Generation Anaplastic Lymphoma Kinase Inhibitor Research Articles

Head to head evaluation of second generation ALK inhibitors brigatinib and alectinib as first-line treatment for ALK+ NSCLC using an in silico systems biology-based approach.

Around 3–7% of patients with non-small cell lung cancer (NSCLC), which represent 85% of diagnosed lung cancers, have a rearrangement in the ALK gene that produces an abnormal activity of the ALK protein cell signaling pathway. The developed ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, ceritinib, alectinib, brigatinib and lorlatinb present good performance treating ALK+ NSCLC, although all patients invariably develop resistance due to ALK secondary mutations or bypass mechanisms. In the present study, we compare the potential differences between brigatinib and alectinib’s mechanisms of action as first-line treatment for ALK+ NSCLC in a systems biology-based in silico setting.Therapeutic performance mapping system (TPMS) technology was used to characterize the mechanisms of action of brigatinib and alectinib and the impact of potential resistances and drug interferences with concomitant treatments.The analyses indicate that brigatinib and alectinib affect cell growth, apoptosis and immune evasion through ALK inhibition. However, brigatinib seems to achieve a more diverse downstream effect due to a broader cancer-related kinase target spectrum. Brigatinib also shows a robust effect over invasiveness and central nervous system metastasis-related mechanisms, whereas alectinib seems to have a greater impact on the immune evasion mechanism.Based on this in silico head to head study, we conclude that brigatinib shows a predicted efficacy similar to alectinib and could be a good candidate in a first-line setting against ALK+ NSCLC. Future investigation involving clinical studies will be needed to confirm these findings. These in silico systems biology-based models could be applied for exploring other unanswered questions.

Acute kidney Injury due to Alectinib, an Anaplastic Lymphoma Kinase inhibitor

Introduction: The array of unintended effects of targeted oncological treatments is still being elucidated. Cognizance of these effects is important since they allow early identification and intervention. Methods: We report a case of progressive reduction in kidney function following the introduction of the oral second generation Anaplastic Lymphoma Kinase (ALK) Inhibitor: Alectinib for the treatment of metastatic non-small cell carcinoma of the lung (NSCLC). Kidney biopsy findings suggested that the predominant manifestation was acute tubular injury, which resolved with cessation of Alectinib therapy and did not recur with the introduction of the third generation ALK inhibitor Lorlatinib. Conclusions: This case report adds to the Onco-Nephrology literature in relation to the potential nephrotoxic effects of ALK inhibitors.

Inflammatory Myofibroblastic Tumor of the Bladder With FN1-ALK Gene Fusion: Different Response to ALK Inhibition

Inflammatory myofibroblastic tumors are rare tumors with an ALK (anaplastic lymphoma kinase) gene rearrangement in up to 65% of all cases. In our patient, the tumor was not primary resectable due to its extension. Under neoadjuvant treatment with the first generation ALK inhibitor crizotinib no tumor response was seen, but the following therapy with the next generation ALK inhibitor lorlatinib led to a rapid and deep response, enabling a complete tumor resection by partial cystectomy. Our case indicates that ALK positive inflammatory myofibroblastic tumors which do not respond to ALK inhibition with crizotinib can be successfully treated with newer agents.

BRAF V600E mutation and MET amplification as resistance pathways of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in lung cancer

BackgroundAnaplastic lymphoma kinase (ALK) targeted therapies have demonstrated remarkable efficacy in ALK-positive lung adenocarcinomas. However, patients inevitably develop resistance to such therapies. To investigate novel mechanisms of resistance to second generation ALK inhibitors, we characterized and modeled ALK inhibitor resistance of ALK-positive patient-derived xenograft (PDX) models established from advanced-stage lung adenocarcinoma patients who have progressed on one or more ALK inhibitors. MethodsWhole exome sequencing was performed to identify resistance mechanisms to ALK inhibitors in PDXs generated from biopsies at the time of relapse. ALK fusion status was confirmed using fluorescent in situ hybridization, immunohistochemistry, RNA-sequencing, RT-qPCR and western blot. Targeted therapies to overcome acquired resistance were then tested on the PDX models. ResultsThree PDX models were successfully established from biopsies of two patients who had progressed on crizotinib and/or alectinib. The PDX models recapitulated the histology and ALK status of their patient tumors, as well as their matched patients’ clinical treatment outcome to ALK inhibitors. Whole exome sequencing identified MET amplification and previously unreported BRAF V600E mutation as independent mechanisms of resistance to alectinib. Importantly, PDX treatment of inhibitors specific for these targets combined with ALK inhibitor overcame resistance. ConclusionsBypass signaling pathway through c-MET and BRAF are independent mechanisms of resistance to alectinib. Individualized intervention against these resistance pathways could be viable therapeutic options in alectinib-refractory lung adenocarcinoma.

A user’s guide to lorlatinib

Rearrangements of the ALK gene are found in approximately 5% of non-small-cell lung cancer. It is of particular importance to test for this rearrangement in patients with metastatic lung adenocarcinoma because these tumors are highly sensitive to therapy with ALK-targeted inhibitors. Lorlatinib is a reversible potent third generation tyrosine kinase inhibitor that is highly selective and targets ALK and ROS1. It was developed to target resistant ALK mutants including the most common G1202R. Lorlatinib has excellent central nervous system (CNS) penetration and its efficacy has also been demonstrated even in patients with intracranial metastases after progression on second generation ALK inhibitors. Potential toxicities include neurocognitive effects and hyperlipidemia. "A User's Guide to Lorlatinib" reviews the mechanism of action, pharmacology and clinical trial data. Also covering the management of adverse events, this "guide" has been prepared to be a practical reference tool to both clinicians and basic researchers.

Efficacy and safety of ALK inhibitors in ALK-rearranged non-small cell lung cancer: A systematic review and meta-analysis

ObjectivesNo overall survival (OS) benefit has been reported from a mature randomized trial with the use of ALK inhibitors. We conducted a systematic review and meta-analysis to assess the efficacy of ALK inhibitors compared to chemotherapy (ALK vs. chemo) and 2nd generation ALK inhibitors compared to 1 st generation ALK inhibitors (ALK-2 G vs. ALK-1 G). MethodsThe electronic databases Medline (PubMed), EMBASE, and the Cochrane Database of Systematic Reviews were searched for relevant randomized trials. Pooled hazard ratios (HR) for OS and progression free survival (PFS), and pooled risk ratios for objective response rates (ORR) and toxicity were meta-analyzed using the generic inverse variance and the Mantel-Haenszel methods. To account for between-studies heterogeneity, random-effect models were used. Subgroup analyses compared PFS by gender, smoking status, brain metastases, race and age. ResultsSix trials were included in the analysis of ALK vs. chemo and four in the analysis of ALK-2 G vs. ALK-1 G. Treatment with ALK inhibitors improved OS compared to chemotherapy (HR: 0.84, 95 %CI 0.72−0.97) while a trend toward a better OS was seen with ALK-2 G vs. ALK-1 G (HR: 0.66, 95 %CI 0.43–1.02). PFS was improved with ALK vs. chemo and ALK-2 G vs. ALK-1 G (HR: 0.44, 95 %CI 0.35−0.54 and HR: 0.38, 95 %CI-0.29−0.51, respectively). ORR was improved with ALK vs. chemo and ALK-2 G vs. ALK-1 G. No difference in toxicity was observed. ConclusionsThis meta-analysis is the first, to our knowledge, to report an OS and PFS benefit with the use of ALK inhibitors compared to chemotherapy from randomized trial data. A trend toward a better OS was also seen with ALK-2 G vs. ALK-1 G and this is likely because of crossover effects and limited OS follow-up. Longer follow up and further research are warranted to directly compare ALK inhibitor sequences and to understand the outcomes of second generation ALK inhibitors as initial therapy.

Repotrectinib (TPX-0005), effectively reduces growth of ALK driven neuroblastoma cells

Neuroblastoma is the most commonly diagnosed extracranial tumor in the first year of life. Approximately 9% of neuroblastoma patients present germline or somatic aberrations in the gene encoding for anaplastic lymphoma kinase (ALK). This increases in high-risk neuroblastomas, which have a 14% frequency of ALK aberrations at the time of diagnosis and show increasing numbers at relapse. Abrogating ALK activity with kinase inhibitors is employed as clinical therapy in malignancies such as non-small cell lung cancer and has shown good results in pediatric inflammatory myofibroblastic tumors and anaplastic large cell lymphomas. A phase I clinical trial of the first generation ALK inhibitor, crizotinib, in neuroblastoma patients showed modest results and suggested that further investigation was needed. Continuous development of ALK inhibitors has resulted in the third generation inhibitor repotrectinib (TPX-0005), which targets the active kinase conformations of ALK, ROS1 and TRK receptors. In the present study we investigated the effects of repotrectinib in a neuroblastoma setting in vitro and in vivo. Neuroblastoma cell lines were treated with repotrectinib to investigate inhibition of ALK and to determine its effect on proliferation. PC12 cells transfected with different ALK mutant variants were used to study the efficacy of repotrectinib to block ALK activation/signaling. The in vivo effect of repotrectinib was also analyzed in a neuroblastoma xenograft model. Our results show that repotrectinib is capable of inhibiting signaling activity of a range of ALK mutant variants found in neuroblastoma patients and importantly it exhibits strong antitumor effects in a xenograft model of neuroblastoma.

NPM-ALK Upregulates Jab1/Csn5 through STAT3 Activation in Anaplastic Large Cell Lymphoma: A Novel Function of NPM-ALK That Contributes to PD1/PD-L1 Immune Checkpoint Regulation

Introduction: ALK+ anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5) resulting in overexpression of NPM-ALK oncoprotein, which activates many oncogenic pathways. The Jab1 (c-Jun activation domain-binding protein-1), initially discovered as a c-Jun co-activator, represents the fifth component of an evolutionary highly conserved 8-subunit protein complex, named COP9 signalosome (CSN). The Jab1/Csn5 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via the CDK inhibitor p27. Recent evidence suggests that Jab1/Csn5 is also involved in immune checkpoint regulation through PD-L1 stabilization by inhibition of PD-L1 proteasomal degradation. Recently, we reported that the protein levels of Jab1/Csn5 are the highest in ALCL as compared to other peripheral T-cell lymphomas (PTCL) and significantly correlate with PD-L1 levels in PTCLs (Drakos et al, HemaSphere 2019; (3):p596, EHA abstract). In this study, we hypothesized that NPM-ALK may regulate Jab1/Csn5 expression and thus contributes to cell proliferation as well as PD-L1/PD1 immune checkpoint regulation. Methods: The in vitro system included 4 ALK+ (Karpas 299, DEL, SUPM2, SUDHL1) and 2 ALK- (Mac1, Mac2a) ALCL cell lines as well as murine Ba/F3 parental and Ba/F3 clones stably transfected with NPM-ALK (Ba/F3-NPM-ALK), EEF1G (Ba/F3-EEF1G-ALK) or control plasmid (Ba/F3-MIG). Transient transfections with Jab1/CSN si-RNAs and STAT3 si-RNAs were also performed in ALCL cells. In addition, ALCL cells were treated with ALK (Crizotonib) or STAT3 (XIII) inhibitors. Two animal models were used in the study: 1) in the ex vivo model, Karpas-299 clones stably transfected with Jab1/CSN5 shRNA constructs were generated and injected in both thighs of SCID-beige immunocompromised mice. The Jab1/CSN5 shRNA and the control mice were followed for tumor development and their tumors were measured and analysed by immunohistochemistry. 2) in the patient derived xenograft (PDX) model of ALK+ ALCL, the mice were treated with the ALK inhibitor Ceritinib or control vehicle and were monitored for changes in tumor characteristics over two weeks. Tumor specimens were taken at early time points (24, 48, and 72 hrs) following treatment in order to obtain viable tumor cells for protein analysis. Results: Jab1/Csn5 was substantially upregulated in the Ba/F3-NPM-ALK and Ba/F3-EEF1G-ALK stable clones as compared to paternal or control Ba/F3-MIG clones. Jab1/Csn5 upregulation was associated with high STAT3 activation (Tyr705-phosphorylation) and increased PD-L1 gene expression in this system. Inversely, inhibition of ALK activity was associated with STAT3 de-activation and decreased protein levels of Jab1/Csn5 and PD-L1 in ALK+ ALCL cells. Knocking down STAT3 by siRNA or inhibition of its activity by the XIII inhibitor resulted in decreased levels of Jab1/Csn5 protein in both ALK+ and ALK- ALCL cell lines. The SCID-beige mice that received Jab1/CSN5-shRNA clones showed significant delay in tumor development and longer survival as compared to control mice, which was associated with significantly decreased PD-L1 protein levels and p27 upregulation in the tumor cells (xenografts). Treatment of the PDX mice with Ceritinib, a potent next generation ALK inhibitor, resulted in substantial tumor necrosis after 72 hrs and decreased tumor size at day 7 post-treatment. At earlier time points, de-activation (de-phosphorylation) of ALK kinase and STAT3 was observed in the Ceritinib-treated mice, which was linked to variably lower levels of Jab1/CSN5 and PD-L1 proteins as compared to control mice. Conclusion: Jab1/CSN5 is a novel downstream target of the NPM-ALK oncogenic kinase that regulates its expression, at least in part, through STAT3 activation. The Jab1/CSN5-mediated stabilization of PD-L1 can be efficiently inhibited by Ceritinib in preclinical animal models of ALK+ ALCL. Disclosures Österborg: BeiGene: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Kancera AB: Research Funding; Gilead: Research Funding. Vega:National Cancer Institute, national Institutes of Health: Other: Grant Funding-R01CA222918.

PCN323 CHANGE IN TREATMENT PARADIGM OF ALK+ NON-SMALL CELL LUNG CANCER (NSCLC) IN THE ITALIAN REAL PRACTICE

ALK rearrangement is observed in 4% to 7% of NSCLC cases. The present analysis aimed to evaluate the evolution of the paradigm of diagnosis and treatment of ALK+ NSCLC in Italy, with a focus on molecular testing adoption and treatment choice. A literature search focused on the main international and national guidelines was conducted in order to identify the diagnostic and therapeutic procedures followed for ALK+ NSCLC patients. The research was enriched with real-world data (monitoring period: MAT 03 2019) supplied by IQVIA (i.e. Oncoview database), in order to achieve a comprehensive overview of current ALK+ NSCLC treatments. Evidences collected were then validated by healthcare physicians to better reflect and understand the Italian real practice. At the time of the present analysis, in Italy, ALK molecular routine assessment was heterogeneous: testing rate was close to 90% in excellence centers, being slightly lower in not specialized centers. Both first and second generation ALK inhibitors were reimbursed representing around 88% of treatments administered in all lines of therapy. Data showed a change in treatment choice within the same class between 2018 and 2019, with first generation ALK inhibitors usage decreasing from 67% to 48% of all treatments and second generation ALK inhibitors increasing from 12% to 39% of all treatments. This analysis showed how the paradigm of diagnosis and treatment for ALK+ NSCLC has changed with the advent of new ALK targeting agents. Overall, second generation ALK inhibitors adoption is increasing, thus confirming a priority role of this therapeutic class as compared with older regimens. Early detection of the ALK rearrangement is needed to ensure prompt initiation of treatment: as a result of new ALK inhibitors introduction, genetic assessment may increase over the time, reaching a homogeneous rate across all hospital centers.

I.17 ALK Genomic Aberrations in NSCLC and Clinical Implications

Anaplastic Lymphoma Kinase (ALK) genomic aberrations in NSCLC was one of the first genomic aberrations, which was successfully targeted with specific drug, e.g. crizotinib, and led to rapid development of personalized therapy in NSCLC. Crizotinib as single agent showed remarkable response (70-80%) in patients with advanced NSCLC, who had the EML-4/ALK- translocation, which occurs in 2-5% of the patients. The genomic abnormality was originally diagnosed by FISH using a defined criterion (split- apart FISH pattern in at least 15% of the cells). However, later the US FDA also approved ALK-IHC for diagnosis using the Ventana ALK-specific antibody, D5F3). Today, this genomic abnormality is mostly detected by NGS. Not only was crizotinib associated to high response rate, but also to long-term outcomes (Solomon BJ et al). However, most of the ALK-positive patients progressed, particularly in the brain. During the studies with crizotinib resistance mutations were seen Second generation ALK- targeted therapies were developed, e. g. alectinib, ceritinib and brigatinib, primarily studied after progression of crizotinib. The new generation ALK-inhibitors showed significant better effect compared to standard chemotherapy and had particularly a good penetration and effect in the CNS. Alectinib was clinically compared to crizotinib as first line therapy in two large randomized studies (J-ALEX and ALEX), and both demonstrated significantly better outcome with alectinib (updated results from ALEX-study showed HR(DFS)= 0.43 with a median disease-free survival of 27.8 months versus 22.8 months for crizotinib), which was approved based on these studies for first line therapy in ALK-positive NSCLC ( Hida T et al, Shaw A et al). Brigatinib was also developed as a second-generation agent and was compared to crizotinib in the ALTA-1 study as first line therapy (HR for PFS=0.49) (Camidge DR et al), but has never been compared to alectinib. Brigatinib was demonstrated to be clearly superior to crizotinib, both with regard to outcome as well as for CNS effect. A question is, however, whether brigatinib is equal or better than alectinib as first line therapy in ALK-positive patients. The third generation ALK-inhibitors have been developed represented by lorlatinib. Ongoing randomized studies have to demonstrate the relative role of lorlatinib compared to other generation ALK-inhibitors. The most important question on this stage is how to sequence the different generations ALK-inhibitors. At most places alectinib is adapted as first line therapy, but there is no clear consensus on what comes after alectinib when the patient is progressing. In the future guidance based on resistant mutation pattern might direct subsequent therapy, but mutation guided therapy for ALK-positive patients has still to be fully developed. The sequencing of different ALK-inhibitors are currently studied in the US national Cancer Institute launched ALK Masterprotocol. 1.Solomon BJ et al: N Engl J Med 2014; 371: 2167-2177 (Crizotinib) 2. Hida T et al: Lancet 2017 (Alectinib) 3. Peters S et al: N Engl J Med 2017: 377(9): 829-838 (Alectinib) 4. Camidge DR et al. J Thorac Oncol 2019: (7): 1233-1243 (Alectinib) 4. Soria JC et al: Lancet 2017; 389: 917-929 (Ceritinib) 5. Camidge DR et al. N Engl J Med 2018; (21): 2027-2039 (Brigatinib) 5. Shaw A et al: J Clin Oncol 2019: 37(16): 1370-1379 (Lorlatinib) Crizotinib, Ceritinib, Alectinib, Brigatinib, Lorlatinib

New generation anaplastic lymphoma kinase inhibitors.

Anaplastic lymphoma kinase (ALK) gene translocations are pro-tumoral driver alterations that encompass 3-7% of non-squamous non-small cell lung cancer (NSCLC) with specific, clinic and histologic features. The therapeutic strategy depends on anti-ALK tyrosine kinase inhibitors (TKIs) of which crizotinib was the first approved for clinical use. Despite its use improved significantly progression-free survival, overall response rate and duration of response of this illness, after a median period of 10.9 months all patients progress due to the development of acquired resistance mutations in the ALK tyrosine kinase domain in approximately one third of patients. Moreover, 60-90% of patients treated with crizotinib has a progression in the central nervous system (CNS) in absence of extracranial worsening of the disease. This is primarily attributed to poor CNS penetration by crizotinib as many pre-clinical and clinical models suggest. For instance, in order to overtake acquired resistance to crizotinib, prolong the control of the disease and manage CNS localizations, several II and III generation TKIs have been developed. Some of them were approved after the failure of crizotinib (ceritinib, alectinib, brigatinib and lorlatinib) and in first line setting (ceritinib, alectinib and brigatinib) while others are still under evaluation for TKI-naive patients such as lorlatinib, ensartinib and entrectinib. In this review we will discuss the most recent results of new TKIs in order to describe a fast growing therapeutic landscape in this setting.

ES09.04 How I Decide 2nd Line Treatment Options for Relapsed/Refractory Metastatic NSCLC

ES09.04 How I Decide 2nd Line Treatment Options for Relapsed/Refractory Metastatic NSCLC

EP1.14-07 Efficacy and Safety of ALK Inhibitors in ALK-Rearranged Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis

First and second generation ALK inhibitors were shown to delay disease progression and improve tumour response in ALK-rearranged advanced non-small cell lung cancer (NSCLC) patients. While a significant progression free survival (PFS) improvement has been consistently reported, no overall survival (OS) benefit was shown in randomized trials. We conducted a systematic review and meta-analysis to assess the efficacy and safety of ALK inhibitors compared to chemotherapy (ALK vs. chemo) and 2nd generation ALK inhibitors compared to 1st generation ALK inhibitors (ALK 2G vs. ALK 1G). The electronic databases PubMed and EMBASE, were searched for relevant randomized trials. Pooled hazard ratios (HR) for overall survival (OS) and progression free survival (PFS), and pooled risk ratios for objective response rates (ORR) and grade 3 or higher toxicity were meta-analyzed using the generic inverse variance and the Mantel-Haenszel methods. To account for between-studies heterogeneity, random-effect models were used. Subgroup analyses compared PFS by gender, smoking status, brain metastases, race and age. Six trials were included in the analysis of ALK vs. chemo and four in the analysis of ALK 2G vs. ALK 1G. Treatment with ALK inhibitors improved OS compared to chemotherapy (HR: 0.84, 95%CI 0.72-0.97) while a trend toward a better OS was seen with ALK 2G vs. ALK 1G without reaching statistical significance (HR: 0.64, 95%CI 0.36-1.16). PFS was improved with ALK vs. chemo and ALK 2G vs. ALK 1G (HR: 0.44, 95%CI 0.35-0.44 and HR: 0.38, 95%CI-0.29-0.51, respectively). Similarly, ORR was improved with ALK vs. chemo and ALK 2G vs. ALK 1G (RR: 2.68, 95%CI 1.89-3.81 and RR: 1.16, 95%CI 1.08-1.24, respectively). The risk of grade 3 or higher toxicity did not differ between treatments (RR 1.08, 95%CI 0.88-1.33 and RR 0.77, 95%CI 0.56-1.06, respectively). Overall the PFS benefit of ALK vs. chemo and ALK 2G vs. ALK 1G was homogenous across all subgroups with a greater degree of benefit within never-smokers when treated with ALK vs. chemo (p for subgroup differences=0.03). This meta-analysis is the first, to our knowledge, to report an OS improvement with the use of ALK vs. chemo. A trend toward a better OS was also seen with ALK 2G vs. ALK 1G and this is likely because of crossover effects and limited OS follow-up. Longer follow up and further research are warranted to directly compare ALK inhibitor sequences and to understand the outcomes of 2nd generation ALK inhibitors as initial therapy.

P1.14-07 Genomic Profiling of Liquid Biopsies During 2nd/3rd Generation ALK Inhibitor Therapy to Identify Novel Mechanisms of Resistance

Second- and third-generation ALK inhibitors each have diverse mechanisms of resistance. Only a fraction of resistance is due to secondary mutations of the ALK gene. Altered bypass tracts are likely the case in some other instances. Genomic alterations of other genes and pathways may be a third mechanism of resistance. Repeat liquid biopsies during the course of patients’ treatments can provide a minimally invasive method for sampling cancer-specific genomic information that leads to improved treatment selection. In the Lung Cancer Clinic of the Princess Margaret Cancer Centre, serial plasma samples were collected from six lung cancer patients with ALK rearrangement at multiple serial clinic visits pre- and post- progression on next-generation ALK inhibitors. We focused on next generation agents, as there has been previous focus on crizotinib resistance mechanisms already. Cell-free DNA (cfDNA) was extracted (median: 50 ng; range: 20-2760 ng) and profiled using a next-generation sequencing (NGS) platform with Geneseeq Prime 425-gene panel at a mean coverage depth of 4747X (and a deduplicated mean coverage depth of 2160X). Somatic alterations from plasma cfDNA were detected in all six patients at various time points with three patients having detectable ALK alterations. Systemic progression (2/2 patients) correlated well with the ability of liquid biopsies to detect somatic mutations, while central nervous system (CNS)-predominant progression did not (4/4 patients). One patient, after disease progression on ceritinib, alectinib and brigatinib, exhibited variable allele fractions (AFs) of ALK G1202R mutation in cfDNA. The levels of G1202R decreased and ultimately became undetectable, corresponding to the patient’s clinical response to lorlatinib. In a patient who exhibited significant systemic progression, a massive increase in mutation AFs and many newly acquired mutations were detected in the cfDNA, including NOTCH1, DICER1, BRCA2, TP53, CDKN2A, ERBB3, and FAT1mutations. However, the increase in the number of co-mutations was not related to increases in the amount of extracted cfDNA. Broad panel-based NGS of plasma cfDNA enabled noninvasive detection of systemic (but not CNS-predominant) progression during second and subsequent generation ALK inhibitor treatment, and can identify known and putative mechanisms of resistance for treatment decision-making.

P1.14-43 A Novel Patient Derived Synchronous Cell Pair with Different Mutations in an ALK-Rearranged Lung Adenocarcinoma Underlines Tumor Heterogeneity

ALK targeted therapy can provide prolonged clinical response rates in ALK-rearranged lung adenocarcinoma (ADC) patients. However, most tumors relapse within a few years of treatment pressure due to a variety of resistance mechanisms, including intratumoral heterogeneity. Understanding these mechanisms is of utmost importance to more precisely tailor future targeted therapies. We established a novel synchronous ALK-translocated lung ADC cell pair from the malignant pleural effusion (PF240-PE) and the pleural carcinosis (PF240-PC) of a 38-year-old female patient following sequential ALK targeted therapy. Immunohistochemistry and mutational analyses were executed in pleural carcinosis tissue specimens and in the tumor cell lines. SRB assays were performed for viability testings following different generations ALK inhibitor treatment alone and combined with SAHA, a pan-HDAC inhibitor. As positive control for all treatment lines we used PF521, another newly established ALK-rearranged but treatment naïve lung ADC cell line. In vivo tumorigenicity was evaluated by performing subcutaneous xenografts. We identified two distinct resistance mutations in both tissue specimens: a so far non-characterized E1161K and the already described L1152R. Strikingly, PF240-PC harbored E1161K and PF240-PE carried L1152R. Immunohistochemistry showed changes from epithelial/carcinomatous to mesenchymal/sarcomatous differentiation following resistance acquisition. In vitro testing revealed that both cell lines were significantly different in morphology and sensitivity to different generation ALK inhibitors including crizotinib, alectinib and lorlatinib. However, the novel tyrosine kinase inhibitor entrectinib was effective in both E1161K and L1152R mutant cells. Importantly, combination treatment of crizotinib or alectinib plus pan-HDAC inhibitor SAHA yielded strong synergism. Of note, both novel cell lines were highly tumorigenic in vivo. In vivo treatment response profiles are currently under evaluation. This is the first evidence of the synchronous establishment of two highly distinct patient-derived ALK translocated lung ADC cell lines carrying different resistant mutations. This concept supports the paramount significance of spatiotemporal intratumoral heterogeneity under targeted therapy. Furthermore, our findings showed that HDAC inhibition could enhance sensitivity of resistant tumor cells to ALK targeted therapy in vitro. Altogether, our findings provide strong evidence for the synchronous emergence of multiple resistance mechanisms and emphasize the importance of multiple site re-biopsies to better identify acquired resistance mechanisms under targeted therapy.

1485PD - Safety and efficacy of WX-0593 in ALK-positive or ROS1-positive non-small cell lung cancer

BackgroundWX-0593 is a potent ALK inhibitor against ALK and ROS1 rearrangement and a series of crizotinib (CRZ)-resistant mutants. We explored the safety and efficacy of WX-0593 in patients (pts) diagnosed with ALK+ or ROS-1+ malignancies, including non-small cell lung cancer (NSCLC). MethodsIn this ongoing phase I, dose-escalation, multicenter trial (NCT03389815), 54 pts (third-grade class-A hospital confirmed ALK+ or ROS-1+) received WX-0593 (30, 60, 90, 120, 180, 240 or 300mg) orally once daily until disease progression or unacceptable toxicity. Activity and safety were evaluated. ResultsFrom 25 September 2017 to 26 December 2018, 54 pts were enrolled, including 46 with ALK+ NSCLC, 10 with ROS1+ NSCLC (2 with both ALK+ and ROS1+, 38.9% with prior CRZ, 9.3% with prior CRZ and second generation ALK inhibitors). Grade 3 QTc prolongation reported in 300mg cohort was identified as dose-limiting toxicity (DLT). Treatment-emergent adverse events (TAEs) associated with WX-0593 in>20% of pts include: hypercholesterolemia (40.7%), nausea (40.7%), hypertension (37.0%) increased ALT (31.5%), increased AST (29.6%), hypertriglyceridemia (25.9%), hyperlipidemia (25.9%), vomiting 25.9%. Serious adverse events (SAE) occurred in 6 pts (11.1%). No treatment related death occurred. 2 pts (3.7%) had SAE related with WX-0593 treatment, including increased ALT (dose of 180mg), increased AST (dose of 180mg) and chronic heart failure (dose of 300mg). For ALK+ NSCLC (n=46), objective response rate (ORR) was 29/44 (65.9%). The ORR in CRZ-naive pts was 17/21 (81.0%), 8/18 (44.4%) in CRZ pre-treated pts. ORR was 3/10 (30.0%) in ROS1+ NSCLC pts. By 26 December 2018, median progression-free survival was not reached, 39 patients remained on WX0593 treatment.Table1485PD Objective response rate in ALK-positive and ROS1-positive NSCLC patientsTableORR, n / m (%)30mg N=360mg N=390mg N=8120mg N=11180mg N=13240mg N=13300mg N=3totalALK+NSCLCall pts2/2 (100.0)2/3 (66.7)2/5 (40.0)8/10 (80.0)7/10 (70.0)7/11 (63.6)1/3 (33.3)29/44 (65.9)CRZ-naive pts2/2 (100.0)2/3 (66.7)06/7 (85.7)3/4 (75.0)3/4 (75.0)1/1 (100.0)17/21 (81.0)pts with prior CRZ002/4 (50.0)1/2 (50.0)3/5 (60.0)2/5 (40.0)0/28/18 (44.4)pts with prior CRZ and other ALK inhibitor000/11/1 (100.0)1/1 (100.0)2/2 (100.0)04/5 (80.0)ROS1+NSCLCall pts0/101/3 (33.3%)0/12/3 (66.7%)0/203/10 (30.0%) ConclusionsThis study revealed the antitumor activity of WX-0593 in ALK- or ROS-positive NSCLC. Safety profile was acceptable. A phase II trial to evaluate the ORR of 120mg and 180mg once daily is ongoing. Clinical trial identificationNCT03389815. Legal entity responsible for the studyQilu Pharmaceutical Co., Ltd. FundingQilu Pharmaceutical Co., Ltd. DisclosureAll authors have declared no conflicts of interest.

1486PD - Exposure-response analyses of ALK-inhibitors crizotinib and alectinib in NSCLC patients

BackgroundCrizotinib and alectinib are first and second generation ALK-inhibitors, respectively, indicated for the treatment of ALK+ NSCLC. At the currently used fixed doses, interpatient variability in exposure is high (40%), while the registration studies suggest that exposure might be linked to efficacy (FDA Reviews). Therefore, the aim of this study was to explore whether exposure to crizotinib and alectinib is related to efficacy in a real life patient (pt) cohort. MethodsA retrospective observational study was performed. ALK+ NSCLC pts who were treated with crizotinib or alectinib and of whom pharmacokinetic (PK) samples were drawn as part of routine care were included. Calculated minimum plasma concentration (Cmin) was taken as a measure of exposure. Efficacy endpoint was progression free survival (PFS). Univariable and multivariable exposure-response analyses were performed using previously proposed thresholds of 235 µg/L for crizotinib and 435 µg/L for alectinib (Verheijen 2017). Variables taken into account were WHO performance status (PS) and previous lines of therapy for crizotinib, and WHO PS and previous treatment with ALK-inhibitors for alectinib. ResultsIn total, 100 pts were included in this study (48 crizotinib, 52 alectinib), of whom 376PK samples were eligible for analysis (235 crizotinib, 141 alectinib). Median number of samples per pt was 3 (range 1 – 15). Median Cmin per pt was 244 µg/L (IQR 176 – 344 µg/L) for crizotinib and 517 µg/L (IQR 369 – 659 µg/L) for alectinib. For crizotinib, median PFS was 5.7 vs 17.4 months (mo) for pts with median Cmin < 235 µg/L (48%) and ≥ 235 µg/L, respectively (log rank test, p=0.08). In multivariable analysis Cmin < 235 µg/L resulted in a HR of 1.79 (95% CI 0.90 – 3.59, p=0.10). For alectinib, median PFS was 12.6mo vs not reached yet after 20mo for pts with Cmin < 435 µg/L (37%) and ≥ 435 µg/L, respectively (log rank test, p=0.04). Multivariable analysis resulted in a HR of 3.86 (95% CI 1.19 – 12.58, p=0.025). Thus far, this could not be explained by bias. ConclusionsThis study shows that exposure to alectinib is associated with efficacy in a real life patient cohort. Therefore, therapeutic drug monitoring might be appropriate to individualize treatment and improve treatment outcomes. Legal entity responsible for the studyNetherlands Cancer Institute - Antoni van Leeuwenhoek Hospital. FundingHas not received any funding. DisclosureS.A. Burgers: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Roche Advisory / Consultancy: MSD. N. Steeghs: Research grant / Funding (institution): AstraZeneca/MedImmune; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): GlaxoSmithKline; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Blueprint Medicines; Research grant / Funding (institution): AB Science; Research grant / Funding (institution): Deciphera; Research grant / Funding (institution): Merck Sharp & Dohme; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Merus; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Incyte. All other authors have declared no conflicts of interest.

P1.14-18 ALK Inhibitor Sequencing and Outcomes Among ALK-Positive (ALK+) NSCLC Patients in the US Community Oncology Setting

Several ALK inhibitors, including 2nd and 3rd generation agents are available for patients with ALK+ NSCLC. Treatment patterns and outcomes with use of multiple sequential ALK inhibitors is limited. A retrospective observational cohort study of patients with ALK+ NSCLC treated with 1st generation (crizotinib) and 2nd generation (alectinib, brigatinib, ceritinib) ALK inhibitors from 1 September 2011 to 31 December 2017. Structured data were obtained via programmatic extraction from the iKnowMed EHR database of the US Oncology Network. Patient demographics and treatment sequences were characterized. Index was the start date of the first ALK. Duration of therapy (DOT) from index to end of the last ALK, and overall survival (OS) were assessed using the Kaplan-Meier method. A total of 410 ALK+ NSCLC patients were included. Median age at index was 62 years, 54% were female, 78% Caucasian, 87% adenocarcinoma histology, and 54% never smokers. 233 (57%), 144 (35%), and 33 (8%) patients received 1, 2, or 3-4 different ALK inhibitors, respectively. Crizotinib monotherapy (50%) was most common. Among patients that received 2 or more ALK inhibitors (n=177), most were crizotinib-led sequences. In 59% of patients, chemotherapy was given prior to the first ALK (median time from start of chemo to index 6.64 mo), and 53% of patients ended their ALK sequence and received subsequent chemo. Median cumulative ALK DOT in the full study population, regardless of line of therapy or sequence was 16 mo (95% CI 6-19). Median OS from index for the full study population was 28 mo (95% CI 24, 36). Median OS among patients who received 1, 2, or 3-4 ALK inhibitors was 15 mo (95% CI 10, 22), 42 mo (95% CI 38, 60) and 56 mo (95% CI 31, 72). Patients received a range of 1 to 4 ALK inhibitors. Crizotinib-led sequences were most common, likely reflecting the approval history of ALK inhibitors during the study period. Longer DOT and OS were observed in patients receiving multiple ALK inhibitors. This study provides an initial view of treatment patterns following the emergence of new ALK inhibitors and suggests feasibility of sequential ALK therapies. Follow-up studies will help improve understanding of outcomes of patients treated with 2nd generation-led sequences.

Alectinib, an Anaplastic Lymphoma Kinase Inhibitor, Abolishes ALK Activity and Growth in ALK-Positive Neuroblastoma Cells.

Oncogenic receptor tyrosine kinases including anaplastic lymphoma kinase (ALK) are implicated in numerous solid and hematologic cancers. ALK mutations are reported in an estimated 9% of neuroblastoma and recent reports indicate that the percentage of ALK-positive cases increases in the relapsed patient population. Initial clinical trial results have shown that it is difficult to inhibit growth of ALK positive neuroblastoma with crizotinib, motivating investigation of next generation ALK inhibitors with higher affinity for ALK. Here, alectinib, a potent next generation ALK inhibitor with antitumor activity was investigated in ALK-driven neuroblastoma models. Employing neuroblastoma cell lines and mouse xenografts we show a clear and efficient inhibition of ALK activity by alectinib. Inhibition of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. The results suggest that alectinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma and should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

ALK resistance mutations (muts) developed in different ALK fusion types.

e14741 Background: Patients (pts) treated with ALK inhibitors (ALKi) inevitably develops resistance. Diverse mutations (muts) confer resistance to ALKis, which may be related to the diverse types of ALK fusion. The objective of this study is to see whether the resistance mechanisms developed during ALKi therapy are ALK fusion types dependent. Methods: The mutation profiles of 38 pts with known ALK fusion partners and positions, who ordered NGS test in our institution after progressed on ≥ 1 ALKis, were analyzed. Results: Resistant muts in ALK kinase domain ( ALK resistant muts) were detected in 53% (20/38) of the resistant pts. 100% (1/1) of the EML4-ALK V5’ variant (V5’), 81% (13/16) of the EML4-ALK V3 variant (V3), 60% (3/5) of the non- EML4-ALK, 23% (3/13) of EML4-ALK V1 variant (V1), and 0% (0/3) of the EML4-ALK V2 variant (V2) had ALK resistant muts. ALK G1202R was detected in 29% of the pts. 100% (1/1) of the V5’, 50% (8/16) of the V3, 20% (1/5, HIP1-ALK) of the non- EML4-ALK, and none of the V1 (0/13) and V2 (0/3) had G1202R. Amplification and gain of function muts in oncogenes and loss of function muts in tumor suppressor gene were detected in 16% (6/38) of the pts, namely 4 V1, 1 V2, and 1 V3. On average, pts treated with 3rd generation ALKis had significantly more muts in ALK (7.5 vs. 3.6, p = 0.0008) and in all targeted regions (9.1 vs. 4.3, p = 0.0005) than pts treated with only 1st and 2nd generation ALKis. Conclusions: The type of resistance mechanisms developed during ALKi therapy may depend on the ALK fusion type of the pt. ALK resistant muts, especially G1202R, developed most frequently in V3/V5’, followed by non- EML4-ALK, and V1/V2, while amplification and grain of function muts in oncogenes and loss of function muts in tumor suppressor genes developed more often in V1/V2 than in V3/V5’. The 3rd generation ALKi may increase the genomic alterations in treated pts.