Summary Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples; at three hotspots plus 13 minor sites – and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from non-oncogenic mutations and allowed robust computational prediction of their effects. We also established differential in vitro crizotinib sensitivity of mutated variants. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma, and will allow tailoring of ALK-targeted therapy to specific mutations.
Neuroblastomas (NBs) harboring activating point mutations in Anaplastic Lymphoma Kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants, and inhibits ALK more effectively than crizotinib in vitro. Most importantly, PF-06463922 induces complete tumor regression in both crizotinib-resistant and sensitive xenograft mouse models of NB, as well as in PDXs harboring the crizotinib-resistant F1174L or F1245C mutations. These studies demonstrate that PF-06463922 has the potential to overcome crizotinib resistance, and exerts unprecedented activity as a single targeted agent against F1174L and F1245C ALK-mutated xenograft tumors, while also inducing responses in a R1275Q xenograft model. Taken together, these results provide the rationale to move PF-06463922 into clinical trials for treatment of patients with ALK-mutated NB.
Purpose The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical. Experimental Design The sensitivity of human neuroblastoma-derived cell lines, cell line-derived and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy. Results In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free-survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification). Conclusions Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multi-agent therapy for ALK-aberrant neuroblastoma patients.
Purpose Anaplastic Lymphoma Kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine if drug combinations could enhance anti-tumor efficacy. Experimental Design We screened combinations of eight molecularly targeted agents against seventeen comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of Ceritinib and Ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the Cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing Ceritinib alone, Ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions. Results The combination of Ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor Ceritinib demonstrated higher cytotoxicity (p=0.008) and synergy scores (p=0.006) in cell lines with ALK mutations as compared to cell lines lacking mutations or alterations in ALK. Compared to either drug alone, combination therapy enhanced growth inhibition, cell cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations, and prevented the emergence of resistance. Murine Ribociclib and Ceritinib plasma concentrations were unaltered by combination therapy. Conclusions This preclinical combination drug screen with in vivo validation has provided the rationale for a first in children trial of combination Ceritinib and Ribociclib in a molecularly selected pediatric population.
Objectives Detrusor underactivity (DU) is diagnosed using urodynamic testing. We hypothesized that nocturia is associated with detrusor underactivity. Methods We performed a retrospective chart review of all women who underwent urodynamic testing at our institution between 2016 and 2018. Uroflowmetry and pressure-flow study parameters were compared between women with nocturia (≥2 voids/night) and without nocturia (0–1 void/night). Detrusor underactivity was diagnosed using 3 different criteria: (1) bladder voiding efficiency (BVE) of <90%, (2) bladder contractility index of <100, and (3) a composite of three urodynamic measures (Gammie criteria). Results Of 358 women, 172 (48%) were in the nocturia group and 186 (52%) were in the no nocturia group. On uroflowmetry, median postvoid residual volume was similar (20 mL) in both groups. Median maximum flow rate (15 vs 17 mL/s, P < 0.05) and average flow rate (6 mL/s vs 7 mL/s, P < 0.05) were significantly lower in the nocturia group compared with the no nocturia group. During pressure-flow study, a significantly greater proportion of women with nocturia were unable to void around the catheter (30% vs 27%, P < 0.01). The overall rate of DU varied with the criteria used: BVE (54%), bladder contractility index (41%), and Gammie criteria (7%). The rate of DU using the BVE criteria was significantly higher in the nocturia group (63% vs 48%, P < 0.01), but no significant differences were noted using the other criteria. Conclusions Nocturia is associated with reduced voiding efficiency in women. The diagnosis of DU using urodynamics is challenging.
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