROWN study. Based on pretreatment ctDNA and tumor tissue analyses, lorlatinib led to strong clinical benefit regardless of the type of ALK rearrangement or presence of potential driver co-mutation. Clinical trial information: NCT03052608.

Alectinib versus crizotinib in ALK-positive advanced non-small cell lung cancer and comparison of next-generation TKIs after crizotinib failure: Real-world evidence.

Anaplastic lymphoma kinase (ALK) fusion is a prognostic indicator for patients with non-small cell lung cancer (NSCLC) receiving tyrosine kinase inhibitors (TKIs). The real-world data of ALK TKIs remain a major concern. Patients with ALK-positive advanced NSCLC, who received crizotinib or alectinib treatment in first line, were retrospectively reviewed. ALK status was detected using immunohistochemistry (IHC) or next-generation sequencing (NGS). Clinical outcomes have been comprehensively analyzed between TKIs, ALK fusions, EML4-ALK variants, and next-generation TKIs after crizotinib failure. One hundred sixty-eight patients were successively enrolled (crizotinib, n=109; alctinib, n=59). Alectinib showed consistent superiority in progressive-free survival (PFS) over crizotinib (hazard ratio [HR]: 0.43, 95% confidential interval [CI]: 0.24-0.77, p=0.004). Multivariate Cox regression showed chemotherapy (CT) prior to TKIs or synchronous chemotherapy seemed not to improve PFS compared to ALK inhibitors alone (p > 0.05). And, alectinib was superior to crizotinib in prolonging intracranial PFS (HR 0.12, 95% CI: 0.03-0.49, p=0.003). Patients in EML4 group had a better prognosis than those in non-EML4 group after alectinib administration (HR 0.13, 95% CI: 0.03-0.60, p=0.009). TP53 co-mutations were relatively common (34.0%) and associated with adverse outcome in ALK-positive patients (adjusted HR 2.22, 95% CI: 1.00-4.92, p=0.049). After crizotinib failure, 33 patients received a sequential application of next-generation ALK TKIs. Compared to ceritinib and brigatinib, alectinib might have better PFS (p=0.043). Our results revealed alectinib had better PFS and higher intracranial efficacy compared to crizotinib in ALK-positive NSCLC, and might improve PFS by comparison with ceritinib and brigatinib after crizotinib failure.

Efficacy and safety of lorlatinib in Asian and non-Asian patients with ALK-positive advanced non-small cell lung cancer: Subgroup analysis of a global phase 2 trial

ObjectivesTo analyze the efficacy and safety of lorlatinib in Asian and non-Asian patients with pretreated anaplastic lymphoma kinase (ALK)-positive, advanced non-small cell lung cancer (NSCLC) from a phase 1/2 study. Materials and methodsIn this ongoing phase 2 part of the trial, patients with ALK- or ROS1-positive, advanced NSCLC enrolled into six expansion cohorts (EXP1–6), based on ALK and ROS1 status and previous therapy, and received lorlatinib 100 mg once daily. The primary endpoint was objective tumor response and intracranial response. Post hoc analyses of activity were conducted in Asian and non-Asian (based on race) ALK-positive patients who received either previous crizotinib with or without chemotherapy (EXP2–3A) or at least one second-generation ALK tyrosine kinase inhibitor with any number of chemotherapy regimens (EXP3B–5). Analysis of safety (adverse events [AEs]) was in the phase 1 and 2 study population who started lorlatinib 100 mg once daily. Results17 Asian patients were enrolled in EXP2–3A and 53 in EXP3B–5; 33 non-Asian patients were enrolled in EXP2–3A and 73 in EXP3B–5. Objective response rates in the Asian and non-Asian subgroups were 82.4% (95% confidence interval [CI]: 56.6–96.2) and 63.6% (95% CI: 45.1–79.6) in EXP2–3A, and 47.2% (95% CI: 33.3–61.4) and 30.1% (95% CI: 19.9–42.0) in EXP3B–5, and median progression-free survival was 13.6 and 12.5 months (EXP2–3A) and 6.9 and 5.5 months (EXP3B–5). Lorlatinib exhibited antitumor activity across ALK resistance mutations, while no differences according to the EML4-ALK variant could be detected. The most common treatment-related AEs were hypercholesterolemia, hypertriglyceridemia, edema, and peripheral neuropathy in both Asian and non-Asian subgroups. ConclusionLorlatinib showed substantial overall and intracranial activity in pretreated patients with ALK-positive NSCLC in both Asian and non-Asian patients. AE profiles were similar between Asian and non-Asian patients. ClinicalTrials.gov NCT01970865.