Arin McKinley

Primary and Secondary Kinase Genotypes Correlate With the Biological and Clinical Activity of Sunitinib in Imatinib-Resistant Gastrointestinal Stromal Tumor

PURPOSE Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor alpha (PDGFRA) kinases, which are imatinib targets. Sunitinib, which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in patients with GIST after they experience imatinib failure. We evaluated the impact of primary and secondary kinase genotype on sunitinib activity. PATIENTS AND METHODS Tumor responses were assessed radiologically in a phase I/II trial of sunitinib in 97 patients with metastatic, imatinib-resistant/intolerant GIST. KIT/PDGFRA mutational status was determined for 78 patients by using tumor specimens obtained before and after prior imatinib therapy. Kinase mutants were biochemically profiled for sunitinib and imatinib sensitivity. RESULTS Clinical benefit (partial response or stable disease for > or = 6 months) with sunitinib was observed for the three most common primary GIST genotypes: KIT exon 9 (58%), KIT exon 11 (34%), and wild-type KIT/PDGFRA (56%). Progression-free survival (PFS) was significantly longer for patients with primary KIT exon 9 mutations (P = .0005) or with a wild-type genotype (P = .0356) than for those with KIT exon 11 mutations. The same pattern was observed for overall survival (OS). PFS and OS were longer for patients with secondary KIT exon 13 or 14 mutations (which involve the KIT-adenosine triphosphate binding pocket) than for those with exon 17 or 18 mutations (which involve the KIT activation loop). Biochemical profiling studies confirmed the clinical results. CONCLUSION The clinical activity of sunitinib after imatinib failure is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinases, which has implications for optimization of the treatment of patients with GIST.

Phase II, Open-Label Study Evaluating the Activity of Imatinib in Treating Life-Threatening Malignancies Known to Be Associated with Imatinib-Sensitive Tyrosine Kinases

Purpose: To evaluate the activity of imatinib in treating advanced, life-threatening malignancies expressing one or more imatinib-sensitive tyrosine kinases. Experimental Design: This was a phase II, open-label, single arm study. Patients ≥15 years old with malignancies showing histologic or molecular evidence of expression/activation of imatinib-sensitive tyrosine kinases were enrolled. Patients were treated with 400 or 800 mg/d imatinib for hematologic malignancy and solid tumors, respectively. Treatment was continued until disease progression or unacceptable toxicity. The primary objective was to identify evidence of imatinib activity with tumor response as the primary end point. Results: One hundred eighty-six patients with 40 different malignancies were enrolled (78.5% solid tumors, 21.5% hematologic malignancies). Confirmed response occurred in 8.9% of solid tumor patients (4 complete, 9 partial) and 27.5% of hematologic malignancy patients (8 complete, 3 partial). Notable activity of imatinib was observed in only five tumor types (aggressive fibromatosis, dermatofibrosarcoma protuberans, hypereosinophilic syndrome, myeloproliferative disorders, and systemic mastocytosis). A total of 106 tumors were screened for activating mutations: five KIT mutations and no platelet-derived growth factor receptor mutations were found. One patient with systemic mastocytosis and a partial response to therapy had a novel imatinib-sensitive KIT mutation (D816T). There was no clear relationship between expression or activation of wild-type imatinib-sensitive tyrosine kinases and clinical response. Conclusion: Clinical benefit was largely confined to diseases with known genomic mechanisms of activation of imatinib target kinases. Our results indicate an important role for molecular characterization of tumors to identify patients likely to benefit from imatinib treatment.

Crenolanib Inhibits the Drug-Resistant PDGFRA D842V Mutation Associated with Imatinib-Resistant Gastrointestinal Stromal Tumors

Purpose: To determine the potential of crenolanib, a potent inhibitor of PDGFRA, to treat malignancies driven by mutant PDGFRA. Experimental Design: The biochemical activity of crenolanib was compared with imatinib using a panel of PDGFRA-mutant kinases expressed in several different cell line models, including primary gastrointestinal stromal tumors (GIST) cells. The antiproliferative activity of crenolanib was also studied in several cell lines with PDGFRA-dependent growth. Results: Crenolanib was significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRA kinases (D842I, D842V, D842Y, DI842-843IM, and deletion I843). For example, crenolanib was 135-fold more potent than imatinib against D842V in our isogenic model system, with an IC50 of approximately 10 nmol/L. The relative potency of crenolanib was further confirmed in BaF3 and primary GIST cells expressing PDGFRA D842V. In contrast, imatinib was at least 10-fold more potent than crenolanib in inhibiting the V561D mutation. For all other tested PDGFRA mutations, crenolanib and imatinib had comparable potency. Conclusions: Crenolanib is a potent inhibitor of imatinib-resistant PDGFRA kinases associated with GIST, including the PDGFRA D842V mutation found in approximately 5% of GISTs. The spectrum of activity of crenolanib suggests that this drug is a type I inhibitor (inhibitor of activated conformation of kinase). Based in part on these results, a phase II clinical study of this agent to treat GIST with the PDGFRA D842V mutation has been initiated. Clin Cancer Res; 18(16); 4375–84. ©2012 AACR.

Multiplex Mutation Screening by Mass Spectrometry: Evaluation of 820 Cases from a Personalized Cancer Medicine Registry

There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.

Sorafenib Inhibits Many Kinase Mutations Associated with Drug-Resistant Gastrointestinal Stromal Tumors

Sorafenib has substantial clinical activity as third- or fourth-line treatment of imatinib- and sunitinib-resistant gastrointestinal stromal tumors (GIST). Because sorafenib targets both angiogenesis-related kinases (VEGFR) and the pathogenetic kinases found in GIST (KIT or PDGFRA), the molecular basis for sorafenib efficacy in this setting remains unknown. We sought to determine the spectrum of activity of sorafenib against different mutant kinases associated with drug-sensitive and drug-resistant GIST. We compared the activity of imatinib and sorafenib against transiently expressed mutant forms of KIT and PDGFRA, including various secondary mutations that have been identified in imatinib-resistant or sunitinib-resistant GISTs. We also examined these drugs against four GIST cell lines, three of which are imatinib resistant. In our in vitro studies, we determined that sorafenib inhibited imatinib-resistant mutations in exons encoding the ATP/drug-binding pocket and in exons encoding the activation loop, with the exception of substitutions at KIT codon D816 and PDGFRA codon 842. Notably our data indicate that sorafenib is more effective than imatinib or sunitinib for inhibiting the kinase activity of drug-resistant KIT mutants (as assessed by biochemical IC50). We hypothesize that a major determinant of the efficacy of sorafenib for treatment of advanced GIST is the activity of this agent against KIT or PDGFRA-mutant kinases. These results have implications for the further development of treatments for drug-resistant GIST. Mol Cancer Ther; 11(8); 1770–80. ©2012 AACR.

Abstract 3586: CP-868,596, a highly potent PDGFR inhibitor, inhibits phosphorylation of the imatinib-resistant PDGFRA D842V activating mutation associated with advanced GIST

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: 8-10% of gastrointestinal stromal tumors (GIST) have activating mutations of the platelet-derived growth factor receptor alpha (PDGFRA) kinase. The most common PDGFRA mutation is the D842V substitution that is encoded by exon 18. This gain-of-function mutation results in auto-phosphorylation and constitutive activation of PDGFRA kinase activity. Type II receptor tyrosine kinase (RTK) inhibitors, such as imatinib and sunitinib, which only bind to the inactive conformation of the RTK, have little or no in vitro activity against this mutation. Clinically, these drugs are not effective for the treatment of GIST patients with D842V mutation. In addition, the D842V mutation can develop as a secondary imatinib resistant mutation during treatment of GISTs with primary imatinib-sensitive PDGFRA mutations (e.g. primary exon 12 mutations), or treatment of patients with hypereosinophillic syndrome with translocations involving PDGFRA. CP-868,596 is an orally bioavailable, highly potent and selective PDGFR TKI. CP-868,596 is a benzimidazole compound that has IC50s of 0.9 nM and 1.8 nM for PDGFRA and PDGFRB, respectively. Phase I trials of CP-868,596 have shown good oral bioavailability, a favorable toxicity profile, and achievable serum concentrations as high as 2000 nM. Methods: Mutant PDGFRA isoforms were expressed by transient transfection of Chinese Hamster ovary cells. The transfected cells were treated with various concentrations of CP-868,596 before preparation of protein lystates. PDGFRA protein was assayed for activation status (phosphorylation) by immunoprecipitation using an anti-PDGFRA antibody, followed by sequential immunoblotting for phospho PDGFRA (using antiphosphotyrosine antibody) or total PDGFRA (anti-PDGFRA monoclonal antibody). IC50 was measured by densitometry of the phospho PDGFRA bands and normalization using total PDGFRA expression (to correct for variations in loading of PDGFRA protein in the various lanes). Results: CP-868,596 inhibited the phosphorylation of wild type PDGFRA at an IC50 of 10 nM and PDGFRA D842V with an IC50 between 10 to 30 nM. Imatinib was ineffective in blocking PDGFRA D842V phosphorylation in these experiments (IC50 > 1000 nM). Profiling of CP-868,596 against other GIST-relevant primary and secondary PDGFRA mutations is ongoing and will be reported. Discussion: GISTs due to D842V activating mutations of PDGFRA gene are clinically resistant to imatinib, sunitinib, and other commercially available tyrosine kinase inhibitors. CP-868,596 blocks phosphorylation of the PDGFRA D842V mutant at clinically achievable concentrations, providing a potentially new therapeutic modality for GIST patients. A clinical trial in GIST patients with primary or secondary PDGFRA D842V mutations is being initiated. 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 3586. doi:10.1158/1538-7445.AM2011-3586