EML4 ALK Research Articles

Abstract 3262: Elucidating the mechanism of resistance to tyrosine kinase inhibitors in ROS1-and ALK-fusion non-small cell lung cancer: The role of CD74

Abstract The therapeutic landscape in treating advanced oncogene-mutated non-small cell lung cancer (NSCLC) was revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Despite the efficacious breakthrough and improved patient outcomes, disease progresses once more due to the seemingly inevitable development of drug resistance in tumor cells. This poses a considerable predicament in the clinical management of oncogene-mutated NSCLC and signifies the importance of elucidating the mechanism of resistance in tumor cells that harbor these genomic alterations which give rise to oncogenic drivers. Recent evidence suggests that drug-tolerant persister (DTP) cells, which are tolerant to initial exposure to TKIs give rise to acquired drug-resistant cells. Currently, the mechanism by which tumor cells undergo a drug-tolerant state is not well understood in NSCLCs driven by ALK or ROS1 fusion. Previously, we identified CD74 as a gene that may play a role in a drug-tolerant state in EGFR-mutated lung cancer via single-cell RNA-sequencing (scRNA-seq). Here, we sought to determine whether the CD74 signaling also leads to resistance to TKIs in NSCLCs with ALK or ROS1 fusion. We showed that CD74 expression increased at both the mRNA and protein levels after treatment with respective TKIs in ALK and ROS1 fusion cells lines. Notably, the ALK and ROS1 fusion cell lines also exhibited an increase in mRNA expression levels of upstream and downstream genes of CD74. Additionally, we generated CD74 knock-out and overexpression models in two cells lines, HCC78 (SLC34A2-ROS1 fusion) and H2228 (EML4–ALK fusion), to further examine the role of CD74 expression in DTP cells. We demonstrated that resistance to TKIs were dependent on CD74 expression. Furthermore, it was determined that anti-CD74 antibody-drug conjugates, in combination with TKIs, synergistically induced apoptosis in these cell lines. In conclusion, our data suggest that CD74 can be a therapeutic target in NSCLC with ALK or ROS1 fusion. Citation Format: Vivian Ho, Motohiro Izumi, Meghan Lee, Daisuke Shibahara, Ikei S. Kobayashi, Susumu S. Kobayashi. Elucidating the mechanism of resistance to tyrosine kinase inhibitors in ROS1-and ALK-fusion non-small cell lung cancer: The role of CD74 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3262.

A Rare Case of High grade Epithelioid Malignant Neoplasm with CAM 5.2 Expression and (EML4–ALK) Fusion in a Pediatric Patient

Abstract Introduction/Objective ALK (anaplastic lymphoma kinase) fusions have been identified in lymphomas and diverse cancer types including carcinomas, melanomas and mesenchymal neoplasms such as inflammatory myofibroblastic tumors (IMT), epithelioid fibrous histiocytoma (EFH). Herein we present a (non IMT, non EFH) ALK rearranged aggressive epithelioid neoplasm. Methods/Case Report We report a 17-year-old male with a 14.0 cm mediastinal mass and extensive metastatic disease involving brain, bones, pleura and lymph nodes. The tumor was made of epithelioid like cell with round nuclei, prominent nucleoli and a moderate amount of cytoplasm, arranged in a nested pattern. Ki-67 showed a high proliferative index, >70%. The tumor cells were positive for CAM 5.2; weakly positive for AE1/3, ALK, CD30; focally positive for p63, p53, S100. Additional stains including CD117, PLAP, OCT-4, Sall-4, glypican-3, synaptophysin, chromogranin, CK5/6, p40, pan-melanoma, myogenin, CD99, WT1, CD34, CD45, CD138, MUM1, CD2, CD4, CD5, CD3, CD7, CD15, CD23, CD31, CD20, CD79a, CD15, PAX8, PAX5, CD43, MPO, desmin, D2-40, Calretinin, ERG, SOX-10, PD-L1 and NUT-1 were all negative. Both BRG and INI1 were retained. The keratin positivity and the lack of “hallmark” cells made anaplastic large cell lymphoma (ALCL) less likely. The absence of desmin and CD30 excluded the rare epithelioid IMT sarcoma. Next-generation sequencing detected an (EML4–ALK) fusion. A diagnosis of high- grade epithelioid malignant neoplasm with (EML4–ALK) fusion was made. Most cases of this category were described as low grade-appearing spindled cells within a myxoid stroma arranged in fascicles (+S100, +CD34) and have unaggressive behavior. Only three cases, were characterized by epithelioid high- grade morphology and metastatic nature. They shared features like little collagenous stroma, conspicuous mitotic activity and tumor necrosis. Results (if a Case Study enter NA) NA Conclusion Identification of kinase fusions was essential for precise classification, better knowledge of this epithelioid high grade malignant neoplasm and for targeted therapy purposes. The significant clinicopathologic heterogeneity of this emerging group requires further investigation.

The Change in Paradigm for NSCLC Patients with EML4-ALK Translocation.

The severe prognosis linked with a lung cancer diagnosis has changed with the discovery of oncogenic molecularly driven subgroups and the use of tailored treatment. ALK-translocated advanced lung cancer is the most interesting model, having achieved the longest overall survival. Here, we report the most important paradigmatic shifts in the prognosis and treatment for this subgroup population occurred among lung cancer.

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 separation of EML4\u2013ALK in firing downstream signaling and promoting lung tumorigenesis

EML4–ALK fusion, observed in about 3%–7% of human lung adenocarcinoma, is one of the most important oncogenic drivers in initiating lung tumorigenesis. However, it still remains largely unknown about how EML4–ALK fusion exactly fires downstream signaling and drives lung cancer formation. We here find that EML4–ALK variant 1 (exon 1–13 of EML4 fused to exon 20–29 of ALK) forms condensates via phase separation in the cytoplasm of various human cancer cell lines. Using two genetically engineered mouse models (GEMMs), we find that EML4–ALK variant 1 can drive lung tumorigenesis and these murine tumors, as well as primary tumor-derived organoids, clearly show the condensates of EML4–ALK protein, further supporting the findings from in vitro study. Mutation of multiple aromatic residues in EML4 region significantly impairs the phase separation of EML4–ALK and dampens the activation of the downstream signaling pathways, especially the STAT3 phosphorylation. Importantly, it also significantly decreases cancer malignant transformation and tumor formation. These data together highlight an important role of phase separation in orchestrating EML4–ALK signaling and promoting tumorigenesis, which might provide new clues for the development of clinical therapeutic strategies in treating lung cancer patients with the EML4–ALK fusion.

P84.07 Distribution and Therapeutic Outcomes of Intergenic Sequence-ALK Fusion and Coexisting ALK Fusions in Lung Adenocarcinoma Patients

ALK-rearranged NSCLC patients show survival benefits from ALK inhibitor. Studies about outcome were mostly focused on EML4–ALK fusion. Here, we performed next-generation sequencing (NGS) of baseline specimens from ALK-positive patients to examine the rearrangement distribution and possible correlation to therapeutic benefit. ALK-positive NSCLC patient(determined by immunohistochemistry) were screened at the West China Hospital, and NGS was performed on baseline samples. Clinical information and therapeutic outcomes were collected and retrospectively analyzed. Among the 89 patients with 22 ALK rearrangements partners, fusions of intergenic sequences with ALK were found in 15 (16.85%). Non-EML4–ALK fusions were present in 18 patients (20.22%), while EML4–ALK fusions in 71 (79.76%). Coexisting rearrangements were present in 16 patients (17.98%). Intergenic–ALK and non EML4–ALK fusions occurred at higher rates in patients with coexisting fusions (62.5% versus 6.85%, and 62.5% versus 10.96%). First-line crizotinib was administered in 41 patients, and the median progression-free survival (mPFS) was 9.7 months. No significant difference in mPFS was found between patients with or without intergenic ALK fusion (12 versus 9.6 months, p = 0.989). In the seven patients who had at least two fusions each, the mPFS was 11.9 months, compared with 9 months among the 24 patients with single (p = 0.336). No significant difference in mPFS was observed between patients with and without EML4–ALK fusions (9.6 versus 12 months, p = 0.924). The hazard ratio(HR) of gender, age and three rearrangements (EML4-, non EML4-, intergenic-ALK) were 0.47, 1.18, 1.09, 1.31, 0.96, respectively.Table 1Characteristics of enrolled patients.CharacteristicTotal (n = 90)TreatmentP valueSurgery (n = 46)Systemic therapy (n = 44)Age (median [range], years)51 (29-78)52.5 (30-78)49 (29-70)0.396Gender (cases, %)0.404Male47 (52.2)26 (56.5)21 (47.7)Female43 (47.8)20 (43.5)23 (52.3)Smoking status (cases, %)0.185Smoker6 (6.7)1 (2.2)5 (11.4)Non-smoker84 (93.3)45 (97.8)39 (88.6)Histology (cases, %)/Adenocarcinoma90 (100)46 (100)44 (100)Non-adenocarcinoma0 (0)0 (0)0 (0)ECOG performance status (cases, %)0.6890-179 (87.8)41 (89.1)38(86.4)2-411 (12.2)5 (10.9)6 (13.6)Stage (cases, %)<0.001I-III49 (54.4)46 (100)3 (6.8)IV41 (45.6)0 (0)41 (93.2)Metastasis (cases, %)<0.001Absent49 (54.4)46 (100)3 (6.8)Present41 (45.6)0 (0)41 (93.2)Brain metastasis13 (14.4)0 (0)13 (29.5)Bone metastasis24 (26.7)0 (0)24 (54.5)Multi-organ metastases20 (22.2)0 (0)20 (45.5)ALK aberrations (cases, %)0.489Rearrangement free Uncertain aberration1 (1.1)0 (0)1 (2.3)ALK rearrangement89 (98.9)46 (100)43 (97.7)Distribution of rearrangements (cases, %)Intergenic sequence-ALK fusion15 (16.9)9 (19.6)6 (14.0)0.480Non-EML4-ALK fusion18 (20.2)10 (21.7)8 (18.6)0.713EML4-ALK fusionVariant 126 (29.2)12 (26.1)14 (32.6)0.502Variant 24 (4.5)3 (6.5)1 (2.3)0.658Variant 330 (33.7)14 (30.4)16 (37.2)0.499Other variants11 (12.4)8 (17.4)3 (7.0)0.136Status of coexisting ALK fusions (cases, %)Absent73 (82.0)37 (80.4)36 (83.7)0.687Intergenic sequence-ALK fusion alone5 (5.6)2 (4.3)3 (7.0)0.938Non-EML4-ALK fusion alone8 (9.0)5 (10.9)3 (7.0)0.787EML4-ALK fusion alone60 (67.4)30 (65.2)30 (69.8)0.647Present16 (18.0)9 (19.6)7 (16.3)0.687Intergenic sequence-ALK fusion + EML4-ALK fusion6 (6.7)4 (8.7)2 (4.7)0.736Intergenic sequence-ALK fusion + non-EML4-ALK fusion1 (1.1)1 (2.2)0 (0)1.000EML4-ALK fusion + non-EML4-ALK fusion3 (3.4)1 (2.2)2 (4.7)0.953Intergenic-ALK fusion + EML4-ALK fusion+ non-EML4-ALK fusion2 (2.2)2 (4.3)0 (0)0.495Intergenic-ALK fusion + intragenic-ALK fusion + non-EML4-ALK fusion1 (1.1)0 (0)1 (2.3)0.483non-EML4-ALK fusion + non-EML4-ALK fusion3 (3.4)1 (2.2)2 (4.7)0.953 Open table in a new tab This is the first to report the occurrence rates and distribution of intergenic–ALK and coexisting of ALK fusions. Intergenic–ALK and non-EML4–ALK were more likely to coexist with other fusions. Neither the type nor number of rearrangements had a significant effect on the therapeutic benefit of treatment with crizotinib.

Comprehensive Multi-Omics Analysis of Gene Fusions in a Large Multiple Myeloma Cohort

Introduction: Gene fusions are the result of genomic rearrangements that create hybrid protein products or bring the regulatory elements of one gene into close proximity of another. Fusions often dysregulate gene function or expression through oncogene overexpression or tumor suppressor underexpression (Gao, Liang, Foltz, et al. Cell Rep 2018). Some fusions such as EML4–ALK in lung adenocarcinoma are known druggable targets. Fusion detection algorithms utilize discordantly mapped RNA-seq reads. Careful consideration of detection and filtering procedures is vital for large-scale fusion detection because current methods are prone to reporting false positives and show poor concordance.Multiple myeloma (MM) is a blood cancer in which rapidly expanding clones of plasma cells spread in the bone marrow. Translocations that juxtapose the highly-expressed IGH enhancer with potential oncogenes are associated with overexpression of partner genes, although they may not lead to a detectable gene fusion in RNA-seq data. Previous studies have explored the fusion landscape of multiple myeloma cohorts (Cleynen, et al. Nat Comm 2017; Nasser, et al. Blood 2017). In this study, we developed a novel gene fusion detection pipeline and post-processing strategy to analyze 742 patient samples at the primary time point and 64 samples at follow-up time points (806 total samples) from the Multiple Myeloma Research Foundation (MMRF) CoMMpass Study using RNA-seq, WGS, and clinical data.Methods and Results: We overlapped five fusion detection algorithms (EricScript, FusionCatcher, INTEGRATE, PRADA, and STAR-Fusion) to report fusion events. Our filtered call set consisted of 2,817 fusions with a median of 3 fusions per sample (mean 3.8), similar to glioblastoma, breast, ovarian, and prostate cancers in TCGA. Major recurrent fusions involving immunoglobulin genes included IGH–WHSC1 (88 primary samples), IGL–BMI1 (29), and the upstream neighbor of MYC, PVT1, paired with IGH (6), IGK (3), and IGL (11).For each event, we used WGS data when available to determine if there was genomic support of the gene fusion (based on discordant WGS reads, SV event detection, and MMRF CoMMpass Seq-FISH WGS results) (Miller, et al. Blood 2016). WGS validation rates varied by the level of RNA-seq evidence supporting each fusion, with an overall rate of 24.1%, which is comparable to previously observed pan-cancer validation rates using low-pass WGS.We calculated the association between fusion status and gene expression and identified genes such as BCL2L11, CCND1/2, LTBR, and TXNDC5 that showed significant overexpression (t-test). We explored the clinical connections of fusion events through survival analysis and clinical data correlations, and by mining potentially druggable targets from our Database of Evidence for Precision Oncology (dinglab.wustl.edu/depo) (Sun, Mashl, Sengupta, et al. Bioinformatics 2018). Major examples of upregulated fusion kinases that could potentially be targeted with off-label drug use include FGFR3 and NTRK1.We examined the evolution of fusion events over multiple time points. In one MMRF patient with a t(8;14) translocation joining the IGH locus and transcription factor MAFA, we observed IGH fusions with TOP1MT (neighbor of MAFA) at all four time points with corresponding high expression of TOP1MT and MAFA. Using non-MMRF single-cell RNA data from different patients, we were able to track cell-type composition over time as well as detect subpopulations of cells harboring fusions at different time points with potential treatment implications.Discussion: Gene fusions offer potential targets for alternative MM therapies. Careful implementation of gene fusion detection algorithms and post-processing are essential in large cohort studies to reduce false positives and enrich results for clinically relevant information. Clinical fusion detection from untargeted RNA-seq remains a challenge due to poor sensitivity, specificity, and usability. By combining MMRF CoMMpass data from multiple platforms, we have produced a comprehensive fusion profile of 742 MM patients. We have shown novel gene fusion associations with gene expression and clinical data, and we identified candidates for druggability studies. DisclosuresVij:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

P3.15-30 Treatment Patterns and Survival of Patients with Stage IV Non-Small Cell Lung Cancer (NSCLC) in the Era of Novel Therapies

Treatment strategies for metastatic non-small cell lung cancer (NSCLC) are evolving rapidly. Real–world evidence (RWE) of treatment patterns and outcomes can further our understanding of the impact of novel therapies. In this population-based study, we investigated treatments and outcomes for stage IV NSCLC in a large Canadian province. Patients diagnosed with de novostage IV NSCLC from April 1st2010 to March 31st2015 were identified. Baseline characteristics, treatments, and outcomes were analyzed. We classified treatments targeting EGFR, EML4–ALK, and ROS1 as targeted therapy and intravenous checkpoint inhibitors as immunotherapy. A total of 6,438 patients were identified with NSCLC, of whom 3,606 (56%) had de novostage IV disease. The median age of diagnosis was 69 years (range 20–100) and 52.4% were male. The median age among those who received targeted therapy and immunotherapy were 63 (27–90) and 61 (37–72) years, respectively, and 41.8% and 61.8% were male, respectively. First line treatments were: 5.7% targeted agents (n= 204), 1% immunotherapy (n = 1), 19.5% palliative chemotherapy (n= 703), 6.8% palliative radiotherapy (n= 246), and 74.8% received supportive care only (n= 2,698). Most frequent subsequent treatments in 2L included: 30.7% targeted agents (n= 125), 1.7% immunotherapies (n= 7), 67.6% palliative chemotherapy (n= 275), 32.2% palliative radiotherapy (n= 131). Median overall survival (mOS) for the whole cohort was 3.8 months (0–not reached [NR]). MOS with targeted therapies was 18 months (1.4–NR), chemotherapy was 9.4 months (1.1–NR) and supportive care only had a mOS of 2.5 months (0–NR) (Figure 1). About 1.0% of patients (n= 34) received immunotherapy at any line. Survival benefit was dependent on type of treatment received, with a trend towards improved survival with newer agents.

Les cancers du poumon de moins de 40 ans

In France, lung cancer induces more than 27,000 related deaths each year. The mean age at diagnosis was 66 years in men and 65 years in women. Lung cancer is rare in young patients under 40 and the incidence in France is around 3 to 4%. Few data are available in this population. Many studies suggested that the disease may represent a specific entity in these young subjects, or at least, is associated with specific characteristics: female over-representation predominance of adenocarcinoma, and advanced stage at diagnosis. Some studies show that young patients with lung cancer have a higher rate of family cancers history and a low educational level. The search for drug-targetable mutations showed in most studies that young patients exhibit a high mutation rate, especially for EGFR and EML4 ALK. It’s mandatory to define the molecular profile of lung tumors, especially in this young population in order to adapt the treatment. In contrast, the question of prognosis is highly controversial.

Association of ALK resistance mutations by EML4-ALK variant (v3 vs. non-v3) in ALK+ non-small cell lung cancer (NSCLC).

9010 Background: ALK rearrangements are established targetable drivers in NSCLC. Recent reports indicate differential progression-free survival to ALK inhibitors according to specific EML4-ALK variant. Methods: We analyzed samples from 634 unique NSCLC patients (704 samples) with tumors harboring ALK rearrangements ( ALK+) detected using hybrid-capture based genomic profiling performed on DNA isolated from FFPE tissue specimens (676 samples) or ctDNA isolated from blood (28 samples) during the course of clinical care. Results: Of the 634 ALK+ cases, we identified 200 (32%) EML4- ALKv1 (E13; A20), 50 (8%) EML4-ALKv2 (E20; A20), 204 (32%) EML4- ALKv3 (E6; A20), 78 (12%) other EML4-ALK, and 102 (16%) non- EML4 ALK rearrangements. Despite relatively equal frequency of EML4-ALK v1 and v3 in this dataset, the presence of a known ALK resistance mutation (n = 40 cases) was significantly associated with v3 as compared to v1 (P &lt; 0.0002). G1202R mutation in particular was significantly associated with EML4-ALK v3 versus v1 (P &lt; 0.002), and as compared to all non-v3 (P = 0.02). The tumor mutation burden (TMB) was generally low (median v1: 1.8, v3: 2.5, non-v3: 1.8 mutations/Mb), and although significantly different between v1 and v3 (P = 0.0008) and v3 and non-v3 (P = 0.003), the difference is not expected to be clinically relevant. Available ALK+ cases with paired pre- and post-treatment samples tested for a single patient will also be evaluated by ALKfusion variant, as well as for novel non-ALK mechanisms of acquired resistance, including a case with MET kinase domain duplication acquired post-ALK targeted therapy. Conclusions: The use of tissue and blood-based next generation sequencing allows for detection of the specific ALK fusion partner, increases the understanding of the biology of ALK+ NSCLC, and may have value to foretell potential mechanisms of resistance and inform the selection of ALK inhibitor therapy. [Table: see text]

Ceritinib plus nivolumab (NIVO) in patients (pts) with anaplastic lymphoma kinase positive (ALK+) advanced non-small cell lung cancer (NSCLC).

2502 Background: Induction of PD-L1 expression due to constitutive oncogenic signaling has been reported in NSCLC models harboring EML4–ALK rearrangements. Here we explore whether the combination of ALKi (ceritinib) and PD1-inhibitor (NIVO) will provide sustained clinical benefit to pts with ALK+ NSCLC. Methods: This phase 1 dose escalation study enrolled previously treated (ALK inhibitor [ALKi] or chemotherapy) or tx-naive pts with stage IIIB/IV ALK+ NSCLC; who received NIVO 3 mg/kg IV Q2W + ceritinib with low-fat meal, at 450 mg/day (group 1) or 300 mg/day (group 2) until progression/unacceptable toxicity. Primary objective: MTD/recommended dose for expansion. Dose escalation was guided by Bayesian logistic regression model with overdose control. Results: Median follow-up: group 1 (n = 14) 13 mos (10-15); group 2 (n = 22) 6 mos (2-10). As of 9 Sep 2016, 16/36 (44%) pts discontinued tx: disease progression (11 [31%] pts), AE’s (3 [8%] pts), and death (2 [6%] pts). In group 1, 4 pts experienced DLT: pancreatitis (n = 2), lipase and transaminase increase (n = 1), and autoimmune hepatitis (n = 1). In group 2, 2 pts experienced DLT: G3 ALT increase. Both dose levels met Bayesian criteria for dose expansion. Overall most frequent (≥40%) AEs (n = 36), were diarrhea (64%), ALT increase (56%), AST increase (44%) and vomiting (42%). Most frequent ( &gt; 10%) grade ≥3 AEs were increases in ALT (22%), GGT (17%), amylase (11%), and lipase (11%), and maculopapular rash (11%). Incidence of rash (grouped term) was 61%; similar in both groups. Grade 3 rash was reported in 29% pts in group 1 and 14% pts in group 2. Preliminary ceritinib steady state PK (AUC0-24 and Cmax) suggested that 300 mg/day exposure was ~ 70-75% of 450 mg/day. Confirmed (c)/unconfirmed (u) ORR: ALKi-pretreated pts (group 1 [n = 8], group 2 [n = 12]) was 63% (4 cPR,1 uPR; 95% CI: 25%, 92%), and 33% (4 uPR) 95% CI: 10%, 65%) respectively; ALKi-naïve pts, (group 1 [n = 6], group 2 [n = 10]) was 83% (5 cPR; 95% CI: 36%, 100%), and 70% (1 cCR, 3 cPR 3uPR; 95% CI: 35%, 93%) respectively. Conclusions: Ceritinib + NIVO is an active combination in ALK+ NSCLC. However, the protocol will be amended to address observed toxicities. Data will be updated for presentation. Clinical trial information: NCT02393625.

Programmed death-ligand 1 expression associated with molecular characteristics in surgically resected lung adenocarcinoma.

BackgroundSeveral clinical trials have shown that immune treatment focus on programmed death-1 and programmed death-ligand 1 (PD-L1) yields a good clinical efficacy in advanced non-small cell lung cancer (NSCLC). We investigated whether the PD-L1 expression was related to clinicopathologic and molecular characteristics in patients with surgically resected NSCLC.MethodsBetween December 2008 and 2013, formalin-fixed, paraffin-embedded samples were obtained from patients with lung adenocarcinoma at Zhejiang Cancer Hospital. RT-PCR was used to analyze EGFR, KRAS, NRAS, PIK3CA, BRAF, HER2 mutations and ALK, ROS1, RET fusion genes. The PD-L1 expression was evaluated by immunohistochemistry and staining of 5 % or more was scored as positive expression. Survival analysis was evaluated using the Kaplan–Meier method. Multivariate regression was performed using the Cox proportional hazards model.ResultsMutations were detected in 76.6 % of the 385 patients tested: EGFR mutation (n = 205, 53.2 %), followed by EML4–ALK rearrangement (n = 18, 4.7 %), KRAS (n = 16, 4.2 %), HER2 (n = 9, 2.3 %), ROS1 rearrangement (n = 8, 2.1 %), PIK3CA (n = 6, 1.6 %), RET rearrangement (n = 6,1.6 %), BRAF (n = 2, 0.5 %), and NRAS mutations (n = 1, 0.2 %). Twenty-four (6.2 %) patients carried coexisting mutations. PD-L1 expression was detected in 48.3 % (186/385) of all the patients. PD-L1 positive patients more frequently carried coexisting mutations (18/24, 75 %), followed by single-gene (145/271, 53.5 %) and pan-negative mutations (23/90, 25.6 %). PD-L1 expression decreased disease-free survival (DFS) in univariate analysis (P = 0.014). Multivariate analysis revealed that PD-L1 expression was not an independent risk factor for poor DFS and overall survival (OS) (P = 0.22 and 0.37, respectively).ConclusionsPD-L1 overexpression is more frequently observed in oncogene-mediated lung adenocarcinoma, especially with coexisting mutation subtypes. PD-L1 expression is not a prognostic factor in surgically resected lung adenocarcinoma patients.

Clinical diagnosis of EML4–ALK mutation in NSCLC by a gold nanoparticle beacon

The existence of EML4–ALK, a mutated fusion gene in non-small-cell lung cancer (NSCLC), is an important consideration in the decision of the treatment options for NSCLC patients. Gold nanoparticle beacons could be applied in clinical detection of the fusion gene visually.

Synergistic effects of crizotinib and radiotherapy in experimental EML4–ALK fusion positive lung cancer

Background and purposeNon-small cell lung cancer (NSCLC) patients with chromosomal rearrangements of the anaplastic lymphoma kinase gene (ALK) are sensitive to the tyrosine kinase inhibitor crizotinib. We aimed to investigate the effects of combined radiotherapy and crizotinib in ALK-positive vs. wild type NSCLC models. Material and methodsClonogenic survival, proliferation and apoptosis of cells exposed to crizotinib and radiotherapy (photon and carbon ions) were evaluated in ALK mutation positive (ALK+; H3122) and negative (ALK−; A549 and LLC) NSCLC lines. The syngeneic mouse (LLC) and human (H3122) xenograft tumor models were further studied in vivo. Tumor growth kinetics, microvascular density (MVD), perfusion and proliferation were assessed. ResultsCrizotinib exerted potent and selective anti-proliferative and pro-apoptotic effects in ALK+ H3122 cells which were augmented by radiotherapy. The synergistic effect of this combination in ALK+ NSCLC was confirmed by isobologram analysis. Crizotinib also sensitized H3122 cells to particle therapy with carbon ions. In H3122 xenografts, dual combination was most effective in reducing tumor proliferation, MVD and perfusion. In contrast, in the LLC model, crizotinib led only to a transient tumor growth inhibition and combined treatment was inferior to radiotherapy alone. ConclusionsCrizotinib elicits beneficial effects in combination with radiotherapy only in ALK-positive NSCLC.

Future of ALK inhibition in non-small-cell lung cancer

Progress in defining molecular targets of oncogenesis and drugs to inhibit cancer growth in specific populations has led to augmented outcomes for patients and new expectations in the development of treatments. The EML4–ALK fusion protein was identified in patients with non-small-cell lung cancer (NSCLC) in 2007.1 Rearrangements in the ALK gene lead to constitutive signalling, triggering transforming properties. Up to now, two drugs have been approved by the US Food and Drug Administration for ALK-rearranged NSCLC—crizotinib and ceritinib.

A Systemic Review of Resistance Mechanisms and Ongoing Clinical Trials in ALK-Rearranged Non-Small Cell Lung Cancer.

The identification of oncogenic driver mutations in non-small cell lung cancer (NSCLC) has led to a paradigm shift and the development of specific molecular treatments. Tumors harboring a rearranged EML4–ALK fusion oncogene are highly sensitive to therapy with ALK-targeted inhibitors. Crizotinib is the first approved treatment for advanced lung tumors containing this genetic abnormality. In this mini review, we discuss the existing data on crizotinib as well as ongoing trials involving this medication. A brief overview of the known resistance mechanisms to crizotinib will also be presented followed by a summary of the ongoing trials involving next-generation ALK-inhibitors or other targeted therapies in patients with ALK+ NSCLC.

EML4–ALK Fusions: Propelling Cancer but Creating Exploitable Chaperone Dependence

The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins.

Optimization of energy transfer between quantum dots and gold nanoparticles in head-to-head configuration for detection of fusion gene

We report simultaneous detection of the variants 3a and 3b in the oncogenic EML4–ALK fusion gene using cDNA from lung cancer patients based on energy transfer between CdSe/ZnS quantum dots (QDs) and gold nanoparticles (AuNPs). To overcome the limitations of the conventional detection methods based on energy transfer, we have applied a head-to-head configuration of QDs and AuNPs. This allowed us not only to use longer probe oligonucleotides than the conventional methods, but also to optimize interparticle distance between QDs and AuNPs for maximum sensitivity and selectivity. By functionalizing two types of QDs (i.e., green- and red-emitting QDs) and 5nm AuNPs with probe oligonucleotides, we demonstrated that energy was selectively transferred from green-emitting QDs to AuNPs when variant 3a was present in target cDNA, or from red-emitting QDs to AuNPs with variant 3b, resulting in fluorescence quenching. Our method could successfully be used to simultaneously detect two variants of EML4–ALK fusion gene at the target DNA concentration (3.45nM) that was lower than the previously reported limit of detection values based on energy transfer phenomenon.

Impact of the new EGF receptor and ALK testing guideline on personalized lung cancer medicine.

All advanced-stage lung adenocarcinoma patients should be tested for EGF receptor (EGFR) mutations and for EML4–ALK translocations as a guide for selecting them for tyrosine kinase inhibitor (TKI) therapy. The new EGFR and ALK guideline provides useful recommendations for appropriate testing [1]. Lung tumors are the leading cause of cancerrelated mortality worldwide [2]. Almost 80% of cases are non-small-cell lung cancer (NSCLC) and extensive disease is present at the time of diagnosis in the majority of patients. Systemic treatment with chemotherapy has reached a plateau of 10–20% response rates and 4–5 months of progression-free survival (PFS), and has not improved in more than a decade [3–5]. A first milestone in a new era of targeted therapy for this disease was reached with the discovery of a subset of tumors carrying activating mutations of EGFR, which can be selectively blocked with TKIs such as erlotinib and gefitinib. Numerous clinical studies have demonstrated the efficacy of these TKIs in the treatment of EGFR-mutated lung tumors, with 55–83% response rates and 9–13 month PFS [6,7]. A second milestone was the discovery, in 2007, of the EML4–ALK translocation in 6.7% of Japanese NSCLC patients [8]. The translocation was later found in 4–7% of Chinese [9], European [10] and US patients [11]. When treated with a novel ALK inhibitor, crizotinib, these patients showed response rates of 57% with a PFS greater than 6 months [12]. As a result of these new developments, many laboratories worldwide are currently testing lung tumor samples for EGFR mutations and EML4–ALK fusions. In an effort to standardize testing practices, the College of American Pathologists, the International Association for the Study of Lung Cancer and the Association for Molecular Pathology constituted writing and advisory panels, presided by three cochairs, to

Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4–ALK-rearranged non–small–cell lung cancer harbored coexisting EGFRmutation

BackgroundThe EML4–ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non–small–cell lung cancers (NSCLCs). The EML4–ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS.Case presentationWe report that a case of EML4–ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor.ConclusionsWe described the first clinical report of a patient with EML4–ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4–ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4–ALK-positive NSCLC.