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353 Protein expression for receptor activator of NFkB (RANK) and its ligand (RANKL) in non-small cell lung cancer (NSCLC)
114 Thursday 20 November 2014 352 POSTER UNC2025, a novel small molecule MerTK and Flt3 tyrosine kinase inhibitor, has therapeutic activity and promotes sensitivity to chemotherapy in animal models of acute leukemia D. Graham1 , D. DeRyckere1 , X. Wang2 , A.A. Hill1 , W. Zhang2 , S.V. Frye3 , H.S. Earp4 . 1 University of Colorado Denver, Department of Pediatrics, Aurora CO, USA; 2 University of North Carolina, Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill NC, USA; 3 University of North Carolina, Center for Integrative Chemical Biology and Drug Discovery and Department of Medicine, Chapel Hill NC, USA; 4 University of North Carolina, Lineberger Comprehensive Cancer Center and Department of Medicine, Chapel Hill NC, USA Background: These studies investigate the potential utility of UNC2025, a dual MerTK and Fms-like tyrosine kinase 3 (Flt3) inhibitor, as a therapy for acute myeloid and acute lymphoid leukemias (AML and ALL). Methods: Inhibition of MerTK and Flt3 phosphorylation was measured using enzymatic and cell-based assays. Induction of apoptosis was determined using flow cytometry and colony-forming potential was assessed in methylcellulose cultures. Orthotopic B-ALL xenografts were generated in NOD/Scid/IL-2 gamma mice using a luciferase-expressing derivative of the 697 cell line. Inhibition of MerTK in bone marrow leukemia cells was determined by immunoblot. Disease burden was assessed using bioluminescence imaging and median survival was determined. Orthotopic patient-derived xenografts (PDX) were generated from primary AML samples and blasts were detected using flow cytometry. Results: UNC2025 inhibited MerTK and Flt3 in enzymatic (Ki = 0.16 and 0.59 nM, respectively) and cell-based (IC50 = 2.7 and 14 nM, respectively) assays and had limited off-target activity against 300 other kinases. Treatment with 100–200 nM UNC2025 induced apoptosis in 40−90% of cells and reduced colony-forming potential by 80–100% in leukemia cell cultures. In animal models, UNC2025 is orally bioavailable and inhibited MerTK phosphorylation in bone marrow leukemic blasts. Treatment with UNC2025 resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 to 70 days after tumor implantation in a B-ALL xenograft model of minimal residual disease (p < 0.0001) and from 27.5 to 45 days when treatment was initiated after establishment of more substantial disease (p < 0.0001). Similarly, in a MerTK-expressing AML PDX model short-term treatment with UNC2025 resulted in a significant decrease in peripheral disease burden (9.3% blasts versus 35.6% for vehicle-treated animals, p = 0.0225) and both the fraction of splenic blasts (28.5 versus 75.7%, p = 0.0005) and spleen mass (170 versus 834 mg, p < 0.0001) were dramatically decreased. UNC2025 was even more effective in a Flt3-ITD mutant, MerTK-expressing AML PDX model, where treatment resulted in disease regression (0.24% peripheral blasts versus 11.4% pre-treatment, p = 0.0094) and prolonged median survival from 16 to >107 days post-treatment (p = 0.0114). In addition, in the B-ALL model treatment with UNC2025 and methotrexate, a chemotherapy currently in clinical use for treatment of ALL, resulted in reduced tumor burden compared to either agent alone and enabled tumor-free survival during an 88 day treatment window. Conclusions: The very high potency, relative selectivity, oral bioavailability, and target inhibition and therapeutic efficacy mediated by UNC2025 in murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of patients with acute leukemia. 353 POSTER Protein expression for receptor activator of NFkB (RANK) and its ligand (RANKL) in non-small cell lung cancer (NSCLC) M. D’Arcangelo1 , S. Ekman2 , W. Dougall3 , D. Branstetter4 , M. Bergqvist2 , P. Liv2 , D. Chan5 , J. Botling6 , F. Hirsch7 . 1 University of Colorado, Division of Medical Oncology, Denver, USA; 2 University of Uppsala, Department of Radiology, Uppsala, Sweden; 3 Amgen Inc., Therapeutic Innovation Unit, Thousand Oaks, USA; 4 Amgen Inc., Pathology, Thousand Oaks, USA; 5 University of Uppsala, Department of Medical Oncology, Denver, USA; 6 University of Uppsala, Department of Immunology, Uppsala, Sweden; 7 University of Colorado, Department of Medical Oncology, Colorado, USA Background: The activation of RANK by its ligand is crucial for bone metastasis in several cancers, including lung tumors. The fully human antiRANKL antibody denosumab reduces the risk of skeletal events. Evidence suggests that denosumab therapy may significantly prolong survival of lung cancer patients with bone metastases versus zoledronic acid. A direct anti-tumor effect of denosumab on lung cancer cells cannot be excluded. Therefore, we investigated RANK/RANKL expression and its prognostic significance in a large cohort of NSCLC patients.
Poster Session – Molecular Targeted Agents I Methods: Immunohistochemistry using the mAbs against RANKL (M366, Amgen) or RANK (N1H8, Amgen) was performed on a tissue microarray including 364 treatment na¨ıve resected NSCLC samples collected at University of Uppsala Sweden (median follow up 7.8 years). Evaluation of EGFR and KRAS mutations were performed with Therascreen EGFR RGQ PCR kit (Qiagen) and ALK overexpression by IHC (D5F3, Cell Signaling). Results were obtained for 352 samples. Median age was 67 years and males were 54%. Pathological stages: I 65%, II 15%, III 17%, IV 3%. Histology: 196 adenocarcinomas, 120 squamous cell carcinomas, 41 large cell carcinomas. A certified pathologist scored the protein expression using H-score, which considers staining intensity and percentage of stained tumor cells (range 0–300). Results: RANK (H-score >5) and RANKL (H-score >10) were detected in 106 (30%) and in 181 (52%) tumors, respectively. Concomitant expression of both proteins was observed in 65 (19%) samples. No correlation was observed between RANK/RANKL expression and histology, gender, smoking status, stage and EGFR mutations. RANK and RANKL positivity were associated with presence of KRAS mutation (p = 0.003 and p<.001, respectively). ALK positive tumors more often expressed RANKL (p = 0.005), while RANK expression seemed not to be affected. RANK and RANKL expression had no prognostic significance in this cohort of patients. Conclusions: RANK and RANKL are expressed at the protein level in a considerable number of early stage NSCLC patients, especially in KRAS mutated and ALK positive tumors, but do not affect survival. These data are focused on the tumor and not on RANKL/RANK expression in normal lung and infiltrating immune cells. These findings support and warrant further investigation on RANKL targeting in NSCLC. 354 POSTER PCR-based assay for BRAFV600 mutation analysis in ctDNA: clinical results from plasma and serum samples M. Gonzalez-Cao1 , C. Mayo de las Casas1 , M.A. Molina-Vila1 , ˜ 2 , J. Cortes2 , L. De Mattos-Arruda2 , J.L. Manzano3 , E. Munoz J.P. Berros4 , M. Sanmamed5 , A. Gonzalez5 , C. Alvarez4 , N. Karachaliou1 , N. Jordana-Ariza1 , S. Martin Algarra5 , R. Rosell1 . 1 Institut Universitari Dexeus Quiron, Instituto Oncologico Dr Rosell, Barcelona, Spain; 2 Hospital Vall d’Hebron, Medical Oncology, Barcelona, Spain; 3 Catalan Institute of Oncology Hospital Germans Trias i Pujol, Medical Oncology, Badalona, Spain; 4 Hospital Central Asturias, Medical Oncology, Oviedo, Spain; 5 Clinica Universitaria de Navarra, Medical Oncology, Pamplona, Spain Background: Determination of BRAFV600 mutation in cell free tumor DNA (ctDNA) in serum (s) or plasma (p) has been studied as a surrogate for tumor tissue biopsy. Our aim was to develop a PCR-based assay to analyze BRAFV600 mutation in s and p, compare results in both samples and to clinically validate ctDNA as a minimally invasive tool. Material and Methods: A quantitative 5 -nuclease PCR-based assay for determination of BRAFV600 mutation in ctDNA was developed and validated in 92 patients previously genotyped in tissue. We compare results from serum and plasma in every patient. Samples along BRAF inhibitor treatment were available from 4 melanoma patients. Results: The assay has a limit detection of 2.5 pg mutated DNA/mL and a ratio of V600 versus wild-type allele of 1:20,000. The assay had a clinical sensitivity of 57.7% and a specificity of 100%. Among patients with the BRAFV600 in tumor tissue (n = 52), 30 (57.7%) had BRAFV600E mutation in ctDNA (s or p), 28 (53.8%) in p (p = 0.05) and 23 (44%) in s (p = 0.016). Four melanoma patients were subjected to longitudinal analysis of BRAFV600 in ctDNA over time. Three out of 4 patients had BRAFV600 detectable in s/p at baseline. Patient 1 presented partial response; the absolute concentration of BRAFV600E increased 48 hours after starting BRAF inhibitor treatment, markedly decreased after 7 days and was not detectable after 30 days. The evolution of the BRAFV600E ratio was parallel in this patient, but remained around 40% in plasma and 10% in serum at 48 hours, to drop afterwards. Patient 2 showed complete response, BRAFV600 was detectable in serum/plasma pretreatment but not at the time of analysis performed after 30 days on treatment, at 240 days of follow-up complete response is ongoing and BRAFV600 is not detectable in s/p. Patient 3 showed progressive disease as best response, he was positive for BRAFV600 in pre-treatment plasma but negative in serum. After two months on BRAF inhibitor treatment, an increase in BRAFV600 quantification was observed both in terms of absolute concentration and also according to mutation load both in plasma and serum. Progressive disease was evident in this patient with rapid evolution and death two weeks after diagnosis of disease progression. Conclusion: Our assay has shown a better sensitivity to capture BRAFV600 mutation in ctDNA from p than from serum. Combination of s and p samples improves likehood ratio of positive test. cfDNA may be a potential minimally invasive alternative tool to help with diagnosis and monitoring of BRAFV600 mutant tumors.
Poster Session – Molecular Targeted Agents I Methods: Immunohistochemistry using the mAbs against RANKL (M366, Amgen) or RANK (N1H8, Amgen) was performed on a tissue microarray including 364 treatment na¨ıve resected NSCLC samples collected at University of Uppsala Sweden (median follow up 7.8 years). Evaluation of EGFR and KRAS mutations were performed with Therascreen EGFR RGQ PCR kit (Qiagen) and ALK overexpression by IHC (D5F3, Cell Signaling). Results were obtained for 352 samples. Median age was 67 years and males were 54%. Pathological stages: I 65%, II 15%, III 17%, IV 3%. Histology: 196 adenocarcinomas, 120 squamous cell carcinomas, 41 large cell carcinomas. A certified pathologist scored the protein expression using H-score, which considers staining intensity and percentage of stained tumor cells (range 0–300). Results: RANK (H-score >5) and RANKL (H-score >10) were detected in 106 (30%) and in 181 (52%) tumors, respectively. Concomitant expression of both proteins was observed in 65 (19%) samples. No correlation was observed between RANK/RANKL expression and histology, gender, smoking status, stage and EGFR mutations. RANK and RANKL positivity were associated with presence of KRAS mutation (p = 0.003 and p<.001, respectively). ALK positive tumors more often expressed RANKL (p = 0.005), while RANK expression seemed not to be affected. RANK and RANKL expression had no prognostic significance in this cohort of patients. Conclusions: RANK and RANKL are expressed at the protein level in a considerable number of early stage NSCLC patients, especially in KRAS mutated and ALK positive tumors, but do not affect survival. These data are focused on the tumor and not on RANKL/RANK expression in normal lung and infiltrating immune cells. These findings support and warrant further investigation on RANKL targeting in NSCLC. 354 POSTER PCR-based assay for BRAFV600 mutation analysis in ctDNA: clinical results from plasma and serum samples M. Gonzalez-Cao1 , C. Mayo de las Casas1 , M.A. Molina-Vila1 , ˜ 2 , J. Cortes2 , L. De Mattos-Arruda2 , J.L. Manzano3 , E. Munoz J.P. Berros4 , M. Sanmamed5 , A. Gonzalez5 , C. Alvarez4 , N. Karachaliou1 , N. Jordana-Ariza1 , S. Martin Algarra5 , R. Rosell1 . 1 Institut Universitari Dexeus Quiron, Instituto Oncologico Dr Rosell, Barcelona, Spain; 2 Hospital Vall d’Hebron, Medical Oncology, Barcelona, Spain; 3 Catalan Institute of Oncology Hospital Germans Trias i Pujol, Medical Oncology, Badalona, Spain; 4 Hospital Central Asturias, Medical Oncology, Oviedo, Spain; 5 Clinica Universitaria de Navarra, Medical Oncology, Pamplona, Spain Background: Determination of BRAFV600 mutation in cell free tumor DNA (ctDNA) in serum (s) or plasma (p) has been studied as a surrogate for tumor tissue biopsy. Our aim was to develop a PCR-based assay to analyze BRAFV600 mutation in s and p, compare results in both samples and to clinically validate ctDNA as a minimally invasive tool. Material and Methods: A quantitative 5 -nuclease PCR-based assay for determination of BRAFV600 mutation in ctDNA was developed and validated in 92 patients previously genotyped in tissue. We compare results from serum and plasma in every patient. Samples along BRAF inhibitor treatment were available from 4 melanoma patients. Results: The assay has a limit detection of 2.5 pg mutated DNA/mL and a ratio of V600 versus wild-type allele of 1:20,000. The assay had a clinical sensitivity of 57.7% and a specificity of 100%. Among patients with the BRAFV600 in tumor tissue (n = 52), 30 (57.7%) had BRAFV600E mutation in ctDNA (s or p), 28 (53.8%) in p (p = 0.05) and 23 (44%) in s (p = 0.016). Four melanoma patients were subjected to longitudinal analysis of BRAFV600 in ctDNA over time. Three out of 4 patients had BRAFV600 detectable in s/p at baseline. Patient 1 presented partial response; the absolute concentration of BRAFV600E increased 48 hours after starting BRAF inhibitor treatment, markedly decreased after 7 days and was not detectable after 30 days. The evolution of the BRAFV600E ratio was parallel in this patient, but remained around 40% in plasma and 10% in serum at 48 hours, to drop afterwards. Patient 2 showed complete response, BRAFV600 was detectable in serum/plasma pretreatment but not at the time of analysis performed after 30 days on treatment, at 240 days of follow-up complete response is ongoing and BRAFV600 is not detectable in s/p. Patient 3 showed progressive disease as best response, he was positive for BRAFV600 in pre-treatment plasma but negative in serum. After two months on BRAF inhibitor treatment, an increase in BRAFV600 quantification was observed both in terms of absolute concentration and also according to mutation load both in plasma and serum. Progressive disease was evident in this patient with rapid evolution and death two weeks after diagnosis of disease progression. Conclusion: Our assay has shown a better sensitivity to capture BRAFV600 mutation in ctDNA from p than from serum. Combination of s and p samples improves likehood ratio of positive test. cfDNA may be a potential minimally invasive alternative tool to help with diagnosis and monitoring of BRAFV600 mutant tumors.