Dose-response Matrix Research Articles

Correlation between tumor voxel dose response matrix and tumor biomarker profile in patients with head and neck squamous cell carcinoma

BackgroundWe have developed a novel imaging analysis procedure that is highly predictive of local failure after chemoradiation in head and neck cancer. In this study we investigated whether any pretreatment biomarkers correlated with key imaging parameters. MethodsPretreatment biopsy material was available for 28 patients entered into an institutional trial of adaptive radiotherapy in which FDG-PET images were collected weekly during treatment. The biopsies were immunohistochemically stained for CD44, EGFR, GLUT1, ALDH1, Ki-67 and p53 and quantified using image analysis. Expression levels were correlated with previously derived imaging parameters, the pretreatment SUVmax and the dose response matrix (DRM). ResultsThe different parameters of the SUVmax and DRM did not correlate with each other. We observed a positive and highly significant (p = 0.0088) correlation between CD44 expression and volume of tumor with a DRM greater than 0.8. We found no correlation between any DRM parameter and GLUT1, p53, Ki-67 and EGFR or ALDH1. GLUT1 expression did correlate with the maximum SUV0 and the volume of tumor with an SUV0 greater than 20. ConclusionsThe pretreatment SUVmax and DRM are independent imaging parameters that combine to predict local recurrence. The significant correlation between CD44 expression, a known cancer stem cell (CSC) marker, and volume of tumor with a DRM greater than 0.8 is consistent with concept that specific foci of cells are responsible for tumor recurrence and that CSCs may be randomly distributed in tumors in specific niches. Dose painting these small areas may lead to improved tumor control.

Effect of uncertainties in quantitative 18 F-FDG PET/CT imaging feedback for intratumoral dose-response assessment and dose painting by number.

Intratumoral dose response can be detected using serial fluoro-2-deoxyglucose-(FDG) positron emission tomography (PET)/computed tomography (CT) imaging feedback during treatment and used to guide adaptive dose painting by number (DPbN). However, to reliably implement this technique, the effect of uncertainties in quantitative PET/CT imaging feedback on tumor voxel dose-response assessment and DPbN needs to be determined and reduced. Three major uncertainties, induced by (a) PET imaging partial volume effect (PVE) and (b) tumor deformable image registration (DIR), and (c) variation of the time interval between FDG injection and PET image acquisition (TI), were determined using serial FDG-PET/CT images acquired during chemoradiotherapy of 18 head and neck cancer patients. PET imaging PVE was simulated using the discrepancy between with and without iterative deconvolution-based PVE corrections. Effect of tumor DIR uncertainty was simulated using the discrepancy between two DIR algorithms, including one with and one without soft-tissue mechanical correction for the voxel displacement. The effect of TI variation was simulated using linear interpolation on the dual-point PET/CT images. Tumor voxel pretreatment metabolic activity (SUV0 ) and dose-response matrix (DRM) discrepancies induced by each of the three uncertainties were quantified, respectively. Adverse effects of tumor voxel SUV0 and DRM discrepancies on tumor control probability (TCP) in DPbN were assessed. Partial volume effect and TI variations of 10 mins induced a mean±standard deviation (SD) of tumor voxel SUV0 discrepancies to be -0.7%±9.2% and 0%±4.8%, respectively. Tumor voxel DRM discrepancies induced by PVE, tumor DIR discrepancy, and TI variations were 0.6%±8.9%, 1.7%±9.1%, and 0%±7%, respectively. Partial volume effect induced SUV0 and DRM discrepancies correlated significantly with the tumor shape and FDG uptake heterogeneity. Tumor DIR uncertainty-induced DRM discrepancy correlated significantly with the tumor volume and shrinkage during treatment. Among the three uncertainties, PVE dominated the adverse effects on the TCP, with a mean±SD of TCP reduction to be 12.7%±9.8% for all tumors if no compensation was applied for. Effect of uncertainties in quantitative FDG-PET/CT imaging feedback on intratumoral dose-response quantification was not negligible. These uncertainties primarily caused by PVE and tumor DIR were highly dependent on individual tumor shape, volume, shrinkage during treatment, and pretreatment SUV heterogeneity, which can be managed individually. The adverse effects of these uncertainties could be minimized by using proper PVE corrections and DIR methods and compensated for in the clinical implementation of DPbN.

Inter/intra-tumoral dose response variations assessed using FDG-PET/CT feedback images: Impact on tumor control and treatment dose prescription

PurposeTo quantify inter/intra-tumoral variations of baseline metabolic activity and dose response. To evaluate their impact on tumor control and treatment dose prescription strategies. Methods and materialsTumor voxel baseline metabolic activity, SUV0, and dose response matrix, DRM, quantified using the pre-treatment and weekly FDG-PET/CT imaging feedback for each of 34 HNSCC patients (25 HPV+ and 9 HVP−) were evaluated. Inter/intra-tumoral variations of tumor voxel (SUV0, DRM) for each of the HPV− and HPV+ tumor groups were quantified and used to evaluate the variations of individual tumor control probabilities and the efficiency of uniform vs non-uniform treatment dose prescription strategies. ResultsTumor voxel dose response variation of all tumor voxels assessed using FDG-PET/CT imaging feedback had the mean(CV) = 0.47(47%), which was consistent with those of previously published in vitro tumor clonogenic assay. The HPV− tumors had the mean(CV) dose response, 0.53(49%), significantly larger than those of the HPV+ tumors, 0.45(43%). However, their baseline SUVs were opposite, 6.5(56%) vs 7.7(65%). Comparing to the inter-tumoral variations, both HPV−/+ tumor groups showed larger intra-tumoral variations, (53%, 58%) vs (20%, 31%) for the baseline SUV and (38%, 37%) vs (31%, 21%) for the dose response. Due to the large dose response variations, treatment dose to control the tumor voxels has very broad range with CV of TCD50 = 97% for the HPV− and 67% for the HPV+ tumor group respectively. As a consequence, heterogeneous prescription dose could potentially reduce the treatment integral dose for 92% of the HPV+ tumors and 78% of the HPV− tumors. ConclusionsThe study demonstrates that tumor dose response assessed using FDG-PET/CT feedback images had a similar distribution to those assessed conventionally using in vitro tumor clonogenic assay. Inter-tumoral dose response variation seems larger for HPV− tumors, but intra-tumoral dose response variations are similar for both HPV groups. These variations cause very large variation on the individual tumor control probability and limit the efficacy of dose escalation and de-escalation in conventional clinical practice. On the other hand, heterogeneous dose prescription guided by metabolic imaging feedback has a potential advantage in radiotherapy.

SynergyFinder: a web application for analyzing drug combination dose–response matrix data

Summary Rational design of drug combinations has become a promising strategy to tackle the drug sensitivity and resistance problem in cancer treatment. To systematically evaluate the pre-clinical significance of pairwise drug combinations, functional screening assays that probe combination effects in a dose-response matrix assay are commonly used. To facilitate the analysis of such drug combination experiments, we implemented a web application that uses key functions of R-package SynergyFinder, and provides not only the flexibility of using multiple synergy scoring models, but also a user-friendly interface for visualizing the drug combination landscapes in an interactive manner. Availability and implementation The SynergyFinder web application is freely accessible at https://synergyfinder.fimm.fi ; The R-package and its source-code are freely available at http://bioconductor.org/packages/release/bioc/html/synergyfinder.html . Contact jing.tang@helsinki.fi.

Higher Anti-Tumor Efficacy of the Dual HER3-EGFR Antibody MEHD7945a Combined with Ionizing Irradiation in Cervical Cancer Cells

The outcome of locally advanced cervical cancer (LACC) is dismal. Biomarkers are needed to individualize treatments and to improve patient outcomes. Here, we investigated whether coexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) could be an outcome prognostic biomarker, and whether targeting both EGFR and HER3 with a dual antibody (MEHD7945A) enhanced ionizing radiation (IR) efficacy. Expression of EGFR and HER3 was evaluated by immunohistochemistry in cancer biopsies (n = 72 patients with LACC). The antitumor effects of the MEHD7945A and IR combotherapy were assessed in 2 EGFR- and HER3-positive cervical cancer cell lines (A431 and CaSki) and in A431 cell xenografts. The mechanisms involved in tumor cell radiosensitization were also studied. The interaction of MEHD7945A, IR, and cisplatin was evaluated using dose-response matrix data. EGFR and HER3 were coexpressed in only in 7 of the 22 biopsies of FIGO IVB cervix cancer. The median overall survival was 14.6 months and 23.1 months in patients with FIGO IVB tumors that coexpressed or did not coexpress EGFR and HER3, respectively. In mice xenografted with A431 (squamous cell carcinoma) cells, MEHD7945A significantly increased IR response by reducing tumor growth and increasing cleaved caspase-3 expression. In A431 and CaSki cells, the combotherapy increased DNA damage and cell death, particularly immunogenic cell death, and decreased survival by inhibiting the MAPK and AKT pathways. An additive effect was observed when IR, MEHD7945A, and cisplatin were combined. Targeting EGFR and HER3 with a specific dual antibody enhanced IR efficacy. These preliminary results and the prognostic value of EGFR and HER3 coexpression should be confirmed in a larger sample.

Tumor Voxel Dose-Response Matrix and Dose Prescription Function Derived Using 18F-FDG PET/CT Images for Adaptive Dose Painting by Number

To construct a tumor voxel dose response matrix (DRM) and dose prescription function (DPF) for adaptive dose painting by number (DPbN) based on treatment feedback of fluoro-2-deoxyglucose (FGD) positron emission tomography (PET)/computed tomography (CT) imaging. FDG-PET/CT images obtained before and after chemoradiation therapy and at weekly chemoradiation therapy sessions for each of 18 patients with head and neck cancer, as well as the treatment outcomes, were used in the modeling. All weekly and posttreatment PET/CT images were registered voxel-to-voxel to the corresponding pretreatment baseline PET/CT image. Tumor voxel DRM was created using serial FDG-PET imaging of each patient with respect to the baseline standardized uptake value (SUV0). A tumor voxel control probability (TVCP) lookup table was created using the maximum likelihood estimation on the tumor voxel (SUV0, DRM) domain of all tumors. Tumor voxel DPF was created from the TVCP lookup table and used as the objective function for DPbN-based inverse planning optimization. Large intertumoral and intratumoral variations on both tumor voxels (SUV0, DRM) were identified. Tumor voxel dose resistance did not show correlation with its baseline SUV0 value and was the major cause of the tumor local failures. Tumor voxel DPF as the function of tumor voxel (SUV0, DRM) values also showed a very large intertumoral and intratumoral heterogeneity. Most human papillomavirus-negative tumorsrequire a treatment dose >100Gy to certain local tumor regions. These treatment doses, which are most unlikely to be implementable in conventional radiation therapy, can be achieved using adaptive DPbN treatment. Clinical feasibility was evaluated by comparing the adaptive DPbN treatment plan with the conventional intensity modulated radiation therapy plan. Tumor voxel (SUV0, DRM) provides an intratumoral prognostic map to target tumor locoregional-resistant regions. The corresponding TVCP or DPF provides a quantitative objective to optimize the intratumoral dose distribution for the individuals. The adaptive DPbN with FDG-PET/CT imaging feedback is feasible to implement in clinics.

High-Throughput Screening for Drug Combinations.

The identification of drug combinations as alternatives to single-agent therapeutics has traditionally been a slow, largely manual process. In the last 10years, high-throughput screening platforms have been developed that enable routine screening of thousands of drug pairs in an in vitro setting. In this chapter, we describe the workflow involved in screening a single agent versus a library of mechanistically annotated, investigation, and approved drugs using a full dose-response matrix scheme using viability as the readout. We provide details of the automation required to run the screen and the informatics required to process data from screening robot and subsequent analysis and visualization of the datasets.

Combined Targeting of BET Family Proteins and BCL2 Is Synergistic in Acute Myeloid Leukemia Cells Overexpressing S100A8 and S100A9

BackgroundThe 5-year survival rate for acute myeloid leukemia (AML) remains poor with most patients succumbing to relapse or refractory disease. Recently, the BCL2 specific inhibitor venetoclax has shown promising anti-leukemia activity in high-risk AML patients. Most patients, however, ultimately develop resistance to monotherapy and novel combination treatments with venetoclax are needed for patients with no other therapy options available. In this study we identified high expression of calcium binding protein S100A8/S100A9 to be associated with venetoclax resistance and looked for drug combinations to overcome the resistance in AML patient samples overexpressing S100A8 and S100A9.MethodsGene expression was assessed by RNA sequencing of AML patient samples and validated by qPCR. Gene enrichment analysis was performed on differentially expressed genes between venetoclax highly sensitive (n=3) and resistant (n=4) samples. Sensitivity of AML patient derived mononuclear cells was assessed to 304 different small molecule inhibitors by measuring cell viability after 72 h incubation with 5 different concentrations (1-10,000 nM) using the CellTiter-Glo (CTG) assay. A drug sensitivity score (DSS) was calculated based on a modified area under the dose response curve calculation. Drug combination studies were performed using AML cell lines resistant to venetoclax and confirmed with primary patient cells (n=15). Data of the drug combination studies were analyzed with the Zero Interaction Potency (ZIP) model by considering a dose-response matrix where two drugs are tested at 8 concentrations in a serially diluted manner. Statistical dependence between gene expression and drug sensitivity data was assessed by Pearson's correlation coefficient modelling.ResultsVenetoclax resistant AML patient samples were found to overexpress genes related to immune responses including inflammatory related proteins S100A8 and S100A9. The expression of S100A8 and S100A9 was upregulated in a sub-group of AML patients with somatic mutations in DNA methylation genes IDH2 and TET2 and chromatin modifier ASXL1. Functional studies with AML cell lines validated high expression of the S100 proteins in cells insensitive to venetoclax (NOMO-1, SKM-1 and SHI-1) whereas sensitive cell lines (MOLM-13, Kasumi-1 and ML-2) did not express the proteins. Integrated analysis of S100A8 and S100A9 expression and ex vivo drug sensitivity data indicated positive correlation of S100 expression with sensitivity to BET inhibitor (birabresib), PI3K inhibitor (TG100-115) and MEK1/2 inhibitor (AZD8330). In contrast, sensitivity to venetoclax and the FLT3 inhibitor quizartinib negatively correlated with S100 gene expression. Subsequently, we combined positively correlating drugs with venetoclax and tested the efficacy of these combinations in AML cell lines and patient samples. From the drug combination studies we found that BET inhibitor birabresib was able to overcome resistance to venetoclax treatment. The BCL2/BET inhibitor combination was synergistic in venetoclax resistant cell lines NOMO-1 and SKM-1, which express high-levels of S100A8 and S100A9 (Figure 1A). Efficacy of the combination on primary AML patient samples correlated with the expression level of the S100 genes. Nine of 11 high expression samples were sensitive to the venetoclax/birabresib combination (Figure 1B-C), whereas no synergy was observed in 3 of 4 samples with a low level of S100 expression.ConclusionsThe calcium binding proteins S100A8 and S100A9 are abundant in myeloid cells and are associated with poor prognosis in AML (Edgeworth et al, J Biol Chem. 1991; Nicolas et al, Leukemia 2011). From ex vivo and in vitro analyses of AML, we found that high expression of S100A8 and S100A9 is highly correlative with resistance to the BCL2 inhibitor venetoclax. In contrast, high S100A8 and S100A9 expression correlates with sensitivity to BET inhibitor birabresib. Interestingly, our studies showed that these two drugs act synergistically in venetoclax resistant AML cell lines and AML patient samples and may be a beneficial novel combination that should be further confirmed in preclinical and clinical investigations. [Display omitted] DisclosuresPorkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Long Term Outcomes of a Positron Emission Tomography (PET) -Based Adaptive Radiation Therapy Protocol in Locally Advanced Head and Neck Cancer.

To report the long-term results of our PET-based adaptive head and neck cancer (HNC) protocol and to confirm outcomes/toxicities are not inferior to historic controls. In addition, we also report PET response parameters that are predictive of locoregional control (LRC). Patients with node positive HNC were eligible for the study. Patients underwent concurrent cisplatin or cetuximab treatment with radiation therapy (RT). The prescription dose was 70 Gy to the gross disease and 60 Gy to uninvolved nodal basins using a concomitant boost in 35 fractions. Simulation employed a thermoplastic mask on a PET-CT simulator and was repeated following fractions 10 and 22. Additionally, weekly PET-CT was obtained for response assessment. Plans were modified at those points taking into account reduction in disease burden and anatomical changes secondary to weight loss. The above protocol patients were matched 1:1 to non-protocol patients based on primary site, performance status, clinical T and N stage, smoking status, treatment dose/technique, chemotherapy use, and P16 status. The matched pairs were then analyzed to compare outcomes and toxicities between the two groups. In addition, the initial and weekly PET scans were analyzed for predictive correlation on LRC using 18FDG retention index (RI) and dose response matrix (DRM). The DRM represents a tumor’s SUV voxel response matrix to treatment. This can map out heterogeneity between and within individual neoplastic sites (either primary site or nodal disease) that may be more or less sensitive/resistant to treatment. A total of 37 patients were included on the adaptive protocol. Baseline characteristics were as follows: The median age was 63 years, 76% were oropharynx, 62% were cT1-2, 81% were cN2, 68% were smokers, and P16 was positive in 65% of patients. 3-year LRC, distant metastasis free survival, and overall survival were 81%, 75%, and 87%. The rates of chronic grade 3 toxicities were 8.1% for dysphagia and esophageal stricture and 2.7% for hoarseness and dental problems. There were 23 matched pairs generated and no significant difference in clinical disease outcomes was noted between the two groups. There was a significant decrease in chronic grade 2 xerostomia in the adaptive group vs the non-adaptive group (27% vs 70% p=0.017). Of interest, mean initial RI and the volume of DRM above certain thresholds were significant predictors of LRC on univariate analysis (p<0.05). Adaptive treatment for patients with locally advanced HNC resulted in significantly reduced rates of xerostomia relative to a matched cohort treated with non-adaptive treatment, and it did so without compromising disease control rates. RI and volume of DRM above certain thresholds are predictive for LRC and may be important factors in clarifying the optimal adaptive RT strategy for HNC in terms of both reducing treatment toxicity and potentially as a mid-treatment response parameter for adaptive dose painting in resistant tumors.

SynergyFinder: a web application for analyzing drug combination dose-response matrix data.

Summary: Rational design of drug combinations has become a promising strategy to tackle the drug sensitivity and resistance problem in cancer treatment. To systematically evaluate the pre-clinical significance of pairwise drug combinations, functional screening assays that probe combination effects in a dose–response matrix assay are commonly used. To facilitate the analysis of such drug combination experiments, we implemented a web application that uses key functions of R-package SynergyFinder, and provides not only the flexibility of using multiple synergy scoring models, but also a user-friendly interface for visualizing the drug combination landscapes in an interactive manner. Availability and Implementation: The SynergyFinder web application is freely accessible at https://synergyfinder.fimm.fi; The R-package and its source-code are freely available at http://bioconductor.org/packages/release/bioc/html/synergyfinder.html.

Abstract 365: High throughput screen to evaluate combinations with ibrutinib in various B-cell malignancies

Abstract The primary objective of this study was to discover synergies leading to mechanistic insights and novel combinations for ibrutinib, a small-molecule inhibitor of Bruton's tyrosine kinase (BTK). Ibrutinib has been approved for relapsed/refractory (R/R) and del(17p) chronic lymphocytic leukemia, and R/R mantle-cell lymphoma (MCL). BTK is part of the B-cell receptor (BCR) signaling pathway, so it is of interest to examine synergy between ibrutinib and agents that target other aspects of the BCR pathway. These include PI3K and IRAK inhibitors and apoptosis inhibitors. Combinations were evaluated in a high-throughput, tumor microenvironment-directed format. Ibrutinib was combined with inhibitors of the BCR pathway and apoptosis, whose targets included MCL-1, BCL2, XPO1, and isoforms of PI3K, IRAK, and BRD. Histologies examined included follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), MCL, acute myeloid leukemia (AML), acute B-lymphoblastic leukemia, and Burkitt's lymphoma. Cell lines were screened in the presence of human marrow stromal-cell-conditioned media and B-cell receptor stimulation via anti-IgG/anti-IgM antibodies in a 72-hour cell viability ATP lite assay (384-well plate, 9×9 optimized matrix, 4 replicates). Dose response matrix screening was used to measure combination effects, which manifest as potency shifts or efficacy boosts. Combination effects can be characterized by comparing each data point to a combination reference model derived from single-agent curves using the Loewe additivity model. Ibrutinib demonstrated varying activity across cell lines. Combination activity was classified based on synergy score raw values. The best combination among tested compounds was with ABT-199, a BCL2i. This combination showed high or medium synergy in 2/5 DLBCL, 3/4 FL, and 2/5 MCL cell lines; this was also the only agent to show good synergy in AML (3/5 lines). Interestingly, ABT-737, a BCL2i that also targets BCL-XL, has previously been shown to be synergistic with ibrutinib in DLBCL lines. Synergy was also seen with other apoptotic agents such as MCL-1i and the epigenetic BETi JQ-1 across B-NHL, but in fewer lines. Among the PI3Ki, PI3Kα/δi did not show much activity, but the PI3Kδ/γi IPI-145 was synergistic in 2/5 DLBCL, 3/4 FL cell lines, and 1/5 MCL cell lines, a pattern very similar to the PI3Kδi CAL-101 (idelalisib). Ibrutinib also combined well with IRAK1/4i and XPO1i selinexor, with high activity in 1/5 DLBCL cell lines each, and medium activity in 1/5 DLBCL, 1/4 FL, and 2/5 MCL (XPO1i) and 1/5 DLBCL, 2/4 FL, and 1/5 MCL cell lines (IRAK1/4i). Synergy was mostly observed in B-cell malignancies, but interesting synergy was observed with ABT-199 in AML. In B-NHL, ibrutinib combined with BCL2i and PI3Ki showed the best combination activity. Ibrutinib was also synergistic with other agents in selected B-NHL lines with no observed antagonism, suggesting that further study in specific histologies is warranted. Citation Format: Shalini Chaturvedi, Michael Schaffer, Cuc Davis, Regina Aquino, Emily Stepanchick, Matthias Versele, Sriram Balasubramanian. High throughput screen to evaluate combinations with ibrutinib in various B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 365.

Coltuximab Ravtansine (SAR3419) Demonstrates Enhanced Activity in Combination with Bendamustine, Gemcitabine and Novel Targeted Agents Such As PI3K Inhibitors in Pre-Clinical Models of Relapsed and/or Refractory Non-Hodgkins Lymphoma (NHL)

Introduction:Relapsed/refractory B-cell NHL remains an area of significant medical need. CD19 is broadly expressed on B-cell malignancies making it an ideal target for antibody-drug conjugate (ADC) based therapy. Coltuximab ravtansine is a CD19-targeting ADC consisting of a CD19-targeting antibody conjugated to the maytansinoid anti-mitotic DM4. In preclinical studies, coltuximab ravtansine has shown potent, targeted activity against NHL cell lines and xenograft models. In early clinical trials, it has been well tolerated and has shown promising signs of efficacy as both a single agent and in combination with rituximab. In the STARLYTE Phase 2 trial coltuximab ravtansine monotherapy resulted in an ORR of 44% in R/R-DLBCL that included an ORR of 21% in hard-to-treat primary refractory patients (NCT01472887). Here we describe studies aimed at the identification of combination partners for coltuximab ravtansine to further optimize clinical benefit to R/R-NHL patients. We are employing a dual approach where we investigate combination of coltuximab ravtansine with multiple, novel targeted therapy partners whilst in parallel also investigating the combination of coltuximab ravtansine with chemotherapies commonly used in the late stage R/R-NHL setting.Methods:Coltuximab ravtansine and the DM4 payload were evaluated in a high throughput screen both as single agents and in combination with a selection of novel, emerging targeted agents across a panel of twenty NHL cell lines. The combinations were evaluated in a dose-response matrix and a statistical method was used to identify combination synergies significantly superseding baseline additivity values. The in vivo efficacy of coltuximab ravtansine was additionally assessed in combination with various clinically relevant chemotherapy agents in subcutaneous xenograft models of NHL.Results:Coltuximab ravtansine and DM4 both showed potent single agent activity against the entire panel of NHL cell lines with median GI50's of 770pM and 100pM, respectively. We observed a significant correlation in the cell line sensitivity of the two compounds suggesting that sensitivity to coltuximab ravtansine is driven, at least in part, by inherent sensitivity of cells to the cytotoxic effects of the DM4 payload. In vitro combination studies for coltuximab ravtansine were performed to identify targets or pathways that result in the most prominent combination effects across the cell line panel. Analysis of the in vitro combination dose-matrix revealed particularly strong synergy between coltuximab ravtansine and various inhibitors of the PI3K/AKT/mTOR axis. Studies to examine the synergism between coltuximab ravtansine and PI3K inhibitors in in vivo models of NHL are ongoing.In order to further determine the utility of coltuximab ravtansine as part of a potential combination regimen for the treatment of R/R-NHL, we assessed the combination of coltuximab ravtansine with the chemotherapy agents bendamustine and gemcitabine in vivo. As gemcitabine is typically used in combination we assessed the efficacy of a coltuximab ravtansine with rituximab and gemcitabine in vivo. In both cases the combination with coltuximab ravtansine was significantly more efficacious than the standard-of-care alone arms.Conclusions:Coltuximab ravtansine demonstrates synergistic activity in combination with multiple PI3K pathway inhibitors across a large panel of NHL cell lines. Additionally, we have shown that combination of coltuximab ravtansine with clinically relevant late stage treatments such as bendamustine and rituximab + gemcitabine is more efficacious than the chemotherapy regimens alone. These results support the continued development of coltuximab ravtansine in R/R-NHL in combination with chemotherapy regimens and suggest that a combination of coltuximab ravtansine with PI3K inhibitors may also be of interest in the clinical setting. DisclosuresSloss:ImmunoGen, Inc.: Employment, Equity Ownership. O'Callaghan:ImmunoGen, Inc.: Employment, Equity Ownership. Deckert:ImmunoGen, Inc.: Employment, Equity Ownership. Tsui:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

Searching for Drug Synergy in Complex Dose–Response Landscapes Using an Interaction Potency Model

Rational design of multi-targeted drug combinations is a promising strategy to tackle the drug resistance problem for many complex disorders. A drug combination is usually classified as synergistic or antagonistic, depending on the deviation of the observed combination response from the expected effect calculated based on a reference model of non-interaction. The existing reference models were proposed originally for low-throughput drug combination experiments, which make the model assumptions often incompatible with the complex drug interaction patterns across various dose pairs that are typically observed in large-scale dose–response matrix experiments. To address these limitations, we proposed a novel reference model, named zero interaction potency (ZIP), which captures the drug interaction relationships by comparing the change in the potency of the dose–response curves between individual drugs and their combinations. We utilized a delta score to quantify the deviation from the expectation of zero interaction, and proved that a delta score value of zero implies both probabilistic independence and dose additivity. Using data from a large-scale anticancer drug combination experiment, we demonstrated empirically how the ZIP scoring approach captures the experimentally confirmed drug synergy while keeping the false positive rate at a low level. Further, rather than relying on a single parameter to assess drug interaction, we proposed the use of an interaction landscape over the full dose–response matrix to identify and quantify synergistic and antagonistic dose regions. The interaction landscape offers an increased power to differentiate between various classes of drug combinations, and may therefore provide an improved means for understanding their mechanisms of action toward clinical translation.

Abstract 4765: Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent

Abstract SAR3419, a novel antibody-drug conjugate (ADC), is composed of a humanized monoclonal IgG1 anti-CD19 antibody (huB4) attached by a cleavable linker to DM4, a derivative of cytotoxic agent maytansine (an inhibitor of microtubule polymerization). CD19 expression is restricted to B-cells and treatment of CD19-positive human lymphoma cells with SAR3419 can result in cell cycle arrest followed by apoptosis. As traditional monotherapies can rarely treat cancer effectively, particularly in non-solid tumours, a combination high throughput screen (cHTS) was performed where SAR3419 was combined with two hundred potential enhancer compounds across a panel of twenty cell lines using a 6x6 combination dose response matrix. The enhancer compounds compose a library of approved, emerging oncology drugs, and well-defined molecular probes. Robust synergies were observed between SAR3419 and PI3K inhibitors and examined further. Target/pathway inhibitor redundancy for the class of compounds served to validate PI3K inhibition as important for combination synergy. Surprisingly, for several of the cell lines, the molecular mechanism important for SAR3419-dependent combination activity is distinct. For example, for the Daudi and REC-1 tumor cell lines, PI3K inhibitor synergy can be attributed to the toxin DM4. In contrast for SU-DHL4 and SU-DHL6 cell lines, little combination activity is observed with toxin alone while the conjugate is strongly synergistic. By examining three-way combination activities (PI3K inhibitor x DM4 x huB4) using high resolution matrices, we show that the anti-CD19 antibody huB4 contributes to the synergy in the SU-DHL6 cell line and linkage of DM4 to huB4 (SAR3419) shifts both the potency and maximum activity for PI3K inhibitor combinations. The results demonstrate that the antibody component of an ADC can contribute to combination activity for specific drug pairings in a drug and cell-type specific manner and has wide ranging implications with respect to combination drug design. Citation Format: Richard J. Rickles, Thomas P. Giordano, Shakira F. Cotard, Jill M. Grenier, Angela Romanelli, Ti Cai. Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2014-4765

Abstract 3405: Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines.

Abstract Background: Resistance to targeted therapies represents a key hurdle in the treatment of cancer. Preclinical data suggest that resistance is frequently driven by redundant growth factor signaling. For example, HGF has been reported to mediate resistance to BRAF inhibitors in V600E BRAF mutant melanoma. Furthermore, clinical data support these findings as circulating and tumor HGF levels have been shown to correlate with outcome in BRAF mutant melanoma patients treated with vemurafenib. In this study, we evaluated the ability of HGF / MET signaling to attenuate the effects of BRAF and MEK inhibitors in the context of V600E BRAF and NRAS mutant cell lines. Our data suggest that combined blockade of MET and MAPK pathway signaling may be required for a robust and durable clinical response in melanoma. Methods: To confirm the ability of HGF to rescue V600E BRAF mutant melanoma cell lines from the effects of vemurafenib, G361 and SKMEL5 cells were treated with vemurafenib alone or in combination with HGF. Time lapse imaging, viability assays and cell cycle analysis were used to measure treatment effects. Analogous experiments were performed with the selective MEK inhibitor PD0325901. Combinations of a potent and selective MET inhibitor (Compound A) with vemurafenib or PD0325901 were tested to evaluate their ability to attenuate the HGF-mediated resistance. To identify other cell lines rescued by HGF, thirteen V600E BRAF mutant melanoma cell lines were treated with a dose response matrix of vemurafenib and HGF. The ability of six additional growth factors to rescue cells from the effects of vemurafenib was also tested. Three NRAS mutant melanoma cell lines were also screened for HGF-mediated resistance to PD0325901. Results: Nine of thirteen V600E BRAF mutant melanoma cell lines were rescued from the effects of vemurafenib by co-treatment with HGF. Detailed analysis of G361 and SKMEL5 cells revealed that HGF was also capable of rescuing the cells from the effects of PD0325901. This HGF-mediated rescue was attenuated by treatment with combinations of vemurafenib + compound A or PD0325901 + compound A. Mechanistic cell cycle analysis of the HGF-mediated rescue of vemurafenib in G361 cells revealed an increase in proliferating cells and a reduction in growth arrested cells, whereas rescue of PD0325901 treatment occurred through a reduction in the percentage of dead or dying cells. Of the seven growth factors tested, HGF was the predominate growth factor capable of conferring resistance to vemurafenib. Furthermore, HGF rescued one of three NRAS mutant melanoma cell lines from the effects of PD0325901, highlighting the breadth of HGF-mediated resistance in melanoma cell lines. Conclusion: These findings demonstrate the potential role for HGF / MET signaling in mediating resistance to BRAF and MEK inhibitors in melanoma, and support the clinical evaluation of MET kinase inhibitors and neutralizing antibodies to HGF in this setting. Citation Format: Sean Caenepeel, Elaina Cajulis, Rick Kendall, Angela Coxon, Paul Hughes. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2013-3405