Computational studies of anaplastic lymphoma kinase mutations reveal common mechanisms of oncogenic activation.

Kinases play important roles in diverse cellular processes, including signaling, differentiation, proliferation, and metabolism. They are frequently mutated in cancer and are the targets of a large number of specific inhibitors. Surveys of cancer genome atlases reveal that kinase domains, which consist of 300 amino acids, can harbor numerous (150 to 200) single-point mutations across different patients in the same disease. This preponderance of mutations-some activating, some silent-in a known target protein make clinical decisions for enrolling patients in drug trials challenging since the relevance of the target and its drug sensitivity often depend on the mutational status in a given patient. We show through computational studies using molecular dynamics (MD) as well as enhanced sampling simulations that the experimentally determined activation status of a mutated kinase can be predicted effectively by identifying a hydrogen bonding fingerprint in the activation loop and the αC-helix regions, despite the fact that mutations in cancer patients occur throughout the kinase domain. In our study, we find that the predictive power of MD is superior to a purely data-driven machine learning model involving biochemical features that we implemented, even though MD utilized far fewer features (in fact, just one) in an unsupervised setting. Moreover, the MD results provide key insights into convergent mechanisms of activation, primarily involving differential stabilization of a hydrogen bond network that engages residues of the activation loop and αC-helix in the active-like conformation (in >70% of the mutations studied, regardless of the location of the mutation).

Preclinical evaluation of the combination of AZD1775 and irinotecan against selected pediatric solid tumors: A Pediatric Preclinical Testing Consortium report.

INTRODUCTION: WEE1 is a serine kinase central to the G2 checkpoint. Inhibition of WEE1 can lead to cell death by permitting cell-cycle progression despite unrepaired DNA damage. AZD1775 is a WEE1 inhibitor that is in clinical development for children and adults with cancer. METHODS: AZD1775 was tested using a dose of 120 mg/kg administered orally for days 1 to 5. Irinotecan was administered intraperitoneally at a dose of 2.5 mg/kg for days 1 to 5 (one hour after AZD1775 when used in combination). AZD1775 and irinotecan were studied alone and in combination in neuroblastoma (n = 3), osteosarcoma (n = 4), and Wilms tumor (n = 3) xenografts. RESULTS: AZD1775 as a single agent showed little activity. Irinotecan induced objective responses in two neuroblastoma lines (PRs), and two Wilms tumor models (CR and PR). The combination of AZD1775 + irinotecan-induced objective responses in two neuroblastoma lines (PR and CR) and all three Wilms tumor lines (CR and 2 PRs). The objective response measure improved compared with single-agent treatment for one neuroblastoma (PR to CR), two osteosarcoma (PD1 to PD2), and one Wilms tumor (PD2 to PR) xenograft lines. Of note, the combination yielded CR (n = 1) and PR (n = 2) in all the Wilms tumor lines. The event-free survival was significantly longer for the combination compared with single-agent irinotecan in all models tested. The magnitude of the increase was greatest in osteosarcoma and Wilms tumor xenografts. CONCLUSIONS: AZD1775 potentiates the effects of irinotecan across most of the xenograft lines tested, with effect size appearing to vary across tumor panels.

Computational algorithms for in silico profiling of activating mutations in cancer.

Methods to catalog and computationally assess the mutational landscape of proteins in human cancers are desirable. One approach is to adapt evolutionary or data-driven methods developed for predicting whether a single-nucleotide polymorphism (SNP) is deleterious to protein structure and function. In cases where understanding the mechanism of protein activation and regulation is desired, an alternative approach is to employ structure-based computational approaches to predict the effects of point mutations. Through a case study of mutations in kinase domains of three proteins, namely, the anaplastic lymphoma kinase (ALK) in pediatric neuroblastoma patients, serine/threonine-protein kinase B-Raf (BRAF) in melanoma patients, and erythroblastic oncogene B 2 (ErbB2 or HER2) in breast cancer patients, we compare the two approaches above. We find that the structure-based method is most appropriate for developing a binary classification of several different mutations, especially infrequently occurring ones, concerning the activation status of the given target protein. This approach is especially useful if the effects of mutations on the interactions of inhibitors with the target proteins are being sought. However, many patients will present with mutations spread across different target proteins, making structure-based models computationally demanding to implement and execute. In this situation, data-driven methods-including those based on machine learning techniques and evolutionary methods-are most appropriate for recognizing and illuminate mutational patterns. We show, however, that, in the present status of the field, the two methods have very different accuracies and confidence values, and hence, the optimal choice of their deployment is context-dependent.

Heterozygosity mapping for human dominant trait variants.

Homozygosity mapping is a well-known technique to identify runs of homozygous variants that are likely to harbor genes responsible for autosomal recessive disease, but a comparable method for autosomal dominant traits has been lacking. We developed an approach to map dominant disease genes based on heterozygosity frequencies of sequence variants in the immediate vicinity of a dominant trait. We demonstrate through theoretical analysis that DNA variants surrounding an inherited dominant disease variant tend to have increased heterozygosity compared with variants elsewhere in the genome. We confirm existence of this phenomenon in sequence data with known dominant pathogenic variants obtained on family members and in unrelated population controls. A computer-based approach to estimating empirical significance levels associated with our test statistics shows genome-wide p-values smaller than 0.05 for many but not all of the individuals carrying a pathogenic variant.

Enrichment of Targetable Mutations in the Relapsed Neuroblastoma Genome.

Neuroblastoma is characterized by a relative paucity of recurrent somatic mutations at diagnosis. However, recent studies have shown that the mutational burden increases at relapse, likely as a result of clonal evolution of mutation-carrying cells during primary treatment. To inform the development of personalized therapies, we sought to further define the frequency of potentially actionable mutations in neuroblastoma, both at diagnosis and after chemotherapy. We performed a retrospective study to determine mutation frequency, the only inclusion criterion being availability of cancer gene panel sequencing data from Foundation Medicine. We analyzed 151 neuroblastoma tumor samples: 44 obtained at diagnosis, 42 at second look surgery or biopsy for stable disease after chemotherapy, and 59 at relapse (6 were obtained at unknown time points). Nine patients had multiple tumor biopsies. ALK was the most commonly mutated gene in this cohort, and we observed a higher frequency of suspected oncogenic ALK mutations in relapsed disease than at diagnosis. Patients with relapsed disease had, on average, a greater number of mutations reported to be recurrent in cancer, and a greater number of mutations in genes that are potentially targetable with available therapeutics. We also observed an enrichment of reported recurrent RAS/MAPK pathway mutations in tumors obtained after chemotherapy. Our data support recent evidence suggesting that neuroblastomas undergo substantial mutational evolution during therapy, and that relapsed disease is more likely to be driven by a targetable oncogenic pathway, highlighting that it is critical to base treatment decisions on the molecular profile of the tumor at the time of treatment. However, it will be necessary to conduct prospective clinical trials that match sequencing results to targeted therapeutic intervention to determine if cancer genomic profiling improves patient outcomes.

Integrative genomics identifies LMO1 as a neuroblastoma oncogene.

Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.

Copy number variation at 1q21.1 associated with neuroblastoma.

Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in human cancer, we performed a genome-wide association study of CNVs in the childhood cancer neuroblastoma, a disease in which single nucleotide polymorphism variations are known to influence susceptibility. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Caucasian controls at approximately 550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. Here we describe the identification of a common CNV at chromosome 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets. This CNV was validated by quantitative polymerase chain reaction, fluorescent in situ hybridization and analysis of matched tumour specimens, and was shown to be heritable in an independent set of 713 cancer-free parent-offspring trios. We identified a previously unknown transcript within the CNV that showed high sequence similarity to several neuroblastoma breakpoint family (NBPF) genes and represents a new member of this gene family (NBPF23). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and the expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a previously unknown neuroblastoma breakpoint family gene in early tumorigenesis of this childhood cancer.

Neuroblastoma in older children, adolescents and young adults: a report from the International Neuroblastoma Risk Group project.

BACKGROUND: Neuroblastoma in older children and adolescents has a distinctive, indolent phenotype, but little is known about the clinical and biological characteristics that distinguish this rare subgroup. Our goal was to determine if an optimal age cut-off exists that defines indolent disease and if accepted prognostic factors and treatment approaches are applicable to older children. PROCEDURE: Using data from the International Neuroblastoma Risk Group, among patients ≥18 months old (n = 4,027), monthly age cut-offs were tested to determine the effect of age on survival. The prognostic effect of baseline characteristics and autologous hematopoietic cell transplant (AHCT) for advanced disease was assessed within two age cohorts; ≥5 to <10 years (n = 730) and ≥10 years (n = 200). RESULTS: Older age was prognostic of poor survival, with outcome gradually worsening with increasing age at diagnosis, without statistical evidence for an optimal age cut-off beyond 18 months. Among patients ≥5 years, factors significantly prognostic of lower event-free survival (EFS) and overall survival (OS) in multivariable analyses were INSS stage 4, MYCN amplification and unfavorable INPC histology classification. Among stage 4 patients, AHCT provided a significant EFS and OS benefit. Following relapse, patients in both older cohorts had prolonged OS compared to those ≥18 months to <5 years (P < 0.0001). CONCLUSIONS: Despite indolent disease and infrequent MYCN amplification, older children with advanced disease have poor survival, without evidence for a specific age cut-off. Our data suggest that AHCT may provide a survival benefit in older children with advanced disease. Novel therapeutic approaches are required to more effectively treat these patients.

Time to disease progression in children with relapsed or refractory neuroblastoma treated with ABT-751: a report from the Children's Oncology Group (ANBL0621).

BACKGROUND: ABT-751, an orally bioavailable sulfonamide binds the colchicine site of beta-tubulin and inhibits microtubule polymerization. Prior phase I studies established the recommended dose in children with solid tumors as 200 mg/m(2) PO daily × 7 days every 21 days and subjects with neuroblastoma experienced prolonged stable disease. We conducted a phase 2 study (NCT00436852) in children and adolescents with progressive neuroblastoma to determine if ABT-751 prolonged the time to progression (TTP) compared to a hypothesized standard based on a historical control population. PROCEDURE: Children and adolescents (n = 91) with a median (range) age 7.7 (2.3-21.5) years and progressive neuroblastoma were enrolled and stratified by disease status into disease measureable by CT/MRI (n = 47) or disease assessable by (123) I-metaiodobenzylguanine scintigraphy (MIBG, n = 44). Response was evaluated using RECIST for measureable disease and the Curie score for MIBG-avid disease. RESULTS: ABT-751 was well tolerated. The objective response rate was 7%. The median TTP was 42 days (95% CI: 36, 56) in the measureable disease stratum and 45 days (95% CI: 42, 85) in the MIBG-avid disease stratum. TTP was similar to the historical control group (n = 136, median TTP 42 days). For the combined strata (n = 91), 1-year progression free survival (PFS) was 13 ± 4% and overall survival (OS) was 48 ± 5%. CONCLUSIONS: The low objective response rate and failure to prolong TTP indicate that ABT-751 is not sufficiently active to warrant further development for neuroblastoma. However, this trial demonstrates the utility of TTP as the primary endpoint in phase 2 trials in children and adolescents with neuroblastoma.

Identification of ALK as a major familial neuroblastoma predisposition gene.

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23-24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

Phenotype restricted genome-wide association study using a gene-centric approach identifies three low-risk neuroblastoma susceptibility Loci.

Neuroblastoma is a malignant neoplasm of the developing sympathetic nervous system that is notable for its phenotypic diversity. High-risk patients typically have widely disseminated disease at diagnosis and a poor survival probability, but low-risk patients frequently have localized tumors that are almost always cured with little or no chemotherapy. Our genome-wide association study (GWAS) has identified common variants within FLJ22536, BARD1, and LMO1 as significantly associated with neuroblastoma and more robustly associated with high-risk disease. Here we show that a GWAS focused on low-risk cases identified SNPs within DUSP12 at 1q23.3 (P = 2.07 × 10⁻6), DDX4 and IL31RA both at 5q11.2 (P = 2.94 × 10⁻6 and 6.54 × 10⁻7 respectively), and HSD17B12 at 11p11.2 (P = 4.20 × 10⁻7) as being associated with the less aggressive form of the disease. These data demonstrate the importance of robust phenotypic data in GWAS analyses and identify additional susceptibility variants for neuroblastoma.

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma.

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children's Oncology Group phase 1 consortium study.

BACKGROUND: Various human cancers have ALK gene translocations, amplifications, or oncogenic mutations, such as anaplastic large-cell lymphoma, inflammatory myofibroblastic tumours, non-small-cell lung cancer (NSCLC), and neuroblastoma. Therefore, ALK inhibition could be a useful therapeutic strategy in children. We aimed to determine the safety, recommended phase 2 dose, and antitumour activity of crizotinib in children with refractory solid tumours and anaplastic large-cell lymphoma. METHODS: In this open-label, phase 1 dose-escalation trial, patients older than 12 months and younger than 22 years with measurable or evaluable solid or CNS tumours, or anaplastic large-cell lymphoma, refractory to therapy and for whom there was no known curative treatment were eligible. Crizotinib was given twice daily without interruption. Six dose levels (100, 130, 165, 215, 280, 365 mg/m(2) per dose) were assessed in the dose-finding phase of the study (part A1), which is now completed. The primary endpoint was to estimate the maximum tolerated dose, to define the toxic effects of crizotinib, and to characterise the pharmacokinetics of crizotinib in children with refractory cancer. Additionally, patients with confirmed ALK translocations, mutations, or amplification (part A2 of the study) or neuroblastoma (part A3) could enrol at one dose level lower than was currently given in part A1. We assessed ALK genomic status in tumour tissue and used quantitative RT-PCR to measure NPM-ALK fusion transcript in bone marrow and blood samples of patients with anaplastic large-cell lymphoma. All patients who received at least one dose of crizotinib were evaluable for response; patients completing at least one cycle of therapy or experiencing dose limiting toxicity before that were considered fully evaluable for toxicity. This study is registered with ClinicalTrials.gov, NCT00939770. FINDINGS: 79 patients were enrolled in the study from Oct 2, 2009, to May 31, 2012. The median age was 10.1 years (range 1.1-21.4); 43 patients were included in the dose escalation phase (A1), 25 patients in part A2, and 11 patients in part A3. Crizotinib was well tolerated with a recommended phase 2 dose of 280 mg/m(2) twice daily. Grade 4 adverse events in cycle 1 were neutropenia (two) and liver enzyme elevation (one). Grade 3 adverse events that occurred in more than one patient in cycle 1 were lymphopenia (two), and neutropenia (eight). The mean steady state peak concentration of crizotinib was 630 ng/mL and the time to reach this peak was 4 h (range 1-6). Objective tumour responses were documented in 14 of 79 patients (nine complete responses, five partial responses); and the anti-tumour activity was enriched in patients with known activating ALK aberrations (eight of nine with anaplastic large-cell lymphoma, one of 11 with neuroblastoma, three of seven with inflammatory myofibroblastic tumour, and one of two with NSCLC). INTERPRETATION: The findings suggest that a targeted inhibitor of ALK has antitumour activity in childhood malignancies harbouring ALK translocations, particularly anaplastic large-cell lymphoma and inflammatory myofibroblastic tumours, and that further investigation in the subset of neuroblastoma harbouring known ALK oncogenic mutations is warranted. FUNDING: Pfizer and National Cancer Institute grant to the Children's Oncology Group.

Podcast on Emerging Treatment Options for Pediatric Patients with ALK-Positive Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic Tumors.

Anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumor (IMT) are rare cancers observed predominantly in children and young adults. ALCL accounts for 10-15% of all pediatric non-Hodgkin lymphomas and is commonly diagnosed at an advanced stage of disease. In children, 84-91% of cases of ALCL harbor an anaplastic lymphoma kinase (ALK) gene translocation. IMT is a rare mesenchymal neoplasm that also tends to occur in children and adolescents. Approximately 50-70% of IMT cases involve rearrangements in the ALK gene. A combination of chemotherapeutic drugs is typically used for children with ALK-positive ALCL, and the only known curative therapy for ALK-positive IMT is complete surgical resection. Crizotinib, a first-generation ALK inhibitor, was approved in the USA in 2021 for pediatric patients and young adults with relapsed or refractory ALK-positive ALCL; however, its safety and efficacy have not been established in older adults. In 2022, crizotinib was approved for adult and pediatric patients with unresectable, recurrent, or refractory ALK-positive IMT. This podcast provides an overview of ALK-positive ALCL and IMT. We discuss the current treatment landscape, the role of ALK tyrosine kinase inhibitors, and areas of future research.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. Highlights: Plasma membrane enriched proteomics defines surfaceome of neuroblastomaMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancersDLK1 expression is driven by a super-enhancer DLK1 silencing in neuroblastoma cells results in cellular differentiation ADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models.

Circulating tumor DNA reveals mechanisms of lorlatinib resistance in patients with relapsed/refractory ALK-driven neuroblastoma.

Activating point mutations in Anaplastic Lymphoma Kinase (ALK) have positioned ALK as the only mutated oncogene tractable for targeted therapy in neuroblastoma. Cells with these mutations respond to lorlatinib in pre-clinical studies, providing the rationale for a first-in-child Phase 1 trial (NCT03107988) in patients with ALK-driven neuroblastoma. To track evolutionary dynamics and heterogeneity of tumors, and to detect early emergence of lorlatinib resistance, we collected serial circulating tumor DNA samples from patients enrolled on this trial. Here we report the discovery of off-target resistance mutations in 11 patients (27%), predominantly in the RAS-MAPK pathway. We also identify newly acquired secondary compound ALK mutations in 6 (15%) patients, all acquired at disease progression. Functional cellular and biochemical assays and computational studies elucidate lorlatinib resistance mechanisms. Our results establish the clinical utility of serial circulating tumor DNA sampling to track response and progression and to discover acquired resistance mechanisms that can be leveraged to develop therapeutic strategies to overcome lorlatinib resistance.

Lorlatinib with or without chemotherapy in ALK-driven refractory/relapsed neuroblastoma: phase 1 trial results.

Neuroblastomas harbor ALK aberrations clinically resistant to crizotinib yet sensitive pre-clinically to the third-generation ALK inhibitor lorlatinib. We conducted a first-in-child study evaluating lorlatinib with and without chemotherapy in children and adults with relapsed or refractory ALK-driven neuroblastoma. The trial is ongoing, and we report here on three cohorts that have met pre-specified primary endpoints: lorlatinib as a single agent in children (12 months to <18 years); lorlatinib as a single agent in adults (≥18 years); and lorlatinib in combination with topotecan/cyclophosphamide in children (<18 years). Primary endpoints were safety, pharmacokinetics and recommended phase 2 dose (RP2D). Secondary endpoints were response rate and 123I-metaiodobenzylguanidine (MIBG) response. Lorlatinib was evaluated at 45-115 mg/m2/dose in children and 100-150 mg in adults. Common adverse events (AEs) were hypertriglyceridemia (90%), hypercholesterolemia (79%) and weight gain (87%). Neurobehavioral AEs occurred mainly in adults and resolved with dose hold/reduction. The RP2D of lorlatinib with and without chemotherapy in children was 115 mg/m2. The single-agent adult RP2D was 150 mg. The single-agent response rate (complete/partial/minor) for <18 years was 30%; for ≥18 years, 67%; and for chemotherapy combination in <18 years, 63%; and 13 of 27 (48%) responders achieved MIBG complete responses, supporting lorlatinib's rapid translation into active phase 3 trials for patients with newly diagnosed high-risk, ALK-driven neuroblastoma. ClinicalTrials.gov registration: NCT03107988 .

Evaluation of the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine in preclinical models of neuroblastoma.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody drug conjugated (ADC) with a pyrrolobenzodiazepine (PBD) dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA sequencing data sets and performed immunohistochemistry (IHC) on neuroblastoma patient derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays (TMAs). We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared anti-tumor activity to expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3/11 and stable disease in another 3/11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for anti-tumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.

Serial Profiling of Circulating Tumor DNA Identifies Dynamic Evolution of Clinically Actionable Genomic Alterations in High-Risk Neuroblastoma.

Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS-MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant-in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. SIGNIFICANCE: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.