- Email: [email protected]
450 Non-invasive Absolute Quantification of EGFR Activating Mutation L858R and Gatekeeper Mutation T790M in NSCLC Plasma Samples Using Droplet Digital PCR
Poster Session – Biomarkers Material and Methods: Archival ovarian carcinomas from 239 patients treated with taxane-platinum chemotherapy were examined immunohistochemically with an anti-14-3-3s antibody. Cytoplasmic staining was evaluated semiquantitatively, scored from 0 (absent) to 3 (strong) and divided into two groups: tumors with low expression (0−1) and high expression (2−3). Univariate and multivariate statistical analyses (Cox proportional hazards model and logistic regression model) were performed to evaluate response to chemotherapy, disease-free survival and overall survival. The analyses were made on the entire group, as well as on subgroups of tumors with (TP53+) and without TP53 accumulation (TP53−). Results: High level of 14-3-3s protein expression was observed in 160 (67%) tumors and was correlated with enhanced probability of complete remission (p = 0.020, OR 2.96, 95% CI 1.19–7.40 − univariate analysis; p = 0.019, OR 3.38, 95% CI 1.22–9.34 − multivariate analysis). This results were observed in the TP53(−) subgroup only. No association was found between 14-3-3s expression and clinicopathological variables. Conclusions: Our results suggest that 14-3-3s protein expression is a good predictor of ovarian cancer response to taxane-platinium chemotherapy in a group of tumors with functional TP53. 448 POSTER Activation of a Specific Metabolic Pathway May Distinguish Aggressive From Indolent Prostate Cancer W. Huang1 , P.A. Young2 , K.W. Eliceiri2 , G. Wilding3 , H.S. Basu3 . 1 University of Wisconsin, Department of Pathology, Madison, USA; 2 University of Wisconsin, Laboratory for Optical and Computational Instrumentation, Madison, USA; 3 University of Wisconsin, Carbone Cancer Center, Madison, USA Background: Persistent increase in serum prostate specific antigen (PSA) levels is an accepted indicator of prostate cancer (PCa). Other prostatic diseases may also elevate serum PSA levels. Thus, it has less than 30% specificity for PCa. Also, a widespread PSA screening is leading to the diagnosis of many low grade and small PCas. Some of these PCas may progress and become lethal, but many of them may remain indolent for the rest of the patients’ lives. There is no approved biomarker that can distinguish the aggressive PCas from the indolent ones. There is strong evidence that reactive oxygen species (ROS) play a key role in the recurrence of androgen-dependent PCa (ADPC) and its progression to often lethal castrate-resistant PCa (CRPC) [1]. It has been shown that androgen induces an increase in ROS levels in cultured ADPC cells via a yet unknown mechanism [2]. We have demonstrated that in certain human PCa cells, androgen causes a marked overexpression and consequent increase in the enzymatic activity of spermidine/spermine acetyl transferase (SSAT) [3]. SSAT converts the polyamines spermidine and spermine to their corresponding acetyl derivatives. An oxidation of acetyl-polyamines by FAD-bound acetyl polyamine oxidase (APAO) is a major contributor to the androgen-induced ROS production in polyaminerich PCa cells. Cellular ROS can set-up an autocrine feed-forward loop of SSAT-ROS-NF-kB-SSAT. This loop keeps on producing ROS activating AR in the absence of androgen and thus helps progression of ADPC to CRPC. Methods: We used multiphoton fluorescence microscopy to determine FAD fluorescence intensity and lifetime in human prostate biopsies containing both PCas and normal prostate tissues. We also used RNA-in situ hybridization (RNA-ISH) study of SSAT gene expression in two human prostate tissue microarrays containing 384 and 462 samples. Results: There is a marked increase in FAD fluorescence intensity and decrease in the ratio of protein-bound FAD to free FAD in PCas as compared to normal epithelia. This suggests increased activity of an FADbound enzyme that recycles FADH2 to increase the cellular free FAD levels. The data showed a marked increase in SSAT expression in PCas as compared to that in the normal prostatic epithelia (P < 0.001). More interestingly, an even higher SSAT expression in metastatic PCas relative to localized PCas (P = 0.014) was also observed. Conclusion: Multiphoton microscopy to determine FAD fluorescence and SSAT gene expression may be employed clinically for PCa prognosis. References [1] Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK. Cancer research, 2008; 68(6): 1777−85. [2] Lamont, K.R. and D.J. Tindall, Advances in cancer research, 2010; 107: 137−62. [3] Basu HS, Thompson TA, Church DR, Clower CC, Mehraein-Ghomi F, Amlong CA, Martin CT, Woster PM, Lindstrom MJ, Wilding G. Cancer Res. 2009; 69(19): 7689−95.
Friday 9 November 2012 139 449 POSTER Molecular and Therapeutic Characterization of 25 Patient Derived Lung Cancer Tumorgrafts and Analysis for Response Markers to Targeted Therapies as Part of a Systems Biological Approach (PREDICT) 3 ¨ , J. Rolff1 , M. Becker1 , R. Yildirimman2 , M. Isau2 , H. Hulsmann R. Kuner3 , O. Politz4 , J. Merk5 , R. Herwig2 , I. Fichtner1 . 1 Experimental Pharmacology & Oncology Berlin-Buch GmbH, EPO, Berlin, Germany; 2 Max-Planck-Istitute for Molecular Genetics (MPI), Vertebrate Genomics, Berlin, Germany; 3 German Cancer Research Center (DKFZ), Molecular Genetics, Heidelberg, Germany; 4 Bayer AG, Therapeutic Research Group Oncology, Berlin, Germany; 5 ELK Berlin Chest Hospital, Chest Surgery, Berlin, Germany
Current treatment of cancer is seriously hampered by the fact that most therapies are not well adapted to the individual responses of tumors which are strongly determined by the individual cellular and genetic background. There is a high molecular variability even among tumors of the same classification. Each tumor is individual and every patient will respond differently to a particular treatment. Therefore, prediction of therapy response by molecular markers is a prerequisite for individualized approaches to cancer treatment. Tumorgrafts derived from patient’s cancer specimen immediately after surgery provide a preclinical research tool considering both heterogeneity and individuality of malignancies. Tumorgrafts allow to test novel anti-tumor agents in a fast and standardized manner and provide sufficient tissue material, even post-treatment, for the search of corresponding predictive biomarkers. For this study we used a panel of 25 stably passagable lung cancer tissuegrafts. These permanent tumor models feature a high coincidence with the original patient tumor regarding histology and genome-wide gene expression profiling. They were subjected to an extensive molecular (genome-scale) and pharmacological characterization. The drug testing resulted in the following response rates (T/C 35%) for single drugs: carboplatin 56%, gemcitabine 56%, etoposide 12%, taxol 80%, erlotinib 20%, and cetuximab 40%. For example, detailed bioinformatic analysis revealed that the response effect of the chimeric EGFR inhibitor cetuximab was correlated to the regulation of the AMPK and mTOR signalling pathways. This finding was underlined by exome sequencing and protein expression (RPPA) analysis performed in parallel. Our investigations are part of a broadly based systems biological project aiming at the individualization of lung cancer therapy through comprehensive genome-scale characterization of preclinical in vitro and in vivo models. The collected data are integrated in a generic computer model which is usable for the prediction of individual therapy success of targeted anti-cancer drugs. 450 POSTER Non-invasive Absolute Quantification of EGFR Activating Mutation L858R and Gatekeeper Mutation T790M in NSCLC Plasma Samples Using Droplet Digital PCR J. Kristof1 , S. Sankar2 , E. Bruening1 , S. Wong1 . 1 MolecularMD, Research and Development, Portland OR, USA; 2 MolecularMD, Scientific Affairs Liaison, Portland OR, USA Background: A high proportion of non-small cell lung cancer (NSCLC) patients treated with erlotinib or gefitinib develop resistance via emergence of the gatekeeper epidermal growth factor receptor (EGFR) T790M mutation. The level and amount of the EGFR T790M resistance mutant in relation to EGFR L858R activating mutation as opposed to simply the presence or absence of the mutations has been shown to be critical for successful therapy selection in a mouse EGFR mutant lung cancer model. Effective treatment of these patients will likely require both high-sensitivity and quantitative detection of EGFR activating and resistance mutations in liquid biopsies, not obtainable with current platforms. We have developed and optimized a droplet digital qPCR assay that enables quantification of both EGFR activating L858R and resistance T790M mutations in circulating nucleic acids isolated from plasma. Methods: The ddPCR assay was developed using the Biorad QX100 platform. Primers and probes were designed to optimize quantification of EGFR L858R and T790M mutations. The Qiagen EGFR Rotor-Gene (RGQ) assay was used according to manufacturer’s protocol. Genomic DNA from the H1975 cell line expressing both EGFR L858R and T790M mutation was diluted into non-amplifiable nucleic acid to determine the assay’s limit of detection (LOD). Patient samples enriched for the likelihood of EGFR T790M mutation were analyzed as well. Results: The ddPCR assay was designed to detect total EGFR in addition to EGFR mutations. Near the critical low level detection range of less than 1000 copies expected in plasma samples, the assay displayed a linear
140 Friday 9 November 2012 dynamic range from 550 copies to a LOD of 5 copies for EGFR T790M. In comparison, the Qiagen EGFR Rotor-Gene (RGQ) assay displayed an average LOD of 55 copies with a dynamic range of 550 copies to 55 copies. In NSCLC patient plasma samples enriched for the likelihood of EGFR mutations, samples that expressed greater than 300 copies were confirmed by the Qiagen EGFR RGQ assay. In addition to quantifying mutant copies, the ddPCR assay determines the ratio of EGFR mutant to total EGFR template within a sample. In the current set of patient plasma samples analyzed we were able to accurately quantify the ratio of T790M mutant compared to total EGFR from as low as 2 ng without pre-amplification. Data on plasma samples measuring the ratio of T790M versus activating mutations L858R will be presented. The assay additionally detected the Q787Q SNP in patient samples that is predicted to have worse prognosis. Conclusion: Droplet digital PCR provides sensitive quantification of low abundance EGFR T790M resistance and L858R activating mutations in plasma without the need for pre-amplification. This assay provides a useful method for selecting treatment, monitoring disease progression, and providing early detection of treatment failure associated with EGFR acquired resistance. 451 POSTER Two Insulin Receptor Isoforms Confer Intrinsic Resistance to the Antibody Against Insulin-like Growth Factor Receptor I A. Forest1 , M. Amatulli1 , C. Damoci1 , D. Ludwig1 , N. Baltes2 , H. Fiebig2 , P. Houghton3 , M. Smith4 , L. Benjamin1 , R. Novosyadlyy1 . 1 ImClone Systems a wholly-owned subsidiary of Eli Lilly and Company, NYC, USA; 2 Oncotest, Freiburg, Germany; 3 Nationwide Children’s Hospital, Columbus, USA; 4 Cancer Therapy Evaluation Program NCI, Bethesda, USA The role of IGF-IR in tumor development and progression has been shown in numerous preclinical studies. Many inhibitors of IGF-IR are currently in clinical development; their antitumor efficacy, however, is limited only to a subset of patients. It remains unclear what molecular markers predict the antitumor activity of these agents. It has been shown previously that insulin receptor may confer resistance to IGF-IR targeting in experimental tumor models. One of the IGF-IR ligands, IGF-II, is capable of activating insulin receptor (IR), which is highly homologous to the IGF-IR. There are two isoforms of IR, IR-A and IR-B, which are thought to mediate mitogenic and metabolic effects, respectively, as a result of different ligand specificity, internalization and downstream signaling. The prevailing hypothesis is that IR-A mediates a primary resistance to anti-IGF-IR antibodies, although the role of individual IR isoforms in the antitumor activity of IGF-IR inhibitors has not been demonstrated. The aim of the present study is to evaluate the impact of total IR and IR isoforms on the antitumor efficacy of anti-IGF-IR monoclonal antibody (mAb), and correlate their expression with the therapeutic outcome in experimental tumor models. Overexpression of IR-A in non-small cell lung carcinoma (NSCLC) cells (A549 and NCI-H1299) confers a complete resistance to anti-IGF-IR mAb both in vitro and in vivo. Surprisingly, overexpression of IR-B, a major mediator of metabolic effects of insulin, results in a partial resistance to anti-IGF-IR mAb. The resistant phenotype in both IR-A- and IR-Boverexpressing cells can be fully reversed in the presence of neutralizing anti-IGF-II antibodies, suggesting that IGF-II is a key driver of resistance to anti-IGF-IR mAb in the setting of IR overexpression. In vivo, expression of total IR rather than individual IR isoforms inversely correlates with the efficacy of anti-IGF-IR mAb in monotherapy in a panel of 34 pediatric solid tumor models. In 9 patient-derived xenograft NSCLC models, total IR, IR-A and IR-B expression negatively affects the outcome of anti-IGF-IR mAb in combination with chemotherapy (pemetrexed/ cisplatin). In conclusion, the present study links IR isoforms, IGF-II and the efficacy of anti-IGF-IR mAb mechanistically and identifies total IR as a candidate biomarker predictive of intrinsic resistance to the anti-IGF-IR antibody. 452 POSTER Preclinical Pharmacokinetics, Radiation Dosimetry and Toxicity of 111 In-BzDTPA-pertuzumab, an Agent for Imaging Early Response to Trastuzumab in Breast Cancer Patients K. Lam1 , C. Chan1 , D.A. Scollard1 , R.M. Reilly1 . 1 University of Toronto, Faculty of Pharmacy, Toronto, Canada Background: We are planning a Phase I/II trial of imaging HER2 downregulation with 111 In-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (BzDTPA)-pertuzumab as an early biomarker of response to trastuzumab (Herceptin® ). Our objective was to conduct preclinical translational bridge studies using BALB/c mice to determine the pharmacokinetics, normal tissue distribution, radiation dosimetry, and toxicity of 111 In-BzDTPApertuzumab.
Poster Session – Biomarkers Methods: 111 In-BzDTPA-pertuzumab (2−3 MBq; 2 mg) was administered intravenously to mice. The blood concentration-time data were fitted to a 2-compartment model. Radiation dosimetry projections in humans were estimated using OLINDA software. Acute toxicity was studied with female BALB/c mice at 25 times the planned human radioactivity dose. Toxicity was assessed by monitoring body weight, hematology and clinical biochemistry parameters, and by morphologic examination of tissues. Results: The highest concentrations of radioactivity were found in the blood (50.4±1.8 percentage injected dose [%ID] at 1 h post injection (p.i.) and decreasing to 12.4±0.5 %ID at 166 h p.i.) and lung (12.6±2.0 at 1 h p.i. and decreasing to 4.5±0.3 %ID at 166 h p.i.). 111 In-BzDTPA-pertuzumab had an a-phase half-life of 3.8 h and a b-phase half-life of 228.2 h. The volume of distribution of the central compartment (V1 ) was 119 mL/kg and the volume of distribution at steady state (Vss ) was 229 mL/kg. The projected whole-body dose in humans was 0.03 mSv/MBq. The projected doses to the liver, kidneys, and lower large intestine were 0.22, 0.31, and 0.03 mSv/MBq, respectively. There was no significant reduction in the ratio of body weight at 15 days compared to pre-treatment weight for mice administered 111 In-BzDTPA-pertuzumab (1.07±0.02) and mice administered unlabelled BzDTPA-pertuzumab (1.07±0.02). Erythrocyte, leukocyte, and platelet counts, and serum alanine aminotransferase and creatinine levels in 111 In-BzDTPA-pertuzumab and BzDTPA-pertuzumab treated mice were not significantly different from control mice. Morphologic examination of tissues is in progress. Conclusion: 111 In-BzDTPA-pertuzumab was well tolerated in mice administered multiples of the dose planned for a Phase I/II trial in breast cancer patients. Supported by the Ontario Institute for Cancer Research with funds from the Province of Ontario. 453 POSTER [F-18] Fluorothymidine (FLT) PET Imaging of Response of Acute Myeloid Leukemia to Chemotherapy J.F. Eary1 , J.M. Link1 , M. Muzi1 , E. Estey2 , K. Kauno1 , K.A. Krohn1 . 1 University of Washington, Nuclear Medicine, Seattle WA, USA; 2 University of Washington, Hematology/Oncology, Seattle WA, USA Background: Ability to predict response to treatment in Acute Myeloid Leukemia (AML) is limited within known prognostic subgroups. This study is evaluating whether FLT PET assessed pre-treatment and mid-treatment will improve prognosis. Methods: This pilot clinical trial is being performed under an FDA IND protocol in newly diagnosed AML patients treated on a cytarabine based regimen. FLT PET imaging is performed at baseline, and mid-therapy. Dynamic acquisitions of the lower spine and pelvis are acquired for 60 minutes, followed by whole body images. Images were reconstructed with CT attenuation correction. Regions of interest for the bone marrow, liver, spleen and normal muscle background were analyzed for FLT uptake at each timepoint. Regional tissue FLT uptake is evaluated as the standard uptake variable (SUV and SUVmax) and as FLTflux using a simple compartmental model analysis applied to the dynamic data. Comparisons are made between images and uptake parameters. Imaging results were compared with clinical response evaluated by day 14 bone marrow examination and peripheral blood count changes. Long term responses will be compared with predicted survival based on pre-therapy AML cytogenetics. Results: Four patients in this pilot study have been enrolled with midtherapy imaging in 3 patients. Baseline SUVmax was consistent between marrow regions in a patient and between patients: mean 13.7, median 13.0, CV 28%. Marrow FLTflux analysis is in progress to compare studies at baseline to those at mid-therapy. Mid-therapy images showed more regional variability than those obtained at baseline. In one patient, SUVmax increased in all marrow regions (average 23%); in two subjects marrow regional uptake decreased by 50% and 35%. Mean marrow to liver uptake ratios were 3.5±1.2 at baseline, whereas the 3 patients at mid-therapy had ratios of 3.5, 1.0, and 1.0, respectively. Spleen uptake was highly variable at baseline but normalized mid-therapy to a spleen/liver ratio of approximately 1:1. Liver and blood pool FLT concentration was modest and did not change with therapy. Two patients (with favorable baseline AML cytogenetics) had complete responses on therapy day 14 examinations, and one (with less favorable baseline cytogenetics) had minimal residual disease. Conclusions: In this ongoing pilot study, FLT PET imaging in AML shows highly increased baseline marrow uptake without significant heterogeneity. At mid-therapy the FLT uptake changes substantially throughout the marrow; in one subject, increased by 23% and the other two subjects, uptake was reduced by >50%. These values were associated with changes in peripheral blood counts and bone marrow exam that indicated early treatment response. Kinetic analysis will reveal whether these imaging changes were associated with transport or flux into the biosynthetic pathway for DNA. Supported by NIH P01 CA 042045 and S10 RR017229.
Friday 9 November 2012 139 449 POSTER Molecular and Therapeutic Characterization of 25 Patient Derived Lung Cancer Tumorgrafts and Analysis for Response Markers to Targeted Therapies as Part of a Systems Biological Approach (PREDICT) 3 ¨ , J. Rolff1 , M. Becker1 , R. Yildirimman2 , M. Isau2 , H. Hulsmann R. Kuner3 , O. Politz4 , J. Merk5 , R. Herwig2 , I. Fichtner1 . 1 Experimental Pharmacology & Oncology Berlin-Buch GmbH, EPO, Berlin, Germany; 2 Max-Planck-Istitute for Molecular Genetics (MPI), Vertebrate Genomics, Berlin, Germany; 3 German Cancer Research Center (DKFZ), Molecular Genetics, Heidelberg, Germany; 4 Bayer AG, Therapeutic Research Group Oncology, Berlin, Germany; 5 ELK Berlin Chest Hospital, Chest Surgery, Berlin, Germany
Current treatment of cancer is seriously hampered by the fact that most therapies are not well adapted to the individual responses of tumors which are strongly determined by the individual cellular and genetic background. There is a high molecular variability even among tumors of the same classification. Each tumor is individual and every patient will respond differently to a particular treatment. Therefore, prediction of therapy response by molecular markers is a prerequisite for individualized approaches to cancer treatment. Tumorgrafts derived from patient’s cancer specimen immediately after surgery provide a preclinical research tool considering both heterogeneity and individuality of malignancies. Tumorgrafts allow to test novel anti-tumor agents in a fast and standardized manner and provide sufficient tissue material, even post-treatment, for the search of corresponding predictive biomarkers. For this study we used a panel of 25 stably passagable lung cancer tissuegrafts. These permanent tumor models feature a high coincidence with the original patient tumor regarding histology and genome-wide gene expression profiling. They were subjected to an extensive molecular (genome-scale) and pharmacological characterization. The drug testing resulted in the following response rates (T/C 35%) for single drugs: carboplatin 56%, gemcitabine 56%, etoposide 12%, taxol 80%, erlotinib 20%, and cetuximab 40%. For example, detailed bioinformatic analysis revealed that the response effect of the chimeric EGFR inhibitor cetuximab was correlated to the regulation of the AMPK and mTOR signalling pathways. This finding was underlined by exome sequencing and protein expression (RPPA) analysis performed in parallel. Our investigations are part of a broadly based systems biological project aiming at the individualization of lung cancer therapy through comprehensive genome-scale characterization of preclinical in vitro and in vivo models. The collected data are integrated in a generic computer model which is usable for the prediction of individual therapy success of targeted anti-cancer drugs. 450 POSTER Non-invasive Absolute Quantification of EGFR Activating Mutation L858R and Gatekeeper Mutation T790M in NSCLC Plasma Samples Using Droplet Digital PCR J. Kristof1 , S. Sankar2 , E. Bruening1 , S. Wong1 . 1 MolecularMD, Research and Development, Portland OR, USA; 2 MolecularMD, Scientific Affairs Liaison, Portland OR, USA Background: A high proportion of non-small cell lung cancer (NSCLC) patients treated with erlotinib or gefitinib develop resistance via emergence of the gatekeeper epidermal growth factor receptor (EGFR) T790M mutation. The level and amount of the EGFR T790M resistance mutant in relation to EGFR L858R activating mutation as opposed to simply the presence or absence of the mutations has been shown to be critical for successful therapy selection in a mouse EGFR mutant lung cancer model. Effective treatment of these patients will likely require both high-sensitivity and quantitative detection of EGFR activating and resistance mutations in liquid biopsies, not obtainable with current platforms. We have developed and optimized a droplet digital qPCR assay that enables quantification of both EGFR activating L858R and resistance T790M mutations in circulating nucleic acids isolated from plasma. Methods: The ddPCR assay was developed using the Biorad QX100 platform. Primers and probes were designed to optimize quantification of EGFR L858R and T790M mutations. The Qiagen EGFR Rotor-Gene (RGQ) assay was used according to manufacturer’s protocol. Genomic DNA from the H1975 cell line expressing both EGFR L858R and T790M mutation was diluted into non-amplifiable nucleic acid to determine the assay’s limit of detection (LOD). Patient samples enriched for the likelihood of EGFR T790M mutation were analyzed as well. Results: The ddPCR assay was designed to detect total EGFR in addition to EGFR mutations. Near the critical low level detection range of less than 1000 copies expected in plasma samples, the assay displayed a linear
140 Friday 9 November 2012 dynamic range from 550 copies to a LOD of 5 copies for EGFR T790M. In comparison, the Qiagen EGFR Rotor-Gene (RGQ) assay displayed an average LOD of 55 copies with a dynamic range of 550 copies to 55 copies. In NSCLC patient plasma samples enriched for the likelihood of EGFR mutations, samples that expressed greater than 300 copies were confirmed by the Qiagen EGFR RGQ assay. In addition to quantifying mutant copies, the ddPCR assay determines the ratio of EGFR mutant to total EGFR template within a sample. In the current set of patient plasma samples analyzed we were able to accurately quantify the ratio of T790M mutant compared to total EGFR from as low as 2 ng without pre-amplification. Data on plasma samples measuring the ratio of T790M versus activating mutations L858R will be presented. The assay additionally detected the Q787Q SNP in patient samples that is predicted to have worse prognosis. Conclusion: Droplet digital PCR provides sensitive quantification of low abundance EGFR T790M resistance and L858R activating mutations in plasma without the need for pre-amplification. This assay provides a useful method for selecting treatment, monitoring disease progression, and providing early detection of treatment failure associated with EGFR acquired resistance. 451 POSTER Two Insulin Receptor Isoforms Confer Intrinsic Resistance to the Antibody Against Insulin-like Growth Factor Receptor I A. Forest1 , M. Amatulli1 , C. Damoci1 , D. Ludwig1 , N. Baltes2 , H. Fiebig2 , P. Houghton3 , M. Smith4 , L. Benjamin1 , R. Novosyadlyy1 . 1 ImClone Systems a wholly-owned subsidiary of Eli Lilly and Company, NYC, USA; 2 Oncotest, Freiburg, Germany; 3 Nationwide Children’s Hospital, Columbus, USA; 4 Cancer Therapy Evaluation Program NCI, Bethesda, USA The role of IGF-IR in tumor development and progression has been shown in numerous preclinical studies. Many inhibitors of IGF-IR are currently in clinical development; their antitumor efficacy, however, is limited only to a subset of patients. It remains unclear what molecular markers predict the antitumor activity of these agents. It has been shown previously that insulin receptor may confer resistance to IGF-IR targeting in experimental tumor models. One of the IGF-IR ligands, IGF-II, is capable of activating insulin receptor (IR), which is highly homologous to the IGF-IR. There are two isoforms of IR, IR-A and IR-B, which are thought to mediate mitogenic and metabolic effects, respectively, as a result of different ligand specificity, internalization and downstream signaling. The prevailing hypothesis is that IR-A mediates a primary resistance to anti-IGF-IR antibodies, although the role of individual IR isoforms in the antitumor activity of IGF-IR inhibitors has not been demonstrated. The aim of the present study is to evaluate the impact of total IR and IR isoforms on the antitumor efficacy of anti-IGF-IR monoclonal antibody (mAb), and correlate their expression with the therapeutic outcome in experimental tumor models. Overexpression of IR-A in non-small cell lung carcinoma (NSCLC) cells (A549 and NCI-H1299) confers a complete resistance to anti-IGF-IR mAb both in vitro and in vivo. Surprisingly, overexpression of IR-B, a major mediator of metabolic effects of insulin, results in a partial resistance to anti-IGF-IR mAb. The resistant phenotype in both IR-A- and IR-Boverexpressing cells can be fully reversed in the presence of neutralizing anti-IGF-II antibodies, suggesting that IGF-II is a key driver of resistance to anti-IGF-IR mAb in the setting of IR overexpression. In vivo, expression of total IR rather than individual IR isoforms inversely correlates with the efficacy of anti-IGF-IR mAb in monotherapy in a panel of 34 pediatric solid tumor models. In 9 patient-derived xenograft NSCLC models, total IR, IR-A and IR-B expression negatively affects the outcome of anti-IGF-IR mAb in combination with chemotherapy (pemetrexed/ cisplatin). In conclusion, the present study links IR isoforms, IGF-II and the efficacy of anti-IGF-IR mAb mechanistically and identifies total IR as a candidate biomarker predictive of intrinsic resistance to the anti-IGF-IR antibody. 452 POSTER Preclinical Pharmacokinetics, Radiation Dosimetry and Toxicity of 111 In-BzDTPA-pertuzumab, an Agent for Imaging Early Response to Trastuzumab in Breast Cancer Patients K. Lam1 , C. Chan1 , D.A. Scollard1 , R.M. Reilly1 . 1 University of Toronto, Faculty of Pharmacy, Toronto, Canada Background: We are planning a Phase I/II trial of imaging HER2 downregulation with 111 In-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (BzDTPA)-pertuzumab as an early biomarker of response to trastuzumab (Herceptin® ). Our objective was to conduct preclinical translational bridge studies using BALB/c mice to determine the pharmacokinetics, normal tissue distribution, radiation dosimetry, and toxicity of 111 In-BzDTPApertuzumab.
Poster Session – Biomarkers Methods: 111 In-BzDTPA-pertuzumab (2−3 MBq; 2 mg) was administered intravenously to mice. The blood concentration-time data were fitted to a 2-compartment model. Radiation dosimetry projections in humans were estimated using OLINDA software. Acute toxicity was studied with female BALB/c mice at 25 times the planned human radioactivity dose. Toxicity was assessed by monitoring body weight, hematology and clinical biochemistry parameters, and by morphologic examination of tissues. Results: The highest concentrations of radioactivity were found in the blood (50.4±1.8 percentage injected dose [%ID] at 1 h post injection (p.i.) and decreasing to 12.4±0.5 %ID at 166 h p.i.) and lung (12.6±2.0 at 1 h p.i. and decreasing to 4.5±0.3 %ID at 166 h p.i.). 111 In-BzDTPA-pertuzumab had an a-phase half-life of 3.8 h and a b-phase half-life of 228.2 h. The volume of distribution of the central compartment (V1 ) was 119 mL/kg and the volume of distribution at steady state (Vss ) was 229 mL/kg. The projected whole-body dose in humans was 0.03 mSv/MBq. The projected doses to the liver, kidneys, and lower large intestine were 0.22, 0.31, and 0.03 mSv/MBq, respectively. There was no significant reduction in the ratio of body weight at 15 days compared to pre-treatment weight for mice administered 111 In-BzDTPA-pertuzumab (1.07±0.02) and mice administered unlabelled BzDTPA-pertuzumab (1.07±0.02). Erythrocyte, leukocyte, and platelet counts, and serum alanine aminotransferase and creatinine levels in 111 In-BzDTPA-pertuzumab and BzDTPA-pertuzumab treated mice were not significantly different from control mice. Morphologic examination of tissues is in progress. Conclusion: 111 In-BzDTPA-pertuzumab was well tolerated in mice administered multiples of the dose planned for a Phase I/II trial in breast cancer patients. Supported by the Ontario Institute for Cancer Research with funds from the Province of Ontario. 453 POSTER [F-18] Fluorothymidine (FLT) PET Imaging of Response of Acute Myeloid Leukemia to Chemotherapy J.F. Eary1 , J.M. Link1 , M. Muzi1 , E. Estey2 , K. Kauno1 , K.A. Krohn1 . 1 University of Washington, Nuclear Medicine, Seattle WA, USA; 2 University of Washington, Hematology/Oncology, Seattle WA, USA Background: Ability to predict response to treatment in Acute Myeloid Leukemia (AML) is limited within known prognostic subgroups. This study is evaluating whether FLT PET assessed pre-treatment and mid-treatment will improve prognosis. Methods: This pilot clinical trial is being performed under an FDA IND protocol in newly diagnosed AML patients treated on a cytarabine based regimen. FLT PET imaging is performed at baseline, and mid-therapy. Dynamic acquisitions of the lower spine and pelvis are acquired for 60 minutes, followed by whole body images. Images were reconstructed with CT attenuation correction. Regions of interest for the bone marrow, liver, spleen and normal muscle background were analyzed for FLT uptake at each timepoint. Regional tissue FLT uptake is evaluated as the standard uptake variable (SUV and SUVmax) and as FLTflux using a simple compartmental model analysis applied to the dynamic data. Comparisons are made between images and uptake parameters. Imaging results were compared with clinical response evaluated by day 14 bone marrow examination and peripheral blood count changes. Long term responses will be compared with predicted survival based on pre-therapy AML cytogenetics. Results: Four patients in this pilot study have been enrolled with midtherapy imaging in 3 patients. Baseline SUVmax was consistent between marrow regions in a patient and between patients: mean 13.7, median 13.0, CV 28%. Marrow FLTflux analysis is in progress to compare studies at baseline to those at mid-therapy. Mid-therapy images showed more regional variability than those obtained at baseline. In one patient, SUVmax increased in all marrow regions (average 23%); in two subjects marrow regional uptake decreased by 50% and 35%. Mean marrow to liver uptake ratios were 3.5±1.2 at baseline, whereas the 3 patients at mid-therapy had ratios of 3.5, 1.0, and 1.0, respectively. Spleen uptake was highly variable at baseline but normalized mid-therapy to a spleen/liver ratio of approximately 1:1. Liver and blood pool FLT concentration was modest and did not change with therapy. Two patients (with favorable baseline AML cytogenetics) had complete responses on therapy day 14 examinations, and one (with less favorable baseline cytogenetics) had minimal residual disease. Conclusions: In this ongoing pilot study, FLT PET imaging in AML shows highly increased baseline marrow uptake without significant heterogeneity. At mid-therapy the FLT uptake changes substantially throughout the marrow; in one subject, increased by 23% and the other two subjects, uptake was reduced by >50%. These values were associated with changes in peripheral blood counts and bone marrow exam that indicated early treatment response. Kinetic analysis will reveal whether these imaging changes were associated with transport or flux into the biosynthetic pathway for DNA. Supported by NIH P01 CA 042045 and S10 RR017229.