Abstract SY14-02: ALK in neuroblastoma

Abstract

Abstract Neuroblastoma is a cancer of early childhood that arises from the developing autonomic nervous system. It is the most common malignancy diagnosed in the first year of life and shows a wide range of clinical phenotypes with some patients having tumors that regress spontaneously, whereas the majority of patients have aggressive metastatic disease. Neuroblastoma remains an important clinical problem as it continues to be a leading cause of childhood cancer mortality despite dramatic escalations in dose-intensive chemo radiotherapy, and long-term survivors experience significant treatment related morbidity. To improve outcome and make paradigm-shifting advances in this disease, it is necessary to discover the key oncogenic drivers of the malignant process and exploit these therapeutically. We recently discovered activating mutations within the tyrosine kinase domain of the anaplastic lymphoma kinase (ALK) oncogene as the major cause of hereditary neuroblastoma, and showed that somatically acquired mutations and gene amplification events often drive the malignant process. Here we report a comprehensive survey of ALK genomic status in all neuroblastoma phenotypic subsets and perform preclinical validation that targeted inhibition of activated ALK kinase is an effective therapeutic strategy. ALK mutations were detected in 7% of cases, were restricted to the tyrosine kinase domain and occurred equally in all patient subsets. The most common germline mutation in familial cases (R1275Q) was also the most common somatic mutation (49% of tumors with a mutation). High-level amplification of ALK was detected in 2.4% of tumors, regional gain of the ALK locus in an additional 19% of cases, and both were associated with differentially increased ALK mRNA expression (P= 0.004). ALK amplification is strongly associated with the high-risk subset (P<0.001) and MYCN amplification (P<0.001). Future studies will need to focus on correlation of pathway activation status and/or sensitivity to targeted interruption of ALK signaling with underlying genomic status. In addition, in order to perform a proper clinical correlative study to determine the independent prognostic impact of ALK aberration, a large meta-analysis will need to be performed that has a sample size 2-3x larger than the number of cases studied here. This is required because relapse and death is rare in the low- and intermediate-risk group of patients, and because other confounders such as MYCN amplification co-occur. A 1500 subject clinical correlative study is ongoing. A major challenge in the field is to distinguish driver mutations that are responsible for malignant transformation from nonfunctional passenger mutations that may arise in the setting of genomic instability. We provide functional evidence of this concept with experimental validation of four of the most common potential driver mutations, each resulting in constitutive kinase activation of the ALK kinase, but in varying degrees. The two most commonly observed mutations resulted in robust constitutive phosphorylation of the ALK kinase, whereas the rare germline mutation G1128A, which has not been observed somatically, resulted in only weak activation, similar to the amount seen with forced over expression of wild-type protein. Pharmacologic inhibition of ALK kinase activity with PF-02341066, a dual c-Met/ALK inhibitor, demonstrated in vitro cytotoxicity in an ALK genomic status dependant manner. Cytotoxicity correlated with abrogation of constitutively activated ALK phosphorylation. Likewise, oral dosing of PF-02341066 in immunocompromised mice xenotransplanted with human neuroblastoma-derived cell lines resulted in differential anti-tumor activity in a genomic status-dependant manner. Xenografts harboring the R1275Q mutation showing complete regression, while those harboring an F1174L mutation showed significant tumor growth delay (P < 0.0001). Xenografts with no evidence for constitutively phosphorylated ALK showed no anti-tumor activity. Computational homology modeling based upon the crystal structure of PF-02341066 bound to c-Met predicted that F1174L destabilizes the preferred protein conformation for PF-02341066 binding, providing in silico evidence for the mutation-specific differential sensitivity to PF-02341066. Our data clearly demonstrate that cytotoxicity to PF-02341066 is highly associated with ALK genomic status and evidence for constitutive activation. If preclinical models are predictive of success in the ongoing and planned clinical trials, and anti-tumor response is restricted to patients with neuroblastomas that have aberrant ALK signaling, it will be imperative to obtain tumor tissue to identify the patients most likely to benefit from ALK-targeted therapy. Understanding the genetic heterogeneity of neuroblastoma tumors and devising strategies to circumvent their rapid selection for resistance to targeted ALK inhibitors will guide the design of second-generation targeted therapies. In addition, knowledge of resistant mutations should permit the development of assays to detect drug-resistant clones before clinical relapse. Citation Format: Yael P. Mosse. ALK in neuroblastoma [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY14-02