- Email: [email protected]
Development of a novel chromogenic RNA in situ hybridization method for detection of somatic mutations
Poster Session – Molecular targeted agents II, Thursday 1 December 2016 sought. Sigma receptors (SR1 and SR2) are members of a class of unique receptors integrated in plasma, mitochondrial and endoplasmatic reticulum membranes of mammalian cells. They have received much attention in the drug-discovery field other than their role in several neurological disorders, also for their probable involvement in cancer cell proliferation and aggressiveness. Therefore, inhibitors or modulators are of great interest as novel therapeutic anti-cancer drugs. In the present work, 3-D primary cultures of GBM endowed with stemness features, were used to evaluate the antitumor activity of a novel sigma receptor modulator RC-106. Materials and Methods: Primary cultures of hGBM have been isolated starting from surgical tumor samples and grown as monolayer or 3Dcell cultures. Growth and morphology of the 3D tumor colonies were monitored by open-source AnaSP and ReViSP software tools. The molecular analyses were performed by flow cytometry and qRT-PCR. Cell viability was measured using CellTiter-Glo® 3D Cell Viability and MTS Assays. The apoptosis and cell cycle were analyzed by flow cytometry. Results: We established a number of cell lines with different growth properties and different stemness gene expression profile. In particular, we evaluated the expression levels of the target genes S1R and S2R, and of tumor propagating cells surface markers as EphA2, CD44 and CD133 by flow cytometric and qRT-PCR analysis. Notwithstanding the inter-tumor heterogeneity observed, the treatment with RC-106 significantly inhibited cell viability and caused a strong apoptosis induction in all cell cultures tested. Conversely, the exposure to different concentrations of temozolomide did not induce significant cytotoxic effet. In particular, RC106 (25mM) induced an impairment of in vitro clonogenic ability of GBM cells, and such effect persist until 42 days, the longest time tested. Conclusions: The high level of expression of S1R and S2R gene detected in patient’s tissues and in their derivative cell lines support the interest for these receptors as potentially druggable targets also in GBM. Notably, RC106 compound showed to be able to induce a strong cytotoxic effect in all the cell lines tested both actively proliferating and in low proliferation rate in response to serum deprivation. No conflict of interest. 449 Poster (Board P128) Impact of circulating biomarkers in patients with metastatic colorectal cancer treated with first-line FOLFOX-aflibercept therapy. Results of the GERCOR VELVET Phase II study A. Tijeras-Raballand1 , A. De Gramont1 , M. Chiron2 , J.B. Bachet3 , T. Andre´ 4 , D. Auby5 , J. Desrame´ 6 , N. Baba-Ahmed7 , C. Lecaille8 , V. Lebrun9 , C. Louvet10 , C. Tournigand11 , S. Benner12 , M. Attia13 , A. De Gramont14 , F. Bonnetain15 , B. Chibaudel14 . 1 AFR Oncology, Preclinical and Translational Department, Paris, France; 2 Sanofi, Translational Medicine, Oncology Department, Vitry sur Seine, France; 3 ˆ ´ ´ ere, ` Hopital Pitie-Salp etri Gastroenterology Department, Paris, France; 4 ˆ Hopital Saint-Antoine, Medical Oncology Department, Paris, France; 5 Centre Hospitalier Mont de Marsan, Gastroenterology Department, Mont ˆ Prive´ Jean Mermoz, Gastroenterology and de Marsan, France; 6 Hopital ˆ Hepatology Department, Lyon, France; 7 Hopital Saint-Joseph, Medical Oncology Department, Paris, France; 8 Polyclinique Bordeaux Nord Aquitaine, Gastroenterology Department, Bordeaux, France; 9 Centre Hospitalo-Universitaire de Limoges, Medical Oncology, Limoges, France; 10 Institut Mutualiste Montsouris, Medical Oncology, Paris, France; 11 Centre ´ Hospitalo-Universitaire Henri Mondor, Medical Oncology, Creteil, France; 12 AFR Oncology, Translational and Clinical Department, Paris, France; 13 GERCOR, Medical Oncology, Paris, France; 14 Institut Hospitalier Franco-britannique, Medical Oncology Department, Levallois-Perret, France; 15 Centre Hopistalo-Universitaire de Besan¸con, Quality of Life in Oncology and Methodology Department, Besan¸con, France Background: The combination of aflibercept to OPTIMOX (VELVET study) was evaluated in patients with previously untreated advanced colorectal cancer (Chibaudel B et al, J Clin Oncol 33, 2015 (suppl; abstr 3567). A biomarker program was set-up to explore the expression of several biomarkers upon treatment cycles to identify the best monitoring biomarkers of the treatment strategy. Methods: VELVET was a prospective, single arm phase II trial. Patient’s plasma samples were collected at baseline, and during induction therapy at day 1 of the first 6 cycles. Circulating biomarkers were analyzed using multiplexing immunoassays (31 biomarkers, 3 panels). All assays were conducted on a Biorad Bioplex platform. Results: Among 49 patients included in the VELVET study from May 2013 to May 2014, 44 (90%) patients were evaluable for circulating biomarkers expression. The proportion of patients with tumor response (CR or PR) was higher in patients with high baseline levels of sVEGFR2, sEGFR, G-CSF, Prolactin and low baseline levels of VEGFA and MIF. Progressionfree survival (PFS) was higher in patients with low baseline levels of sIL6Ra (HR: 0.52; P = 0.045) and Osteopontin (HR: 0.53; P = 0.045) and in
Poster abstracts S147
patients with high baseline levels of VEGF-C (HR: 0.45; P = 0.014) and sVEGFR3 (HR: 0.50; P = 0.045). Overall survival was higher in patients with high sVEGFR3 (HR: 0.36; P = 0.030) and IL-8 (HR: 0.32; P = 0.014) levels. In both responders and non-responders, sVEGFR-1 dramatically increased upon exposure to aflibercept and remained overexpressed for the all course of induction therapy. Induction therapy also comes with increased expression of VCAM (+42%), SDF-1 (+62%) and SP-D along with decreased expression of sVEGFR-3, VEGF-C, Ang1, Ang2, PDGF and IL-8 Tumor expression of some of these markers will be presented at the meeting. Conclusions: Exposure to aflibercept is associated with an increase of sVEGFR-1 at cycle 1. Elevated baseline expression levels of on-target sVEGFR3 predict favorable outcome in patients treated with aflibercept. Conflict of interest: Advisory Board: Pr C. Tournigand (Sanofi); Pr C. Louvet (Sanofi, Roche, Celgene); Dr JB Bachet (Amgen, Lilly, Celgene, Roche); Pr A. Thierry (Roche, Boeringher); Dr B. Chibaudel (Sanofi); Pr A. de Gramont (Sanofi). Other Substantive Relationships: Marielle Chiron is an employee of Sanofi. 450 Poster (Board P129) Development of AVID100, a novel antibody–drug conjugate for the treatment of EGFR expressing solid tumors M. O’Connor-McCourt1 , J. Koropatnick2 , S. Maleki2 , R. Figueredo2 , I. Tikhomirov3 , M. Jaramillo4 . 1 Formation Biologics, R&D, Montreal, Canada; 2 Lawson Health Research Institute, Cancer Research Program, London, Canada; 3 Formation Biologics, R&D, Houston, USA; 4 National Research Council Of Canada, Human Health Therapeutics, Montreal, Canada Background: AVID100 is a novel epidermal growth factor receptor (EGFR)targeting antibody–drug conjugate (ADC). EGFR is an important oncogene overexpressed by many types of solid tumors, including lung, breast, head and neck, and others. Unlike currently marketed anti-EGFR therapeutics that depend on EGFR blockade for anti-cancer activity, AVID100 has an additional mechanism of action of direct cytotoxicity via conjugated payload. This is expected to result in significantly enhanced anti-cancer activity of AVID100 compared to currently marketed anti-EGFR agents. We conducted in vitro and in vivo studies to evaluate the anticancer activity of AVID100. Methods: Effects of AVID100 were tested against EGFR+ cancer cell lines, including those resistant to marketed anti-EGFR agents. Activity of AVID100 against non-transformed EGFR+ keratinocytes was also evaluated. Pharmacology studies investigating the anti-cancer activity of AVID100 were performed in mice bearing human tumor xenograft models, including breast and head and neck cancers. Results: AVID100 demonstrated potent and broad activity against multiple cell lines with IC50 values in the pM to nM range. The ADC was also active against cell lines resistant to marketed to anti-EGFR therapeutics. In vivo, AVID100 demonstrated significant anti-cancer activity in multiple cancer models including complete remissions in breast and head and neck cancer xenografts. Importantly, AVID100 was demonstrated to be minimally toxic against normal EGFR+ keratinocytes. Skin toxicity is a class effect of anti-EGFR therapeutics and this result suggests AVID100 skin toxicity will be comparable to other agents in the class, despite significantly higher potency of AVID100 on tumors. Tolerability of AVID100 was subsequently confirmed in non-human primate studies. Conclusion: AVID100 is a promising anti-cancer therapeutic for the treatment of EGFR expressing tumors, including tumor types resistant to currently marketed anti-EGFR agents. AVID100 is currently undergoing IND-enabling development with clinical trials planned for 2016. Conflict of interest: Ownership: Formation Biologics. Advisory Board: Formation Biologics. Board of Directors: Formation Biologics. Corporatesponsored Research: Formation Biologics. 451 Poster (Board P130) Development of a novel chromogenic RNA in situ hybridization method for detection of somatic mutations X.M.M. Wang1 , X. Wu1 , L. Pan1 , N. Su1 , E. Park1 , R. Monroe1 , Y. Luo1 , X.J. Ma1 . 1 Advanced Cell Diagnostics, R&D, Newark, USA Background: The identification of somatic mutations in tumors is becoming increasingly important for patient selection for targeted therapies. While high throughput sequencing technologies allow for comprehensive mutation-profiling, sequencing does not permit assessment of intratumoral heterogeneity or the association of genetic alterations with cellular morphology. In addition, DNA mutational status does not predict expression of the mutant allele which may provide information connecting genotype to phenotype. Therefore, a technology for mutation detection directly in the tumor context is desirable. We present a chromogenic in situ hybridization
S148 Poster abstracts
Poster Session – Molecular targeted agents II, Thursday 1 December 2016
(ISH) method for the detection of mutations directly in gene transcripts. Improving upon the double Z (ZZ) probe design strategy of RNAscope® , we have developed a next generation ISH technology to detect single nucleotide variations and small indels. Materials and Methods: We selected 8 common mutations in the BRAF, KRAS, EGFR, APC, and PIK3CA genes (BRAF V600E; KRAS G12D & G12V; EGFR T790M & E746-A750 del; APC 853G>T; PIK3CA H1074R & E545K) and designed double Z probes specific for each mutation and the corresponding wild-type (wt) sequence. We then performed chromogenic RNA ISH to test the probe specificity in FFPE sections prepared from known cell lines. We subsequently tested the assay’s ability to detect BRAF V600E mutations in blinded FFPE samples of human colorectal carcinoma (CRC) previously characterized for BRAF status by PCR. Results: Mutation specific probes generated distinct chromogenic dot signals, representing individual mutant transcripts, in histologic sections from mutant but not wt cell lines. Probes for wt sequences at the same positions resulted in signals in wt and heterozygous mutant cell lines, but not in homozygous mutant cell lines. Blinded analysis of 10 CRC samples (5 mutant & 5 wt) correctly identified the BRAF V600E status consistent with PCR findings. Evaluation of the spatial distribution of mutant transcripts revealed similar signals across the tumors in 4 of 5 cases. One tumor showed a decrease in the signals for mutant transcripts in poorly differentiated areas of the tumor. Conclusions: The described novel RNA ISH technology enables detection of somatic mutations as well as mapping of genetically distinct subpopulations within tumors. The technique also allows for direct visualization of allele-specific expression. This functionality has the potential to further our understanding of tumor heterogeneity and the evolution of resistance mutations. Furthermore, as the mutation status can be visualized chromogenically, this technology has the potential for incorporation into the current anatomic pathology workflow for light microscopic assessment of patients’ tumors for specific mutations that would permit selection for targeted therapies. No conflict of interest. 452 Poster (Board P131) Phase 1 results of PTC596, a novel small molecule targeting cancer stem cells (CSCs) by reducing levels of BMI1 protein G. Shapiro1 , P. Bedard2 , J. Infante3 , T. Bauer3 , A. Prawira2 , O. Laksin4 , M. Weetall5 , J. Baird4 , A. Branstrom5 , E. O’Mara4 , R. Spiegel4 . 1 Dana Farber Cancer Institute, Early Drug Development Center, Boston, USA; 2 Princess Margaret Cancer Centre, Division of Medical Oncology, Toronto, Canada; 3 Sarah Cannon Research Institute, Drug Development Program, Nashville, USA; 4 PTC Therapeutics, Clinical, South Plainfield, USA; 5 PTC Therapeutics, Research, South Plainfield, USA Background: PTC596 is a first-in-class, oral investigational new drug that reduces levels of BMI1, a protein required for CSC survival. PTC596 reduced the number of CSCs in preclinical models and slowed growth rates of several tumor xenografts. The primary objectives of this first-inhuman trial of PTC596 are to determine safety, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and pharmacokinetics (PK). Secondary objectives include initial assessment of biological efficacy, pharmacodynamic changes and anti-tumor activity. Methods: A 2-stage, Phase I multi-center study is being conducted in patients with advanced solid tumors. PTC596 is administered in 4 week cycles using bodyweight-adjusted twice-per-week (biw) oral dosing. Stage 1 involves dose escalation using a modified 3+3 design. Anti-tumor activity is assessed by RECIST 1.1. Additional cohorts are being enrolled to obtain pre- and post-treatment (C2D1) tumor biopsies for markers of target engagement. In stage 2, up to 40 patients with a limited number of tumor types will be enrolled at the recommended Phase 2 dose (RP2D). Patients who derive benefit may remain on PTC596. Results: To date, 18 patients have been enrolled at doses of 0.65 (N = 3), 1.3 (N = 3), 2.6 (N = 3), 5.2 (N = 6) and 10 mg/kg (N = 3). No DLTs or drug related grade 2 toxicities were observed in the first 3 cohorts. Grade 1 nausea and vomiting were seen at these lower doses. Grade 2 diarrhea was seen in 2 patients dosed at 5.2 mg/kg. Though a protocol defined MTD was not reached, the dose of 10 mg/kg was deemed intolerable due to Grade 2 nausea, vomiting, and diarrhea in 2 of 3 patients. One Grade 4 DLT of neutropenia and thrombocytopenia was observed in a patient receiving 10.0 mg/kg. An intermediate dosing cohort at 7.0 mg/kg has been initiated. Over the dosing range, plasma concentrations of PTC596 increased in an approximately dose-proportional manner. Though no objective responses have been observed to date, at doses of 2.6 mg/kg biw, plasma concentrations of PTC596 exceed those demonstrated to be efficacious in mouse xenograft models. Conclusion: PTC596 lowers levels of BMI1 in preclinical tumor models and is generally well tolerated as a monotherapy at doses that result in
preclinical target plasma concentrations. Full study information is available on ClinicalTrials.gov, Identifier NCT02404480. Conflict of interest: Ownership: John Baird, Art Branstrom, Marla Weetall, Edward O’Mara are financially compensated as employees of PTC Therapeutics. Oscar Laskin and Robert Spieling are consultants for PTC Therapeutics. 453 Poster (Board P132) Development of selective MELK kinase inhibitors for breast cancer treatment P. Wegrzyn1 , P. Kowalczyk1 , M. Dobrzanska1 , M. Knop1 , M. Obacz1 , K. Gluza1 , C. Commandeur1 , A. Sabiniarz1 , K. Wiklik1 , A. Golas1 , F. Vaillancourt2 , N. Larsen2 , J. Wang3 , D. Reynolds2 , D. Ito3 , J. Zou3 , M. Aicher2 , P. Smith2 , P. Zhu2 , K. Brzozka1 . 1 Selvita S.A., R&D, Krakow, Poland; 2 H3 Biomedicine, R&D, Cambridge, MA, USA; 3 Eisai, R&D, Tokyo, Japan Maternal embryonic leucine zipper kinase (MELK) is an atypical member of the AMPK family of serine-threonine kinases that has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. Its activity is correlated with its phosphorylation level, is cell cycle dependent, and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. In recent years MELK has been identified as a novel oncogenic protein that is overexpressed in several types of solid cancers with low levels in normal tissues. MELK expression level correlates intensively with poor prognosis in colon, breast, ovary, lung, prostate cancer and glioblastoma. Recent findings underlying the oncogenic role of this kinase in tripe negative breast cancer (TNBC), a category of high-grade and invasive tumors. Due to the lack of estrogen and progesterone receptors TNBC remains difficult to treat with hormonal therapies. Additionally, therapies targeting HER2, such as Herceptin, are also inefficient in this type of tumors. Despite the fact that the exact MELK function is not known, selective targeting of this kinase may be an effective cancer treatment strategy. The knockdown of MELK decreases cell-cycle progression, proliferation and tumor growth. In this study we are reporting development of a series of selective MELK kinase inhibitors. Synthetized compounds exert excellent selectivity and potency in MELK inhibitions with the low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of breast cancer cell lines with different genetic background as well as with different MELK kinase levels. We identified several cell lines in which MELKi induced cell death with nanomolar ED50 values. Potent MELK inhibitors exhibited significant tumor growth suppression in xenograft studies using breast cancer cell lines in mice. Taken altogether, the presented data supports our rationale of using MELK kinase inhibitors as a novel and interesting approach for the cancer therapy. Conflict of interest: Ownership: Selvita S.A. Board of Directors: Selvita S.A. 454 Poster/Poster in the spotlight (Board P133) Exploratory biomarker analysis of first-line cobimetinib (C) + paclitaxel (P) in patients (pts) with advanced triple-negative breast cancer (TNBC) from the phase 2 COLET study M. Wongchenko1 , D. Miles2 , S.B. Kim3 , N. Xu4 , L. Amler1 , Y. Yan1 , B. Simmons5 , V. McNally6 , A. Brufsky7 . 1 Genentech, Inc., Oncology Biomarker Development, South San Francisco, USA; 2 Mount Vernon Cancer Centre, Medical Oncology, London, United Kingdom; 3 Asan Medical Center, University of Ulsan College of Medicine, Oncology, Seoul, South Korea; 4 Genentech, Inc., PD Biostatistics, South San Francisco, USA; 5 Genentech, Inc., Product Development Oncology, South San Francisco, USA; 6 Roche Products Ltd., PDC, Welwyn Garden City, United Kingdom; 7 University of Pittsburgh, Division of Hematology–Oncology, Department of Medicine, Pittsburgh, USA Background: Resistance to standard taxane-based chemotherapy is common in TNBC. Preclinical data suggest that MEK inhibition may overcome taxane resistance and enhance antitumor immune response. The safety and efficacy of combining C, a specific and allosteric inhibitor of MEK1/2, with P was explored in pts with metastatic/locally advanced TNBC and no prior systemic therapy for metastatic disease. Here, we present a retrospective exploratory biomarker analysis of nonrandomized pts. Material and Methods: The COLET study (ClinicalTrial.gov ID, NCT02322814; sponsored by F. Hoffmann-La Roche, Ltd.) consisted of a safety run-in (n ≈ 12) followed by a blinded 1:1 randomized stage (n ≈ 100 pts) to C + P or placebo (PBO) + P. Pts were treated with P 80 mg/m2 on days 1, 8, and 15 and C/PBO 60 mg/day on days 3−23 of each 28-day cycle. Tumor biopsies were collected prior to treatment, optionally at cycle 1 day 15 (C1D15), and at disease progression. FoundationOne genomic profile testing was performed by Foundation
Poster abstracts S147
patients with high baseline levels of VEGF-C (HR: 0.45; P = 0.014) and sVEGFR3 (HR: 0.50; P = 0.045). Overall survival was higher in patients with high sVEGFR3 (HR: 0.36; P = 0.030) and IL-8 (HR: 0.32; P = 0.014) levels. In both responders and non-responders, sVEGFR-1 dramatically increased upon exposure to aflibercept and remained overexpressed for the all course of induction therapy. Induction therapy also comes with increased expression of VCAM (+42%), SDF-1 (+62%) and SP-D along with decreased expression of sVEGFR-3, VEGF-C, Ang1, Ang2, PDGF and IL-8 Tumor expression of some of these markers will be presented at the meeting. Conclusions: Exposure to aflibercept is associated with an increase of sVEGFR-1 at cycle 1. Elevated baseline expression levels of on-target sVEGFR3 predict favorable outcome in patients treated with aflibercept. Conflict of interest: Advisory Board: Pr C. Tournigand (Sanofi); Pr C. Louvet (Sanofi, Roche, Celgene); Dr JB Bachet (Amgen, Lilly, Celgene, Roche); Pr A. Thierry (Roche, Boeringher); Dr B. Chibaudel (Sanofi); Pr A. de Gramont (Sanofi). Other Substantive Relationships: Marielle Chiron is an employee of Sanofi. 450 Poster (Board P129) Development of AVID100, a novel antibody–drug conjugate for the treatment of EGFR expressing solid tumors M. O’Connor-McCourt1 , J. Koropatnick2 , S. Maleki2 , R. Figueredo2 , I. Tikhomirov3 , M. Jaramillo4 . 1 Formation Biologics, R&D, Montreal, Canada; 2 Lawson Health Research Institute, Cancer Research Program, London, Canada; 3 Formation Biologics, R&D, Houston, USA; 4 National Research Council Of Canada, Human Health Therapeutics, Montreal, Canada Background: AVID100 is a novel epidermal growth factor receptor (EGFR)targeting antibody–drug conjugate (ADC). EGFR is an important oncogene overexpressed by many types of solid tumors, including lung, breast, head and neck, and others. Unlike currently marketed anti-EGFR therapeutics that depend on EGFR blockade for anti-cancer activity, AVID100 has an additional mechanism of action of direct cytotoxicity via conjugated payload. This is expected to result in significantly enhanced anti-cancer activity of AVID100 compared to currently marketed anti-EGFR agents. We conducted in vitro and in vivo studies to evaluate the anticancer activity of AVID100. Methods: Effects of AVID100 were tested against EGFR+ cancer cell lines, including those resistant to marketed anti-EGFR agents. Activity of AVID100 against non-transformed EGFR+ keratinocytes was also evaluated. Pharmacology studies investigating the anti-cancer activity of AVID100 were performed in mice bearing human tumor xenograft models, including breast and head and neck cancers. Results: AVID100 demonstrated potent and broad activity against multiple cell lines with IC50 values in the pM to nM range. The ADC was also active against cell lines resistant to marketed to anti-EGFR therapeutics. In vivo, AVID100 demonstrated significant anti-cancer activity in multiple cancer models including complete remissions in breast and head and neck cancer xenografts. Importantly, AVID100 was demonstrated to be minimally toxic against normal EGFR+ keratinocytes. Skin toxicity is a class effect of anti-EGFR therapeutics and this result suggests AVID100 skin toxicity will be comparable to other agents in the class, despite significantly higher potency of AVID100 on tumors. Tolerability of AVID100 was subsequently confirmed in non-human primate studies. Conclusion: AVID100 is a promising anti-cancer therapeutic for the treatment of EGFR expressing tumors, including tumor types resistant to currently marketed anti-EGFR agents. AVID100 is currently undergoing IND-enabling development with clinical trials planned for 2016. Conflict of interest: Ownership: Formation Biologics. Advisory Board: Formation Biologics. Board of Directors: Formation Biologics. Corporatesponsored Research: Formation Biologics. 451 Poster (Board P130) Development of a novel chromogenic RNA in situ hybridization method for detection of somatic mutations X.M.M. Wang1 , X. Wu1 , L. Pan1 , N. Su1 , E. Park1 , R. Monroe1 , Y. Luo1 , X.J. Ma1 . 1 Advanced Cell Diagnostics, R&D, Newark, USA Background: The identification of somatic mutations in tumors is becoming increasingly important for patient selection for targeted therapies. While high throughput sequencing technologies allow for comprehensive mutation-profiling, sequencing does not permit assessment of intratumoral heterogeneity or the association of genetic alterations with cellular morphology. In addition, DNA mutational status does not predict expression of the mutant allele which may provide information connecting genotype to phenotype. Therefore, a technology for mutation detection directly in the tumor context is desirable. We present a chromogenic in situ hybridization
S148 Poster abstracts
Poster Session – Molecular targeted agents II, Thursday 1 December 2016
(ISH) method for the detection of mutations directly in gene transcripts. Improving upon the double Z (ZZ) probe design strategy of RNAscope® , we have developed a next generation ISH technology to detect single nucleotide variations and small indels. Materials and Methods: We selected 8 common mutations in the BRAF, KRAS, EGFR, APC, and PIK3CA genes (BRAF V600E; KRAS G12D & G12V; EGFR T790M & E746-A750 del; APC 853G>T; PIK3CA H1074R & E545K) and designed double Z probes specific for each mutation and the corresponding wild-type (wt) sequence. We then performed chromogenic RNA ISH to test the probe specificity in FFPE sections prepared from known cell lines. We subsequently tested the assay’s ability to detect BRAF V600E mutations in blinded FFPE samples of human colorectal carcinoma (CRC) previously characterized for BRAF status by PCR. Results: Mutation specific probes generated distinct chromogenic dot signals, representing individual mutant transcripts, in histologic sections from mutant but not wt cell lines. Probes for wt sequences at the same positions resulted in signals in wt and heterozygous mutant cell lines, but not in homozygous mutant cell lines. Blinded analysis of 10 CRC samples (5 mutant & 5 wt) correctly identified the BRAF V600E status consistent with PCR findings. Evaluation of the spatial distribution of mutant transcripts revealed similar signals across the tumors in 4 of 5 cases. One tumor showed a decrease in the signals for mutant transcripts in poorly differentiated areas of the tumor. Conclusions: The described novel RNA ISH technology enables detection of somatic mutations as well as mapping of genetically distinct subpopulations within tumors. The technique also allows for direct visualization of allele-specific expression. This functionality has the potential to further our understanding of tumor heterogeneity and the evolution of resistance mutations. Furthermore, as the mutation status can be visualized chromogenically, this technology has the potential for incorporation into the current anatomic pathology workflow for light microscopic assessment of patients’ tumors for specific mutations that would permit selection for targeted therapies. No conflict of interest. 452 Poster (Board P131) Phase 1 results of PTC596, a novel small molecule targeting cancer stem cells (CSCs) by reducing levels of BMI1 protein G. Shapiro1 , P. Bedard2 , J. Infante3 , T. Bauer3 , A. Prawira2 , O. Laksin4 , M. Weetall5 , J. Baird4 , A. Branstrom5 , E. O’Mara4 , R. Spiegel4 . 1 Dana Farber Cancer Institute, Early Drug Development Center, Boston, USA; 2 Princess Margaret Cancer Centre, Division of Medical Oncology, Toronto, Canada; 3 Sarah Cannon Research Institute, Drug Development Program, Nashville, USA; 4 PTC Therapeutics, Clinical, South Plainfield, USA; 5 PTC Therapeutics, Research, South Plainfield, USA Background: PTC596 is a first-in-class, oral investigational new drug that reduces levels of BMI1, a protein required for CSC survival. PTC596 reduced the number of CSCs in preclinical models and slowed growth rates of several tumor xenografts. The primary objectives of this first-inhuman trial of PTC596 are to determine safety, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and pharmacokinetics (PK). Secondary objectives include initial assessment of biological efficacy, pharmacodynamic changes and anti-tumor activity. Methods: A 2-stage, Phase I multi-center study is being conducted in patients with advanced solid tumors. PTC596 is administered in 4 week cycles using bodyweight-adjusted twice-per-week (biw) oral dosing. Stage 1 involves dose escalation using a modified 3+3 design. Anti-tumor activity is assessed by RECIST 1.1. Additional cohorts are being enrolled to obtain pre- and post-treatment (C2D1) tumor biopsies for markers of target engagement. In stage 2, up to 40 patients with a limited number of tumor types will be enrolled at the recommended Phase 2 dose (RP2D). Patients who derive benefit may remain on PTC596. Results: To date, 18 patients have been enrolled at doses of 0.65 (N = 3), 1.3 (N = 3), 2.6 (N = 3), 5.2 (N = 6) and 10 mg/kg (N = 3). No DLTs or drug related grade 2 toxicities were observed in the first 3 cohorts. Grade 1 nausea and vomiting were seen at these lower doses. Grade 2 diarrhea was seen in 2 patients dosed at 5.2 mg/kg. Though a protocol defined MTD was not reached, the dose of 10 mg/kg was deemed intolerable due to Grade 2 nausea, vomiting, and diarrhea in 2 of 3 patients. One Grade 4 DLT of neutropenia and thrombocytopenia was observed in a patient receiving 10.0 mg/kg. An intermediate dosing cohort at 7.0 mg/kg has been initiated. Over the dosing range, plasma concentrations of PTC596 increased in an approximately dose-proportional manner. Though no objective responses have been observed to date, at doses of 2.6 mg/kg biw, plasma concentrations of PTC596 exceed those demonstrated to be efficacious in mouse xenograft models. Conclusion: PTC596 lowers levels of BMI1 in preclinical tumor models and is generally well tolerated as a monotherapy at doses that result in
preclinical target plasma concentrations. Full study information is available on ClinicalTrials.gov, Identifier NCT02404480. Conflict of interest: Ownership: John Baird, Art Branstrom, Marla Weetall, Edward O’Mara are financially compensated as employees of PTC Therapeutics. Oscar Laskin and Robert Spieling are consultants for PTC Therapeutics. 453 Poster (Board P132) Development of selective MELK kinase inhibitors for breast cancer treatment P. Wegrzyn1 , P. Kowalczyk1 , M. Dobrzanska1 , M. Knop1 , M. Obacz1 , K. Gluza1 , C. Commandeur1 , A. Sabiniarz1 , K. Wiklik1 , A. Golas1 , F. Vaillancourt2 , N. Larsen2 , J. Wang3 , D. Reynolds2 , D. Ito3 , J. Zou3 , M. Aicher2 , P. Smith2 , P. Zhu2 , K. Brzozka1 . 1 Selvita S.A., R&D, Krakow, Poland; 2 H3 Biomedicine, R&D, Cambridge, MA, USA; 3 Eisai, R&D, Tokyo, Japan Maternal embryonic leucine zipper kinase (MELK) is an atypical member of the AMPK family of serine-threonine kinases that has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. Its activity is correlated with its phosphorylation level, is cell cycle dependent, and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. In recent years MELK has been identified as a novel oncogenic protein that is overexpressed in several types of solid cancers with low levels in normal tissues. MELK expression level correlates intensively with poor prognosis in colon, breast, ovary, lung, prostate cancer and glioblastoma. Recent findings underlying the oncogenic role of this kinase in tripe negative breast cancer (TNBC), a category of high-grade and invasive tumors. Due to the lack of estrogen and progesterone receptors TNBC remains difficult to treat with hormonal therapies. Additionally, therapies targeting HER2, such as Herceptin, are also inefficient in this type of tumors. Despite the fact that the exact MELK function is not known, selective targeting of this kinase may be an effective cancer treatment strategy. The knockdown of MELK decreases cell-cycle progression, proliferation and tumor growth. In this study we are reporting development of a series of selective MELK kinase inhibitors. Synthetized compounds exert excellent selectivity and potency in MELK inhibitions with the low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of breast cancer cell lines with different genetic background as well as with different MELK kinase levels. We identified several cell lines in which MELKi induced cell death with nanomolar ED50 values. Potent MELK inhibitors exhibited significant tumor growth suppression in xenograft studies using breast cancer cell lines in mice. Taken altogether, the presented data supports our rationale of using MELK kinase inhibitors as a novel and interesting approach for the cancer therapy. Conflict of interest: Ownership: Selvita S.A. Board of Directors: Selvita S.A. 454 Poster/Poster in the spotlight (Board P133) Exploratory biomarker analysis of first-line cobimetinib (C) + paclitaxel (P) in patients (pts) with advanced triple-negative breast cancer (TNBC) from the phase 2 COLET study M. Wongchenko1 , D. Miles2 , S.B. Kim3 , N. Xu4 , L. Amler1 , Y. Yan1 , B. Simmons5 , V. McNally6 , A. Brufsky7 . 1 Genentech, Inc., Oncology Biomarker Development, South San Francisco, USA; 2 Mount Vernon Cancer Centre, Medical Oncology, London, United Kingdom; 3 Asan Medical Center, University of Ulsan College of Medicine, Oncology, Seoul, South Korea; 4 Genentech, Inc., PD Biostatistics, South San Francisco, USA; 5 Genentech, Inc., Product Development Oncology, South San Francisco, USA; 6 Roche Products Ltd., PDC, Welwyn Garden City, United Kingdom; 7 University of Pittsburgh, Division of Hematology–Oncology, Department of Medicine, Pittsburgh, USA Background: Resistance to standard taxane-based chemotherapy is common in TNBC. Preclinical data suggest that MEK inhibition may overcome taxane resistance and enhance antitumor immune response. The safety and efficacy of combining C, a specific and allosteric inhibitor of MEK1/2, with P was explored in pts with metastatic/locally advanced TNBC and no prior systemic therapy for metastatic disease. Here, we present a retrospective exploratory biomarker analysis of nonrandomized pts. Material and Methods: The COLET study (ClinicalTrial.gov ID, NCT02322814; sponsored by F. Hoffmann-La Roche, Ltd.) consisted of a safety run-in (n ≈ 12) followed by a blinded 1:1 randomized stage (n ≈ 100 pts) to C + P or placebo (PBO) + P. Pts were treated with P 80 mg/m2 on days 1, 8, and 15 and C/PBO 60 mg/day on days 3−23 of each 28-day cycle. Tumor biopsies were collected prior to treatment, optionally at cycle 1 day 15 (C1D15), and at disease progression. FoundationOne genomic profile testing was performed by Foundation