EML4-ALK Fusion Variant Research Articles

A novel double fusion of EML4-ALK and PLEKHA7-ALK contribute to rapid progression of lung adenocarcinoma: a case report and literature review

A 40-year-old male with EML4-ALK (E6:A20) fusion variant 3 and previously unreported PLEKHA7-ALK (P3:A20) fusion in lung adenocarcinoma exhibited resistance to alectinib and chemotherapy. Subsequent next-generation sequencing (NGS) from the plasma specimen revealed the co-existing mutation in the KEAP1 gene, which may represent an intrinsic resistance to ALK-TKI. Furthermore, the presence of double fusion PLEKHA7-ALK (P3:A20) may also have played a critical role in the resistance to alectinib. KEAP1 mutation (p.E244K) was also founded in this patient which may lead to resistance to standard chemotherapy. The patient was then treated with brigatinib, which effectively halted the rapid progression. Unfortunately, the patient deceased to uncontrollable, rapidly progressing pleural effusion and pulmonary embolism, resulting in an overall survival of 9 months. This represents the rare case of NSCLC with a double fusion of EML4-ALK and PLEKHA7-ALK, exhibiting resistance to alectinib and chemotherapy. Our case suggests that the double fusion of EML4-ALK and PLEKHA7-ALK and co-existing KEAP1 mutation may serve as an adverse prognostic factor. Additionally, brigatinib may offer a potential treatment option for lung adenocarcinoma patients with PLEKHA7-ALK (P3:A20) fusion.

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

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.

The mesenchymal morphology of cells expressing the EML4–ALK V3 oncogene is dependent on phosphorylation of Eg5 by NEK7

Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressingEML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes withEg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 andEg5 phosphorylation promotes the mesenchymal morphology typical of these cells.

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.

Impact of EML4-ALK fusion variant and co-occurring TP53 mutation on treatment duration of first-line next-generation ALK TKIs in ALK fusion+ NSCLC.

9029 Background: Molecular biomarkers such as EML4-ALK fusion variant 3 (V3) and TP53 mutation are associated with poor prognosis in patients (pts) with anaplastic lymphoma kinase fusion positive ( ALK+) non–small cell lung cancer (NSCLC). We evaluated the effect of EML4-ALK fusion variants and TP53 mutation status on time to treatment discontinuation (TTD) of first-line (1L) treatment with a next-generation ALK tyrosine kinase inhibitor (TKI) in pts with ALK+ NSCLC in the real-world setting. Methods: Pts with advanced/metastatic NSCLC who were ALK+ in circulating tumor DNA by Guardant360 (G360) liquid biopsy test from Jan 1, 2018, to Dec 31, 2021, and received 1L monotherapy with a next-generation ALK TKI (alectinib, brigatinib, ceritinib, lorlatinib) were identified in the Guardant INFORM clinical-genomics database. Median TTD of 1L ALK TKI treatment was determined by Kaplan-Meier (KM) methods. The impact of EML4-ALK fusion variant and TP53 mutation status on TTD was evaluated. Results: Database search identified 495 pts with advanced/metastatic NSCLC who had ALK fusion by G360; of these, 214 pts received 1L treatment with a next-generation ALK TKI; 164 had G360 testing before treatment (alectinib, n=147; brigatinib, n=7; ceritinib, n=2; lorlatinib, n=8). Among these 164 pts, median age was 58.6 y, 53% were female, and 29% had baseline brain metastases. Median follow-up from start of 1L treatment was 14.8 mo. Median TTD was 19.6 mo (95% CI: 13.6–not reached [NR]). TP53 mutations were detected in 66 pts (40%). Median (95% CI) TTD in pts with TP53 mutation vs WT was 17.1 (9.5–28.5) vs 21.9 mo (14.3–NR; log-rank P=0.1712). Of 130 pts with EML4-ALK fusion variant type detected, 63 (48%) had V1, 54 (42%) V3, 8 (6%) V2, 4 (3%) V5, and 1 (1%) V8. In pts with EML4-ALK fusion V1 vs V3, median (95% CI) TTD was 19.0 (13.6–NR) vs 21.8 mo (8.7–NR; HR: 0.73; log-rank P=0.3039), with the KM plot showing numerically longer TTD in pts with V1 vs V3 during the first 18 mo and lines crossing at the tail end. Of 117 pts with both an EML4-ALK fusion variant (V1/V3) and TP53 data, 21 (18%) had both V3 and a TP53 mutation. Pts with co-occurring EML4-ALK V3 and TP53 mutation had the shortest TTD (median TTD 8.7 [3.9–NR; HR: 2.59; P=0.0219]; Table). Conclusions: Among pts with NSCLC and ALK fusion, co-occurrence of EML4-ALK V3 and TP53 mutation is common and associated with poor prognosis and high risk of early discontinuation of 1L treatment with a next-generation ALK TKI, most likely due to disease progression. Additional novel or combination therapies are warranted for this subpopulation. [Table: see text]

Coexistence of a novel SETD2-ALK, EML4-ALK double-fusion in an advanced lung adenocarcinoma patient after alectinib resistant and response to immunotherapy combined with chemotherapy: a case report

The single echinoderm microtubule-associated protein-like 4 (EML4) gene and anaplastic lymphoma kinase (ALK) gene fusion is the most common variant of ALK rearrangements in non-small cell lung cancer (NSCLC). Herein, we firstly report that coexistence of a novel histone methyltransferase (SETD2)-ALK, EML4-ALK double-fusion is sensitive to alectinib as first-line therapy, and response to immunotherapy combined with chemotherapy after resistant. The patient responded to alectinib as a first-line therapy and achieved progression-free survival (PFS) for 26 months. After resistance, liquid biopsy showed that the reason of drug resistance was the disappearance of SETD2-ALK and EML4-ALK fusion variants. In addition, chemotherapy combined with immunotherapy subsequently achieved a survival benefit of more than 25 months. Therefore, alectinib may be a viable therapeutic option for NSCLC patients with double ALK fusion and immunotherapy combined with chemotherapy may be a viable therapeutic option when double ALK fusion loss may be the mechanism of alectinib resistance.

Abstract 313: Mechanisms of oncogenic tyrosine kinase fusion formation and selection in human cancers

Abstract Introduction: Tyrosine kinase (TK) fusions are common events in the development of resistance to targeted therapy for non-small cell lung cancer (NSCLC) and others. The mechanisms by which recurrent breakpoints are generated in solid tumors and by which they are selected are unclear. APOBEC3 proteins are the main factors for mutations in solid cancers and the mutagenic activity of APOBEC3 can activate the DNA damage response by contributing to double-strand break (DSB) formation. However, the possibility that APOBEC3 proteins could induce chromosomal translocations in solid tumors has not been investigated. In addition, mechanistic explanations on how recurrent chromosomal translocation occur in lung cancer and solid tumors in precise genomic breakpoints and with specific partners are lacking. In this work, we present a newly developed functionally active chromosomal translocation sequencing (FACTS) approach to provide insights on the mechanisms that determine the selection of oncogenic fusions in human cancers. Methods: We applied FACTS to lung cells to map genome-wide translocations when DNA DSB is introduced in TK genes by the CRISPR/Cas9 system. We generated genomic maps of DNA breakpoints joining to anaplastic lymphoma kinase (ALK) before and after selection by high-throughput genome-wide translocation sequencing (HTGTS). We tested the effect of APOBEC3 proteins in an overexpression system or in an all seven APOBEC3 gene knockout lung cells by FACTS. Results: Overexpression of APOBEC3 proteins significantly increased the formation of functional fusions compared to control cells. In addition, deletion of APOBEC3 genes significantly decreased the formation of functional fusions compared to APOBEC3 wild-type cells. With the FACTS data and bioinformatics analyses, we found that gene expression rather than locus accessibility dictates the selection of TK fusions and that TK fusions are likely selected from a pool of translocations occurring at the right position in transcribe genes. In addition, we found that active transcription in silent genes was sufficient to introduce the spontaneous formation of functional fusions. By using HTGTS approach, we found that breakpoints before selection did not show a preferential strand bias, but after selection the breakpoints showed a strong bias. By comparing oncogenic potential of EML4-ALK fusion variants, we found that protein stability, but not subcellular localization, is key to determining the exon usage of EML-ALK fusions. In addition, we revealed that the selection of fusion partners for each TK is more likely dictated by protein stability rather than by mRNA expression. Conclusion: FACTS allows rapid and comprehensive mapping of functional chromosomal translocations and provides insights into the mechanisms of their formation and selection. The FACTS approach could be used to predict the oncogenic activity of fusion proteins to guide clinical decisions. Citation Format: Taek-Chin Cheong, Ahram Jang, Qi Wang, Giulia C. Leonardi, Maria K. Lehtinen, Marian H. Harris, Roberto Chiarle. Mechanisms of oncogenic tyrosine kinase fusion formation and selection in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 313.

The impact of theALK fusion variant on clinical outcomes in EML4-ALK patients with NSCLC: a systematic review and meta-analysis.

Background: Recent studies showed that ALK-fusion variants are associated with heterogeneous clinical outcomes. However, contradictory conclusions have beendrawn inother studies showing no correlation between ALK variants and prognoses. Methods: A systematic review and meta-analysis wasperformed to evaluate the prognostic value of EML4-ALK fusion variants for patientoutcomes. Results: 28 studies were included in the analysis. According to the pooled results, patients harboring variant 1 showed equivalent progression-free survival(PFS)and overall survival (OS) with non-v1 patients(hazard ratio [HR] for PFS: 0.91 [0.68-1.21];p=0.499; OS: 1.12 [0.73-1.72];p=0.610). Similarly, patients with v3 showed the same disease progress as non-v3 patients (pooled HR for PFS=1.07 [0.72-1.58];p=0.741). However, pooled results for OS suggested that patients with v3 had worse survival than non-v3 patients(HR=3.44 [1.42-8.35];p=0.006). Conclusion: Results suggestthat patients with v1 exhibited no significant difference from non-v1 in terms of OS and PFS, while v3 was associated with shorter OS in ALK-positive patients withnon-small cell lung cancer.

Coexistence of a novel NBEA-ALK, EML4-ALK double-fusion in a lung adenocarcinoma patient and response to alectinib: A case report

ObjectivesThe echinoderm microtubule-associated protein-like 4 gene (EML4) and anaplastic lymphoma kinase gene (ALK) fusion is the most common ALK rearrangements in non-small cell lung cancer (NSCLC). Herein, we firstly report that coexistence of a novel Neurobeachin (NBEA)-ALK, EML4-ALK double-fusion is sensitive to alectinib. Materials and methodsHematoxylin-eosin staining (HE), fluorescent in situ hybridization (FISH), and next-generation sequencing (NGS) was performed on the biopsy sample. ResultsThe patient responded to alectinib as a second-line treatment and achieved stable disease for 11 months, without significant symptoms of toxicity. Significantly, the liquid biopsy also validated clinical benefit, with the disappearance of NBEA-ALK and EML4-ALK fusion variants. We also provided a comprehensive review of all 50 ALK fusion genes in NSCLC. ConclusionThis is the first report on one patient with a novel NBEA-ALK, EML4-ALK double-ALK fusion beneficial from alectinib. Alectinib may be a viable therapeutic option for NSCLC patients with double-ALK fusion, and liquid biopsy could dynamically monitor clinical curative effect.

P45.11 Co-occurring CDKN2A/B Alteration Is Associated With Worse Survival Outcomes in Advanced ALK-Positive Non-Small Cell Lung Cancer

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have demonstrated promising activity in ALK-positive (ALK+) non-small cell lung cancer (NSCLC). However, co-occurring genetic alterations, especially those occurring downstream of tumor initiation oncogenes, may influence the efficacy of target therapies. This study analyzed whether co-occurring alterations influence survival outcomes to second-generation anti-ALK therapy in metastatic ALK+ NSCLC patients. From March 2018 to February 2020, in a single center, we analyzed prospectively 44 tumor specimens of patients with metastatic ALK+ NSCLC. Those tumor samples with positive immunohistochemistry, assessed by D5F3 assay, underwent genotyping using the Next-Generation Sequencing platform (Foundation One CDx). Progression-free survival (PFS) and overall survival (OS) were estimated for the total cohort and main commutations. Forty-two patients were analyzed, mean age was 52.5±10.7 years, females in 59.5%, never smokers in 73.8%, and ECOG PS (0-1) in 90.5%. The lung adenocarcinoma subtype was observed in 97.5% of tumor samples, and the predominant solid pattern was the most frequently reported in 23.8%. All patients received a second-generation ALK-TKI in the first or second-line, 82.5% and 17.5%, respectively. Alectinib, brigatinib, and ceritinib were administered in 79%, 13.1%, and 7.9% of the cases. EML4-ALK 3a/b variant was the most common (47.6%), followed by variant 1 in 38.1%. The median PFS in the overall cohort was 22.3 months. No significant differences were observed in the progression-free intervals according to the distinct variants, neither when the variable was dichotomized (3a/b variant vs. others). The most frequent co-occurrent alterations were TP53 (23.8%) and CDKN2A/B (14.3%). Loss of the CDKN2A/B gene was the most common genetic aberration (83.3%) within this subgroup. Among patients with co-current CDKN2A/B alterations, median PFS was significantly shorter than CDKN2A/B wild-type patients (9.3 vs. 28.2 months), p=0.047. The median PFS was shorter in patients harboring a commutation in TP53; however, this difference was not significant (10.7 vs. 22.8 months), p=0.147. The presence of CDKN2A/B co-occurring alteration was correlated significantly with higher baseline serum carcinoembryonic antigen (CEA) levels (p=0.008), and other EML4-ALK fusion variants rather than 3a/b (p=0.02). After adjusting for confounding variables, CDKN2A/B co-occurring alterations remained strongly associated with poorer PFS outcomes. Co-occurring genetic alterations in CKN2A/B confers a poor prognosis in patients treated with a second-generation ALK-TKI in metastatic ALK+ NSCLC patients. The loss of the CDKN2A/B gene was the most frequent alteration seen in this subgroup of patients.

Brigatinib Versus Crizotinib in ALK Inhibitor–Naive Advanced ALK-Positive NSCLC: Final Results of Phase 3 ALTA-1L Trial

IntroductionIn the phase 3 study entitled ALK in Lung cancer Trial of brigAtinib in 1st Line (ALTA-1L), which is a study of brigatinib in ALK inhibitor–naive advanced ALK-positive NSCLC, brigatinib exhibited superior progression-free survival (PFS) versus crizotinib in the two planned interim analyses. Here, we report the final efficacy, safety, and exploratory results. MethodsPatients were randomized to brigatinib 180 mg once daily (7-d lead-in at 90 mg once daily) or crizotinib 250 mg twice daily. The primary end point was a blinded independent review committee–assessed PFS. Genetic alterations in plasma cell-free DNA were assessed in relation to clinical efficacy. ResultsA total of 275 patients were enrolled (brigatinib, n = 137; crizotinib, n = 138). At study end, (brigatinib median follow-up = 40.4 mo), the 3-year PFS by blinded independent review committee was 43% (brigatinib) versus 19% (crizotinib; median = 24.0 versus 11.1 mo, hazard ratio [HR] = 0.48, 95% confidence interval [CI]: 0.35–0.66). The median overall survival was not reached in either group (HR = 0.81, 95% CI: 0.53–1.22). Posthoc analyses suggested an overall survival benefit for brigatinib in patients with baseline brain metastases (HR = 0.43, 95% CI: 0.21–0.89). Detectable baseline EML4-ALK fusion variant 3 and TP53 mutation in plasma were associated with poor PFS. Brigatinib exhibited superior efficacy compared with crizotinib regardless of EML4-ALK variant and TP53 mutation. Emerging secondary ALK mutations were rare in patients progressing on brigatinib. No new safety signals were observed. ConclusionsIn the ALTA-1L final analysis, with longer follow-up, brigatinib continued to exhibit superior efficacy and tolerability versus crizotinib in patients with or without poor prognostic biomarkers. The suggested survival benefit with brigatinib in patients with brain metastases warrants future study.

Patient-derived lung cancer organoids for the selection of therapeutic options in an ALK-rearranged tumor.

e21014 Background: ALK rearrangements are key targets in non-small-cell lung cancer (NSCLC). Unfortunately, the optimal sequential strategy of ALK-tyrosine kinase inhibitors (TKI) remains to be defined. Testing drug sensitivity in patient derived organoids (PDOs) could support a rational drug selection in this setting. Methods: We designed an observational study assessing the correlation between drug sensitivity of PDOs established in Invitrocue and the clinical outcome in our institution. To date, forty cases have been included, of which nine were lung cancers. Results: PDO was sucessfully established in 7/9 cases (77%). Three patients did not receive any of the drugs tested in vitro due to clinical deterioration. One patient was deemed as sensitive to carboplatin but tumor showed to be resistant. A meaningful correlation was observed in an oncogenic addicted tumor. A 54-year-old never-smoker man who had been diagnosed with lung adenocarcinoma stage IVa (T3N1M1a). He received standard first-line therapy with platinum-based chemotherapy, immunotherapy and antiangiogenics achieving tumor progression. A next-generation sequencing (NGS) panel revealed the presence of the EML4-ALK fusion variant 3a/b. The patient started alectinib but showed progression after 12 months. A second NGS panel did not identify any new ALK resistance mutation but acquiring TP53. Treatment was switched to brigatinib with no response. Finally, fresh tumor tissue was obtained from a liver biopsy to establish PDOs and drug sensitivity to 8 compounds was tested. The In vitro results demonstrated no activity of lorlatinib but a strong response to crizotinib. The patient started Crizotinib 250mg BID achieving partial response after 8 weeks and treatment duration of 6 months. A third liver biopsy for PDOs and NGS revealed acquired ALK C1156Y and I1171T mutations that confer resistance to crizotinib but sensitive to ceritinib, respectively. Conclusions: We present clinical evidence that PDOs are an alternative tool in oncogenic addicted tumors helping to guide treatment decisions and increasing a more personalized sequential treatment approach.

Therapeutic Sequencing in ALK+ NSCLC.

Anaplastic lymphoma kinase-rearranged non-small-cell lung cancer (ALK+ NSCLC) is a model disease for the use of targeted pharmaceuticals in thoracic oncology. Due to higher systemic and intracranial efficacy, the second-generation ALK tyrosine kinase inhibitors (TKI) alectinib and brigatinib have irrevocably displaced crizotinib as standard first-line treatment, based on the results of the ALEX and ALTA-1L trials. Besides, lorlatinib and brigatinib are the preferred second-line therapies for progression under second-generation TKI and crizotinib, respectively, based on the results of several phase II studies. Tissue or liquid rebiopsies at the time of disease progression, even though not mandated by the approval status of any ALK inhibitor, are gaining importance for individualization and optimization of patient management. Of particular interest are cases with off-target resistance, for example MET, HER2 or KRAS alterations, which require special therapeutic maneuvers, e.g., inclusion in early clinical trials or off-label administration of respectively targeted drugs. On the other hand, up to approximately half of the patients failing TKI, develop anatomically restricted progression, which can be initially tackled with local ablative measures without switch of systemic therapy. Among the overall biologically favorable ALK+ tumors, with a mean tumor mutational burden uniquely below 3 mutations per Mb and the longest survival among NSCLC currently, presence of the EML4-ALK fusion variant 3 and/or TP53 mutations identify high-risk cases with earlier treatment failure and a need for more aggressive surveillance and treatment strategies. The potential clinical utility of longitudinal ctDNA assays for earlier detection of disease progression and improved guidance of therapy in these patients is a currently a matter of intense investigation. Major pharmaceutical challenges for the field are the development of more potent, fourth-generation TKI and effective immuno-oncological interventions, especially ALK-directed cell therapies, which will be essential for further improving survival and achieving cure of ALK+ tumors.

Abstract 2400: Strong biological bias for ALK intron 19 breakpoints in NSCLC

Abstract Introduction: EML4-ALK translocations are detected in 4-8 % of non-small cell lung cancer (NSCLC). While different EML4-ALK variants are defined by different breakpoints in the EML4 gene, most frequently located in intron 6 or 13, ALK breakpoint is almost invariably in intron 19. Rare reports describe EML4-ALK translocations with breakpoints in intron 17 or intron 18 of the ALK gene. Despite all these ALK breakpoints potentially generate oncogenic fusions, the reasons of this strong imbalance toward intron 19 (exon 20) breakpoints in ALK positive NSCLC are currently unknown. The aim of this study is to investigate the mechanisms that underlie ALK translocation in NSCLC. Methods: Eight different EML4-ALK fusions were either overexpressed with a retroviral system or generated with CRISPR/Cas9 lentivirus from the endogenous loci. EML4 exons 1-6 (E6) or exons 1-13 (E13) with different ALK exons: E6;A18, E6;A19, E6;A20 or E6;A21 and E13;A18, E13;A19, E13;A20, E13;A21. The fusions E6;A21 and E13;A21 contained an early stop codon not producing functional proteins. These EML4-ALK fusions were expressed in NIH3T3 and PC9 by retroviral vectors or engineered in PC9 (an EGFR-dependent lung cancer cell line that is sensitive to inhibition with the EGFR inhibitor osimertinib) and BEAS-2B cells by CRISPR/Cas9. HTGTS was performed on engineered PC9 cells. NSG immunodeficient mice were used for in vivo experiments with NIH3T3 and BEAS-2B cells. Results: The retroviral overexpression system showed that all EML4-ALK fusion proteins were expressed leading to an actively phosphorylated ALK in NIH3T3 and PC9 cells, except E6;A21 and E13;A21 fusions where no protein was detected, as expected. Accordingly, all active variants were able to induce NIH3T3 cell transformation and tumor formation in vivo and conferred resistance to osimertinib in PC9 cells. In contrast, when EML4-ALK translocations were induced by CRISPR/Cas9 in PC9 cells, only E6;A20 and E13;A20 variants rapidly overcame osimertinib inhibition while other variants rescued osimertinib-inihibited cells slowly and less efficiently. Surprisingly, the E6;A20 and E13;A20 fusions were invariably found in all resistant clones independently of the translocation induced by CRISPR/Cas9. Similarly, all tumors formed in vivo by BEAS-2B cells contained the E6;A20 or E13;A20 variants irrespective of the translocation induced originally. By cloning translocation junctions at large scale with HTGTS, we found that clones initially forced to break into ALK intron 17 eventually acquired a second breakpoint in ALK intron 19. Conclusions: Our data show that all EML4-ALK fusion variants were equally oncogenic when overexpressed. In contrast, when EML4-ALK variants were generated from the endogenous loci there was a strong selection bias toward ALK fusions originating in intron 19 suggesting that intron 19 variants have the strongest oncogenic potential in lung epithelial cells. Citation Format: Giulia C. Leonardi, Taek-Chin Cheong, Chiara Ambrogio, Tao Chen, Wei-Tien Tai, Elif Karaca, Ines Mota, Massimo Libra, Mark M. Awad, Rafael Blasco-Patino, Roberto Chiarle. Strong biological bias for ALK intron 19 breakpoints in NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2400.

Abstract CT025: Impact of the EML4-ALK fusion variant on the efficacy of lorlatinib in patients (pts) with ALK-positive advanced non-small cell lung cancer (NSCLC)

Abstract BACKGROUND: Lorlatinib is a selective, potent, brain-penetrant, 3rd-generation (gen) ALK/ROS1 tyrosine kinase inhibitor (TKI) preclinically active against most known ALK resistance mutations. In pts with ALK+ advanced NSCLC with disease progression following 2nd-gen ALK TKIs, lorlatinib has shown robust clinical activity and we found that tumor genotyping for ALK mutations may identify such pts more likely to respond to lorlatinib treatment. Prior analyses have suggested that EML4-ALK variant type may influence ALK TKI treatment benefit. To further identify molecular correlates of response, we performed an exploratory subgroup analysis by EML4-ALK variant type and ALK resistance mutations in pts previously treated with 2nd-gen ALK TKIs and who received the recommended Ph 2 dose of lorlatinib (100 mg once daily). METHODS: Baseline plasma samples were collected from pts with ALK+ NSCLC with ≥1 prior 2nd-gen ALK TKI enrolled in the ongoing registrational Ph 2 study (NCT01970865). Circulating free DNA (cfDNA) was analyzed using Guardant360 (Guardant Health, Inc., CA, USA) to determine EML4-ALK variant and ALK kinase domain mutations. Objective response rate (ORR) and duration of response (DOR), by independent central review, were evaluated according to EML4-ALK variant type and ALK resistance mutation status. RESULTS: ALK fusions were detectable in 64 (41.0%) of 156 pt plasma samples. EML4-ALK variants 1, 2, and 3 were detected at frequency of 17.3%, 2.6% and 15.4%, respectively. Other EML4-ALK variants (including the variant types 4, 5, 7, and 8) were also detected in 3.2% of pts, as well as some other less frequent fusion partners (e.g., KIF5B) in 2.6% of pts. Based on cfDNA, ALK resistance mutations were detected in 40 pts; of whom, 6 pts harbored EML4-ALK variant 1, 1 pt had variant 2 and 18 had variant 3. The G1202R/Del mutation was detected in 23 pts samples; of which, 15 (65.2%) also harbored EML4-ALK variant 3. ORR was 33.3% (95% CI 16.5-54.0), 75.0% (95% CI 19.4-99.4) and 45.8% (95% CI 25.6-67.2) for variants 1, 2 and 3, respectively, while median DOR was similar for pts with variant 1 or 3 (both 6.9 months). Median DOR was not reached for variant 2. Of note, no responses were observed in the pts with other types of ALK rearrangements detected. Finally, ALK fusions were not detected in the cfDNA of 92 pts (59.0%), including 12 pts who had detectable ALK mutations. Among these 92 pts, ORR was 39.1% (95% CI 29.1-49.9) and median DOR was 7.1 months (95% C 5.5-not reached). Confirmation of these results in the tumor tissue is ongoing. CONCLUSION: In this heavily pretreated group of ALK+ NSCLC pts, the presence of an ALK resistance mutation might enrich for EML4-ALK variants 1 and 3. Lorlatinib exhibited antitumor activity irrespective of EML4-ALK variant and across a variety of ALK resistance mutations. Citation Format: Todd M. Bauer, Jean-François Martini, Benjamin Besse, Chia-Chi Lin, Ross A. Soo, Gregory J. Riely, Sai-Hong Ignatius Ou, Francesca Toffalorio, Antonello Abbattista, Holger Thurm, D. Ross Camidge, Steven Kao, Rita Chiari, Shirish Gadgeel, Enriqueta Felip, Alice T. Shaw, Benjamin J. Solomon. Impact of the EML4-ALK fusion variant on the efficacy of lorlatinib in patients (pts) with ALK-positive advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT025.

Abstract A21: Longitudinal analysis of cell-free DNA for therapy monitoring of ALK-positive non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) patients with ALK rearrangements are routinely treated with tyrosine kinase inhibitors (TKIs), leading to improved survival. However, clinical courses vary widely and the disease remains incurable. Therefore, early detection and molecular characterization of treatment failure is important for therapy guidance. To identify indicators of therapy response and resistance, we performed genotyping of circulating cell-free DNA (cfDNA), including panel-based deep sequencing (&amp;gt;4,200x coverage) and shallow whole-genome sequencing (sWGS; 0.5x coverage) from serial plasma samples (n=282) of 73 patients with ALK rearrangements. Variable mutation levels were marked in all patients and correlated with clinical features. At progression, individual dynamics of resistance mutations were seen: while some mutations became undetectable upon therapy switch, variant fractions of other alterations further increased. We also found mutated TP53 at the time of progression in patients with initially TP53 wild-type tumors. The progression-free survival of patients with acquired TP53 mutations was comparable to that of primarily TP53 mutated and shorter than that of persistently TP53 wild-type cases. Shallow WGS of cfDNA identified copy number variations (CNVs), some of which might contribute to disease progression. To quantitatively assess CNVs, trimmed median absolute deviation from copy number neutrality (t-MAD) scores were calculated from sWGS data. Longitudinal t-MAD assessment reflected the kinetics of mutant allele frequencies in cfDNA and allowed disease monitoring with increasing t-MAD and mutation levels at tumor progression. Moreover, EML4-ALK fusion variant 3 (V3) and TP53 mutations define molecular determinants of adverse outcome in ALK+ NSCLC. Shallow WGS of cfDNA identified increased t-MAD levels in patients with EML4-ALK V3 compared to those with fusion V1. Similarly, patients with TP53 mutations showed a higher t-MAD than TP53 wild-type cases. In summary, overall survival of patients with detectable mutations in plasma DNA at any time point during clinical follow-up was shorter than that of patients without detectable mutations in cfDNA. Similarly, overall survival time from the first detection of mutations cfDNA until death/end of follow-up was shorter than in cases without detectable mutations. Our data suggest that liquid biopsies could help to improve the care of ALK+ NSCLC patients through early detection of disease progression and tailored treatment of resistant tumors. Although individual courses were seen, rising mutation levels could indicate the need of radiologic reassessment earlier than scheduled. Detection of CNVs as surrogate for genomic instability and acquired TP53 mutations in liquid identified high-risk cases and suggests the potential clinical utility of cfDNA monitoring for this disease beyond profiling of ALK resistance mutations. Citation Format: Steffen Dietz, Petros Christopoulos, Lisa Gu, Volker Endris, Zhao Yuan, Simon J. Ogrodnik, Tomasz Zemojtel, Marc A. Schneider, Anna-Lena Volckmar, Michael Meister, Thomas Muley, Martin Reck, Matthias Schlesner, Michael Thomas, Albrecht Stenzinger, Holger Sültmann. Longitudinal analysis of cell-free DNA for therapy monitoring of ALK-positive non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A21.

Correlation of baseline molecular and clinical variables with ALK inhibitor efficacy in ALTA-1L.

9517 Background: Efficacy of ALK TKIs in patients (pts) with ALK+ non-small cell lung cancer (NSCLC) varies. We evaluated the impact of EML4-ALK fusion variants and other baseline (BL) molecular and clinical variables on clinical efficacy of brigatinib (BRG) vs crizotinib (CRZ) as first ALK TKI therapy in pts with ALK+ NSCLC in the phase 3 ALTA-1L (NCT02737501) trial. Methods: Plasma samples were collected at screening for molecular genetic analysis of ALK and other genes implicated in NSCLC by next-generation sequencing. Exploratory analyses were performed to identify associations of clinical outcomes with oncogenic alterations including ALK fusion variants and TP53 status. Results: 124 BL samples were collected from 136 BRG-treated pts and 127 from 137 CRZ-treated pts. Pts with plasma samples were representative of the intent-to-treat population. BL ALK fusion detection rate was 52% (65/124) and 54% (68/127) in the BRG and CRZ arms, respectively, of which 83% (54/65) and 93% (63/68) were EML4-ALK fusions. In pts with detectable EML4-ALK fusions, the three predominant EML4-ALK fusion variants (V1, V2, V3) were equally distributed between arms; V1 and V3 were most prevalent (BRG/CRZ: V1, 42%/47%; V3, 42%/33%) but V1 was more frequent than V3 in pts without BL brain metastasis (47% vs 36%) or prior chemotherapy (45% vs 35%). Gender and age did not impact variant type. BRG showed higher ORR and improved mPFS vs CRZ in all variant subgroups; pts with V3 had poorer PFS compared with V1 and V2 regardless of treatment (Table). In pts with V3, BRG showed significantly improved PFS (HR=0.273, 95% CI 0.125, 0.597) and higher ORR (84% vs 67%) vs CRZ. TP53 mutation was detected in 30% (37/124) of pts in BRG arm and 26% (33/127) in CRZ arm. In pts with detectable ALK fusion, TP53 mutation showed poorer PFS in both arms than nonmutant/undetected cases (Table). BRG had better ORR and PFS vs CRZ in pts regardless of TP53 mutation status. Additional analyses of BL variables are ongoing. Conclusions: EML4-ALK fusion variant 3 and TP53 mutation were identified as poor prognosis biomarkers in ALK+ NSCLC. BRG demonstrated better efficacy than CRZ as first-line therapy in pts regardless of EML4-ALK fusion variant and TP53 mutation status. These findings may help define areas of greatest unmet need. Clinical trial information: NCT02737501 . [Table: see text]

Defining molecular risk in ALK+ NSCLC.

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancers (NSCLC) have the best prognosis among metastatic pulmonary malignancies, with a median patient survival currently exceeding 5 years. While this is definitely a major therapeutic success for thoracic oncology, it may not be entirely attributable to rapid drug development and the strenuous clinical efforts. At the genetic level, ALK+ disease is also unique, distinguished by the lowest tumor mutational burden (mean below 3 mutations/Mbp), the lowest frequency of TP53 mutations (20–25%) and very few other co-mutations compared to other NSCLC. The relative simplicity and stability of the genetic landscape not only contribute to the relatively favourable clinical course, but also make study of the effects from individual molecular features easier. EML4-ALK fusion variant 3 (E6;A20) and TP53 mutations were recently identified as main molecular determinants of adverse outcome: they occur in about 30–40% and 20–25% of newly-diagnosed cases, respectively, have possibly synergistic effects and are independently associated with more aggressive disease, shorter progression-free survival under treatment with ALK inhibitors and worse overall survival. Secondary detection of TP53 mutations at disease progression in previously negative patients defines another subset (about 20%) with similarly poor outcome, while detection of ALK resistance mutations guides next-line therapy. As our biological understanding deepens, additional molecular risk factors will be identified and refine our concepts further. The translation of clinical risk at the molecular level and the ability to predict early events are of key importance for individualized patient management and preclinical modeling in order to advance therapeutic options.

Updated Efficacy and Safety Data and Impact of the EML4-ALK Fusion Variant on the Efficacy of Alectinib in Untreated ALK-Positive Advanced Non–Small Cell Lung Cancer in the Global Phase III ALEX Study

IntroductionAt the prior data cutoff (February 9, 2017) the ALEX trial showed superior investigator-assessed progression-free survival (PFS) for alectinib versus crizotinib in untreated, anaplastic lymphoma kinase (ALK)-positive, advanced NSCLC (hazard ratio = 0.47, 95% confidence interval: 0.34–0.65, p < 0.001). The median PFS in the alectinib arm was not reached versus 11.1 months with crizotinib. Retrospective analyses suggest that the echinoderm microtubule-associated protein-like 4 gene-ALK variant (EML4-ALK) may influence ALK-inhibitor treatment benefit. We present updated analyses, including exploratory subgroup analysis by EML4-ALK variant, after an additional 10 months’ follow-up (cutoff December 1, 2017). MethodsPatients were randomized to receive twice-daily alectinib, 600 mg, or crizotinib, 250 mg, until disease progression, toxicity, death, or withdrawal. PFS was determined by the investigators. Baseline plasma and tissue biomarker samples were analyzed by using hybrid-capture, next-generation sequencing to determine EML4-ALK variant. ResultsBaseline characteristics were balanced. Investigator-assessed PFS was prolonged with alectinib (stratified hazard ratio = 0.43, 95% confidence interval: 0.32–0.58). The median PFS times were 34.8 months with alectinib and 10.9 months with crizotinib. EML4-ALK fusions were detectable in 129 patient plasma samples and 124 tissue samples; variants 1, 2, and 3/ab did not affect PFS, objective response rate, or duration of response. Investigator-assessed PFS was longer for alectinib than for crizotinib across EML4-ALK variants 1, 2, and 3a/b in plasma and tissue. Despite longer treatment duration (27.0 months in the case of alectinib versus 10.8 months in the case of crizotinib), the safety of alectinib compared favorably with that of crizotinib. ConclusionAlectinib continues to demonstrate superior investigator-assessed PFS versus crizotinib in untreated ALK-positive NSCLC, irrespective of EML4-ALK variant.