Anaplastic Lymphoma Kinase Aberrations Research Articles

Anaplastic Lymphoma Kinase Aberrations in Rhabdomyosarcoma: Clinical and Prognostic Implications

The aim of this study is to investigate anaplastic lymphoma kinase (ALK) protein expression and underlying genetic aberrations in rhabdomyosarcoma (RMS), with special attention to clinical and prognostic implications. A total of 189 paraffin-embedded RMS tumor specimens from 145 patients were collected on tissue microarray. ALK protein expression was evaluated by immunohistochemistry. ALK gene (2p23) copy number and translocations were determined by in situ hybridization. cDNA sequencing of the receptor tyrosine kinase domain of the ALK gene was assessed in 43 samples. Strong cytoplasmic ALK protein expression was more frequently observed in alveolar RMS (ARMS) than in embryonal RMS (ERMS) (81% v 32%, respectively; P < .001). ALK gene copy number gain was detected in the vast majority of ARMS (88%), compared with 52% of ERMS (P < .001). ALK copy number correlated with protein expression in primary tumors (n = 107). We identified one point mutation (2%) and seven tumors harboring whole exon deletions (16%). In ERMS, specific ALK gain in the primary tumor correlated with metastatic disease (100% in metastatic disease v 29% in nonmetastatic disease; P = .004) and poor disease-specific survival (5-year disease-specific survival: 62% v 82% for nonspecific or no gain; P = .046). Because ALK aberrations on genomic and protein levels are frequently found in RMSs, in particular ARMS, and are associated with disease progression and outcome in ERMS, ALK may play a role in tumor biology and may provide a potential therapeutic target for these tumors. Future research should aim at the oncogenic role of ALK and the potential effect of ALK inhibitors in RMS.

Abstract 742: Mechanisms of resistance to small molecule inhibition of anaplastic lymphoma kinase in human neuroblastoma

Abstract The purpose of this study was to identify therapeutically targetable mechanisms of resistance to anaplastic lymphoma kinase (ALK) inhibition in neuroblastoma. ALK is a receptor tyrosine kinase (RTK) frequently activated in neuroblastoma, a malignancy of the autonomic nervous system representing almost 10% of pediatric cancers. ALK aberrations, including mutation and amplification, lead to increased autophosphorylation and activation in neuroblastoma. PF-02341066, an orally available small-molecule inhibitor of the ALK tyrosine kinase, has shown dramatic activity in non-small lung cancers harboring an ALK translocation, and is currently in phase 1 trials in pediatrics. Although pharmacological inhibition of kinases in diseases such as chronic myelogenous leukemia has significantly improved survival, resistance inevitably develops. Mechanisms of resistance to tyrosine kinase inhibitors have included mutation of the targeted receptor as well as up-regulation and dependence switch to other RTKs. We hypothesize that patients with neuroblastoma who successfully respond to ALK inhibition will eventually relapse and that in vitro models can be used to predict the underlying mechanisms of resistance. To mimic the development of resistance, we treated ALK-expressing human neuroblastoma-derived cell lines in vitro with PF-02341066 for several months. Once resistance was established, serial dilutions were used to generate clonal populations of PF-02341066-resistant cells. Western blotting was used to confirm inhibition of phospho-ALK and rule out drug efflux as a resistance mechanism. DNA analysis revealed no additional PF-02341066-induced mutations or copy number variations within ALK. We next used Western blot and phoshpho-array technology to evaluate changes in the activation state of a panel of RTKs as well as their downstream signaling molecules. Interestingly, in comparison to the untreated parental line, PF-02341066-resistant cells had up-regulated activated epidermal growth factor receptor (EGFR) as well as other downstream signaling molecules. Dependence of the PF-02341066-resistant cells on these pathways was verified by showing increased sensitivity of the resistant versus parental cell lines to inhibition of these up-regulated pathways. Taken together, these data suggest that ALK inhibition in neuroblastoma leads to a dependence switch to EGFR and implies the need to develop therapeutic strategies to target this receptor when ALK inhibition is used clinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 742. doi:10.1158/1538-7445.AM2011-742

Abstract 4563: Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma

Abstract The purpose of this study was to provide the first pre-clinical evidence for antibody targeting of anaplastic lymphoma kinase (ALK) in human neuroblastoma. ALK is a receptor tyrosine kinase expressed by the majority of neuroblastoma tumors. Germline mutations of ALK, shown to account for most cases of familial neuroblastoma, as well as somatically acquired ALK aberrations, induce increased auto-phosphorylation and constitutive ALK activation and increased downstream signaling. Antibody targeting of receptor tyrosine kinases, as monotherapy or together with small-molecule inhibitors, is highly effective in other cancers such as breast and lung cancer. While antibody-mediated immunotherapy targeting of disialoganglioside GD2 has recently been reported to significantly increase 2-year survival in high-risk neuroblastoma patients, over half the patients receiving this therapy ultimately succumb to their disease and therapeutic alternatives are urgently needed to improve survival rates of this devastating pediatric cancer. Therefore, we hypothesized that antibody targeting of ALK in neuroblastoma was a therapeutically appropriate strategy. To first confirm the potential of anti-ALK antibody-mediated immunotherapy, we used in vitro assays to demonstrate enhanced immune-cell induced cytotoxicity of antibody-treated human neuroblastoma-derived cell lines. We next showed that in vitro antibody treatment of neuroblastoma cell lines expressing activated ALK led to growth inhibition and cell death. These effects were enhanced by treatment with PF-02341066, an orally available small-molecule inhibitor of the ALK tyrosine kinase. To identify the mechanism behind this enhanced combined effect, we used flow cytometry to show that PF-02341066 sensitizes cells to antibody treatment by inducing accumulation of cell-surface ALK, thus increasing the accessibility of antigen for antibody binding. Finally, to further predict in vivo cytotoxic mechanisms of dual ALK targeting, we used flow cytometry to demonstrate enhanced apoptosis and proliferation inhibition resulting from combined antibody and inhibitor treatment as compared to either drug alone. Taken together, these findings provide strong evidence for the therapeutic relevance of antibody targeting of ALK in neuroblastoma and argue for the rapid development and further testing of a clinical grade anti-ALK antibody. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2011-4563

Inhibition of ALK Signaling for Cancer Therapy

Paradigm shifting advances in cancer can occur after discovering the key oncogenic drivers of the malignant process, understanding their detailed molecular mechanisms, and exploiting this transdisciplinary knowledge therapeutically. A variety of human malignancies have anaplastic lymphoma kinase (ALK) translocations, amplifications, or oncogenic mutations, including anaplastic large cell lymphoma, inflammatory myofibroblastic tumors, non-small cell lung cancer, and neuroblastoma. This finding has focused intense interest in inhibiting ALK signaling as an effective molecular therapy against diseases with ALK-driven pathways. Recent progress in the elucidation of the major canonical signaling pathways postulated to be activated by NPM-ALK signaling has provided insight into which pathways may present a rational therapeutic approach. The identification of the downstream effector pathways controlled by ALK should pave the way for the rational design of ALK-inhibition therapies for the treatment of a subset of human cancers that harbor ALK aberrations.