Tumor Drug Response Research Articles

Omics-based nanomedicine: The future of personalized oncology

The traditional “one treatment fits all” paradigm disregards the heterogeneity between cancer patients, and within a particular tumor, thus limit the success of common treatments. Moreover, current treatment lacks specificity and therefore most of the anticancer drugs induce severe adverse effects. Personalized medicine aims to individualize therapeutic interventions, based on the growing knowledge of the human multiple ‘-oms’ (e.g. genome, epigenome, transcriptome, proteome and metabolome), which has led to the discovery of various biomarkers that can be used to detect early stage cancers and predict tumor progression, drug response, and clinical outcome. Nanomedicine, the application of nanotechnology to healthcare, holds great promise for revolutionizing disease management such as drug delivery, molecular imaging, reduced adverse effects and the ability to contain both therapeutic and diagnostic modalities simultaneously termed theranostics. Personalizednanomedicine has the power of combining nanomedicine with clinical and molecular biomarkers (“OMICS” data) achieving improve prognosis and disease management as well as individualized drug selection and dosage profiling to ensure maximal efficacy and safety. Tumor’s heterogeneity sets a countless challenge for future personalized therapy in cancer, however the use of multi-parameter ‘omic’s data for specific molecular biomarkers recognition together with versatile drug delivery nanocarriers, which could target concomitantly and specifically tumor cells subpopulations, might heralds a brighter future for personalized cancer management. In this review, we present the current leading technologies available for personalized oncology. We discusses the immense potential of combining the best of these two worlds, nanomedicine and high throughput OMICS technologies to pave the way towards cancer personalized medicine.

Vascular endothelial growth factor-dependent spatiotemporal dual roles of placental growth factor in modulation of angiogenesis and tumor growth

Placental growth factor (PlGF) remodels tumor vasculatures toward a normalized phenotype, which affects tumor growth, invasion and drug responses. However, the coordinative and spatiotemporal relation between PlGF and VEGF in modulation of tumor angiogenesis and vascular remodeling is less understood. Here we report that PlGF positively and negatively modulate tumor growth, angiogenesis, and vascular remodeling through a VEGF-dependent mechanism. In two independent tumor models, we show that PlGF inhibited tumor growth and angiogenesis and displayed a marked vascular remodeling effect, leading to normalized microvessels with infrequent vascular branches and increased perivascular cell coverage. Surprisingly, elimination of VEGF gene (i.e., VEGF-null) in PlGF-expressing tumors resulted in (i) accelerated tumor growth rates and angiogenesis and (ii) complete attenuation of PlGF-induced vascular normalization. Thus, PlGF positively and negatively modulates tumor growth, angiogenesis, and vascular remodeling through VEGF-dependent spatiotemporal mechanisms. Our data uncover molecular mechanisms underlying the complex interplay between PlGF and VEGF in modulation of tumor growth and angiogenesis, and have conceptual implication for antiangiogenic cancer therapy.

Tissue slice grafts of human renal cell carcinoma: An authentic preclinical model with high engraftment rate and metastatic potential

ObjectiveDiscovery of curative therapies for renal cell carcinoma (RCC) is hampered by lack of authentic preclinical models. Tumorgrafts, generated by direct implantation of patient-derived tissues into mice, have demonstrated superior ability to predict therapeutic response. We evaluated “tissue slice grafts” (TSGs) as an improved tumorgraft model of RCC. Materials and methodsCores of fresh RCC were precision-cut at 300µm and implanted under the renal capsule of RAG2−/−γC−/− mice. Engraftment rate, histology, biomarker expression, genetic fidelity, and metastatic potential were evaluated. Magnetic resonance imaging (MRI) was tested as a noninvasive method to measure tumor volume, and response to a targeted therapy was determined. ResultsAll 13 cases of RCC engrafted and displayed characteristic histology and biomarkers. TSG volume quantified noninvasively by MRI highly correlated with graft weights, providing a unique tool for monitoring orthotopic growth. Moreover, in 2 cases, cancer cells from TSGs metastasized to clinically relevant sites, including bone. Microarray analysis and DNA sequencing demonstrated a high degree of correlation of global gene expression and von Hippel-Lindau (VHL) status between TSGs and parental tumors. Treatment of TSGs with sunitinib significantly decreased graft weight and mean vessel density compared with controls. ConclusionThe TSG model of RCC faithfully recapitulates tumor pathology, gene expression, genetic mutation, and drug response. The high engraftment rate and metastatic potential of this authentic model, in conjunction with the ability to generate large first-generation animal cohorts and to quantitate tumor volume at the orthotopic site by MRI, proffer significant advantages compared with other preclinical platforms.

Abstract 5589: Molecular signatures of in vitro drug response in lung cancer.

Abstract We are developing in vitro drug response signatures based on profiling of mRNA (Illumina WG6-V3 arrays), DNA mutation (COSMIC and deep sequencing), DNA copy number (Illumina Human1M-Duov3 SNP array) and DNA methylation (Illumina HumanMethylation450) from lung cancer cell lines to predict which drugs a patient's tumor is most likely to respond to. We have generated drug response phenotypes (MTS colorimetric assays) for ∼25 standard, targeted, and new chemotherapy agents and combinations for ∼ 100 non-small cell lung cancer (NSCLC) lines. All assays were done in triplicates or more and were very reproducible over time (r > 0.8). More than 10,000 MTS assays were generated and we designed a high-throughput database software named DIVISA (Database of In VItro Sensitivity Assays) for the purpose of storing and analyzing these assays. Some drugs showed a wide range of sensitivities (> 10,000-fold in IC50 values) and IC50 clustering indicates that drug response phenotypes can be grouped according to drug types. As part of a joint NCI SPORE, NCI SPECS, and DOD PROSPECT effort we have collected 275 clinically annotated frozen tumors with drug response information including 94 that represent lung cancer resection followed by adjuvant treatment. These specimens have also been profiled on Illumina expression arrays to formally test the clinical relevance of the tumor cell line signatures, and to verify that the signatures predict for response only in the presence of treatment and thus are not prognostic of survival in the absence of treatment. In addition, we have 3 primary tumor datasets totaling 96 specimens with EGFR mutation information, thus providing a validation set for EGFR tyrosine kinase inhibitor signatures. Using a weighted voting classification, cell line signatures predicted drug response in primary tumors with accuracies of ∼65% for targeted therapy (EGFR) but with somewhat lower accuracies for platin/taxane therapies suggesting that cell line predictive signatures may be better suited for targeted drugs. To facilitate translation to clinical trials we are working with High Throughput Genomics (HTG) to develop quantitative mRNA profiles that are performed on formalin fixed paraffin embedded (FFPE) material on a platform that can be transferred to a CLIA certified environment. These studies thus provide a preclinical human tumor model platform for systematically testing new drugs and for developing signatures to guide their most efficient use in early clinical tests. Funded by University of Texas SPORE in Lung Cancer (P50CA70907) and NCI SPECS Lung Cancer (CA114771). Citation Format: Luc Girard, Michael Peyton, Ignacio Wistuba, Yang Xie, Rachel Greer, Milind B. Suraokar, Carmen Behrens, Guanghua Xiao, John Heymach, David A. Wheeler, Caleb F. Davis, Kenneth Huffman, David S. Shames, Kevin R. Coombes, Adi F. Gazdar, David CL Lam, David G. Beer, John D. Minna. Molecular signatures of in vitro drug response in lung cancer. [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 5589. doi:10.1158/1538-7445.AM2013-5589

Abstract SY11-02: Detection of tumor-associated mutations in circulating DNA: clinical applications and experiences.

Abstract The detection of tumor-associated mutations is of paramount importance in the era of personalized medicine. Mutational testing is now a prerequisite for the use of some approved therapies (e.g., KRAS for cetuximab in colorectal cancer [CRC]; BRAF for vemurafenib in melanoma), and these clearly established correlations between tumor mutational status and drug response elevate the importance and urgency of evaluating such associations in clinical trials of investigational drugs. While archival primary tumor tissue is often used for mutational evaluation, such material has inherent limitations, which may be overcome by recent technological developments enabling the detection of tumor-associated mutations using plasma-derived DNA. For example, when a tumor tissue specimen is unavailable, use of plasma DNA would allow mutational status to be ascertained without the need for an invasive procedure to obtain a new tumor sample. In addition, it is now apparent that most patients treated with targeted therapies will eventually develop drug resistance, often via the acquisition of new tumor-associated mutations; these mutations may vary not only between patients but also between metastases within an individual patient. As such, the mutational status of an archival primary tumor specimen may not be relevant to guide the selection of subsequent therapies, and obtaining fresh tumor tissue from each metastasis that arises following the development of drug resistance is impractical. In such instances, mutational analysis of DNA derived from a real-time plasma sample obtained after the onset of drug resistance may offer advantages in terms of both availability and biological relevance, since new mutations acquired in response to a particular targeted therapy may be detectable in plasma DNA. Finally, mutational analysis of plasma DNA may be useful in clinical trials to evaluate potential correlations between mutational status and clinical outcome. For such exploratory analyses, the collection of archival tumor specimens from a high proportion of enrolled patients can be logistically and ethically difficult to achieve, not to mention of questionable relevance given that acquired mutations would not be detectable in these specimens. Collection of fresh tumor tissue samples at study entry would provide biologically relevant material, but can be problematic and costly to obtain in large, global clinical trials. Thus, the utility of plasma DNA for real-time mutational analysis in the clinical-trial setting offers several distinct advantages. Since DNA derived from both normal and tumor cells exists in the circulation, the detection of tumor-associated mutations in plasma DNA requires the ability to identify a relatively small number of mutant alleles among an excess of wild-type alleles. With the goal of identifying the most suitable technology for this purpose, we conducted a comparison of available methodologies and found that BEAMing technology (Beads, Emulsions, Amplification, and Magnetics) offered very sensitive detection of known tumor-associated mutations using plasma DNA, although this technique is not well suited for the discovery of previously unknown mutations. We have now used BEAMing to analyze more than 2,000 patient samples collected from oncology clinical trials, allowing us to evaluate a number of genes (e.g., KRAS, NRAS, HRAS, BRAF, PIK3CA, AKT1, EGFR, KIT, and PDGFRA) in different cancer types (e.g., CRC, gastrointestinal stromal tumors, hepatocellular carcinoma, non-small-cell lung cancer, and breast cancer). We have used BEAMing of plasma DNA both prospectively, to enroll patients into a phase I trial based on a molecular profile of interest, and retrospectively, to evaluate potential associations between mutational status and clinical outcome in phase II and III trials. In many of these trials, collection of both fresh plasma and archival tumor tissue from a subset of patients has enabled us to compare mutational status in patient-matched plasma and tumor samples. Our experiences with BEAMing of plasma DNA to determine tumor-associated mutational status will be discussed. Citation Format: Michael Jeffers, Chetan D. Lathia, Scott M. Wilhelm, Dimitris Voliotis, Dirk Laurent, Carol E. Pena. Detection of tumor-associated mutations in circulating DNA: clinical applications and experiences. [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 SY11-02. doi:10.1158/1538-7445.AM2013-SY11-02

Abstract 1636: Altering the function of cancer-associated fibroblasts via activation of Notch signaling to inhibit tumor growth.

Abstract Objectives. Cancer-associated fibroblasts (CAF) play critical roles in determining tumor growth, invasion, metastasis, drug response and tumor angiogenesis. We have previously demonstrated that activation of the Notch1 pathway confers normal human dermal fibroblasts a suppressive phenotype to melanoma growth. Here, we further investigated whether manipulation of Notch signaling in melanoma-associated fibroblasts (MAF) alter their regulatory phenotype by which affects melanoma growth. Methods. MAF were isolated from human metastatic melanoma and characterized. Notch pathway activity in MAF versus normal human dermal fibroblasts was examined using Notch pathway RT2-PCRArray and immunoblotting. Enforced activation of Notch pathway was achieved by transducing MAF with lentiviral vector encoding active form of Notch1 (NIC). The effect of active Notch1-engineered MAF on melanoma growth was tested by in vitro co-culture and in vivo co-xenografting animal model. Results. Isolated MAF are α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) positive. MAF exhibited a relatively lower Notch activity compared to normal human dermal fibroblasts. Enforced activation of Notch1 pathway suppressed cellular activities of MAF. Active Notch1-engineered MAF significantly inhibited melanoma cell growth in vitro and retarded xenografted human melanoma cell growth on mouse skin. Conclusions. Notch pathway activity is lower in MAF. Constitutive activation of Notch pathway confers MAF to a suppressive phenotype in regulating melanoma growth. Our study demonstrated that Notch signaling functions as a critical molecular switch in determining the regulatory role of tumor stromal fibroblasts and provided a novel approach to target tumor microenvironment through modulating stromal fibroblasts by manipulation of Notch signaling. Citation Format: Hongwei Shao, Mecker G. Moller, Omaida C. Velazquez, Zhao-Jun Liu. Altering the function of cancer-associated fibroblasts via activation of Notch signaling to inhibit tumor growth. [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 1636. doi:10.1158/1538-7445.AM2013-1636

Stem cells of the mammary epithelium and their role in breast cancer

Breast carcinogenesis is a multistage process in which genetic mutations in oncogenes and tumor suppressor genes cooperate with epigenetic alterations. Mammary tumor tissue is composed of normal and tumor cells. The tumor cells themselves seem to be clonal in origin, but heterogeneous in appearance. This suggests the existence of cancer stem cells (CSCs). They can originate from transformed tissue stem cells or progenitor cells with acquired self-renewal capacity and are able to escape from conventional therapy. Normal mammary stem cells (MaSCs) are required for the generation and maintenance of the mammary epithelium during the cyclical developmental stages of the mammary gland. They can reconstitute a functional gland upon transplantation into a mammary fat pad cleared from endogenous epithelium. CSCs share features with their normal counterparts; they are able to initiate tumor growth upon transplantation and express stem cell associated surface proteins, which can be utilized for their enrichment. Oncogenic transformation of MaSCs and their orthotopic transplantation in mice provides a promising strategy to model the early stages of mammary tumors and to follow the progression and drug responsiveness of these tumors in vivo. Oncogenic pathways e.g. estrogen and progesterone receptor (ER and PR), and signal transducers and activators of transcription (Stat) 3 and 5 signaling play important roles in normal development and malignant transformation of mammary cells. We are investigating the crosstalk between Stat5, Stat3 and ER signaling in the initiation of luminal tumors in mouse tumor models and their roles in tumor progression, metastasis formation and drug responsiveness. 1. Mammary stem cells in normal development and tumor formation The mammary gland is a unique organ, distinguished by its largely postnatal development. The mouse mammary gland shares many structural and functional features with the human breast and represents a valuable model for the study of organogenesis and tumorigenesis. During puberty, under control of the ovarian hormones, small rudimentary ductal structures invade into stromal fat pad. The mammary ducts grow at the terminal end buds (TEBs) and branch under the influence of steroid hormones during pregnancy. They are composed of an inner layer of luminal epithelial and an outer layer of myoepithelial located on the basement membrane. The expression of surface markers like EpCAM (epithelial cell adhesion molecule), CD24 (heat stable antigen,

Abstract LB-111: Mastospheres as a new 3D ex vivo breast cancer microtumors for preclinical drug testing

Abstract There is increasing evidence that three-dimensional (3D) tissue culture technologies have many uses within the preclinical assays in cancer. The 3D tumor model accurately reproduces the in vivo tumor phenotype and represents an additional tool for studying tumor biology and allowing better preclinical evaluation of anticancer drugs. One of the used models involves small tumor aggregates, termed spheres, that are obtained from mechanical tumor dissociation and that have shown the superiority of 3D culture over standard two-dimensional cell culture for mimicking the tumor biology and drug response observed in vivo. The aim of this study was to generate new ex vivo 3D models from breast (BC) cancer xenografts established from patients’ tumor fragments. These ex vivo mastospheres are easily obtained from mechanically dissociated fresh human BC tissue xenografted in Nude mice, in a similar way as colospheres from human colon cancer (Weiswald et al, Br J Cancer 2009, 101:473). In contrast to mammospheres described in the literature, 3D mastospheres are obtained from tumor fragments, without enzymatic tumor dissociation, without matrix substratum and in SVF supplemented medium. From a large panel of patient-derived BC xenografts already well characterized (Marangoni et al, Clin Cancer Res 2007, 13:3989; Cottu et al, Breast Cancer Res Treat 2011; Reyal et al, Breast Cancer Res 2012, 14:R11), we get mastospheres from 26 out of 36 (72%) xenografts. Mastosphere formation is scored on day 1 after culture according to the number of spheres/mg of dissociated xenograft tissue. Within mastospheres, we clearly distinguish 3 distinct morphologies: round, grape-like and aggregates. Histological analyses show also that mastospheres were formed only with proliferating cancer cells. In a reproducible way, dissociation of a given xenograft leads to a similar score and to a similar morphology. All BC subtypes (luminal, triple negative and HER2+ tumors) give rise to mastospheres. We noted that the 5 out of 5 xenografts (100%) able to metastasise in Nude mice (lung metastasis) form mastospheres, suggesting that the capacity to give mastospheres could be related to tumour aggressiveness, as already reported with colospheres and colon cancer. These different features prompted us to test the potential of these mastospheres in chemosensitivity assays. We first demonstrated that mastospheres can be kept viable a couple of days, consistent with ex vivo assay aim. For this purpose, we tested the sensitivity to cisplatin in two xenograft models with different in vivo response. We found that the paired mastospheres tested in viability assays mimicked these different response profiles (mean of IC50: 1.2 µM versus 15.5 µM). In conclusion, according to these preliminary data, mastospheres deserve additional investigation because of their interest as new ex vivo microtumour model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-111. doi:1538-7445.AM2012-LB-111

Modeling the Biomechanical Roles of Cancer Stem Cell Niche in Myeloma Development

Multiple myeloma, currently an incurable common hematological malignancy, is notorious for its recurrence and drug resistance. Our work led to the hypothesis that intercellular communications between myeloma initiating cells (MICs, a.k.a. myeloma stem cell) and bone marrow stromal cells (BMSCs) during the biomechanical remodeling of cancer stem cell niches play a key role in myeloma initiation and drug resistance. MICs prime BMSCs via SDF-1 paracrine; the activated BMSCs provide stiffer micro-environment, which boosts sustainable tumor growth under drug treatments. Understanding this pro-oncogenetic positive-feedback loop is thus crucial to the cure of the disease.In this study we used a 3D multi-scale agent-based modeling (ABM) approach to investigate the role of MIC-BMSC interactions in tumor growth and drug responses. The model is composed of four agent types, each representing a cell type: MIC, BMSC, PC (cancer progenitor cells), and MM (matured cancer cells). At intracellular level, specific signaling pathways were inferred from functional proteomics (reverse phase protein array) and gene expression data, and encapsulated into corresponding agent as ODEs. Intercellular communications were simulated on the agent-to-agent interfaces. The SDF-1 paracrine was described by PDEs of mass transfer. Intercellular events were extended to tissue level as tumor growth by a cancer stem cell lineage model (which includes MIC, PC, and MM agents). By seamlessly incorporating multi-scale events, our model quantitatively illustrated the interruption of BMSC contraction reduces the change of forming drug-resistant MIC strains and suppresses MIC colonogenesis, which was experimentally validated. Our work answered how the biomechanical remodeling of cancer stem cell niches via intercellular communications between tumor and stromal cells affects myeloma drug responses and prognosis, provided insights into myeloma development mechanisms, and cast new light on novel drug candidates and treatment strategies targeting the MIC-BMSC interactions.

The Roles of VHL-Dependent Ubiquitination in Signaling and Cancer

The function of tumor suppressor VHL is compromised in the vast majority of clear cell renal cell carcinoma, and its mutations or loss of expression was causal for this disease. pVHL was found to be a substrate recognition subunit of an E3 ubiquitin ligase, and most of the tumor-derived mutations disrupt this function. pVHL was found to bind to the alpha subunits of hypoxia-inducible factor (HIF) and promote their ubiquitination and proteasomal degradation. Proline hydroxylation on key sites of HIFα provides the binding signal for pVHL E3 ligase complex. Beside HIFα, several other VHL targets have been identified, including activated epidermal growth factor receptor (EGFR), RNA polymerase II subunits RPB1 and hsRPB7, atypical protein kinase C (PKC), Sprouty2, β-adrenergic receptor II, and Myb-binding protein p160. HIFα is the most well studied substrate and has been proven to be critical for pVHL’s tumor suppressor function, but the activated EGFR and PKC and other pVHL substrates might also be important for tumor growth and drug response. Their regulations by pVHL and their relevance to signaling and cancer are discussed.

Modeling CML Development and Drug Resistance Using iPSC Technology,

Abstract 3767Reprogramming of neoplastic cells to pluripotency provides a unique tool to personalize the exploration of tumor pathogenic mechanisms and drug resistance using iPSCs with patient-specific chromosomal abnormalities. We have developed a technology to generate transgene-free iPSCs from bone marrow and cord blood cells employing episomal vectors. Using this approach we created transgene-free iPSCs from a patient with CML in the chronic phase. CMLiPSCs showed a unique complex chromosomal translocation identified in the patinet's marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. Importantly, these CMLiPSCs are devoid of genomic integration and expression of reprogramming factors, which are incompatible for modeling tumor development and drug response (Hu et al. Blood 117:e109). We have also shown that these CMLiPSCs contain the BCR-ABL oncogene without any detectable mutations in its kinase domain. By coculture with OP9, we generated APLNR+ mesodermal cells, MSCs, and lin-CD34+CD45+ hematopoietic progenitors from CMLiPSCs, and control BMiPSCs from a normal subject and analyzed the levels of BCR-ABL protein and tyrosine-phosphorylated (pTyr) cellular proteins in the different cell populations. The highest level of BCR-ABL protein expression was found in the in undifferentiated iPSCs, however, the overall cellular pTyr levels was lower than the control BMiPSCs, suggesting that BCR-ABL kinase activity was suppressed in the CMLiPScs. Consistent with these findings, imatinib does not inhibit the growth and survival of these CMLiPSCs. The levels of BCR-ABL protein decreased upon differentiation with a major reduction observed when cells became mesoderm. Following differentiation of CMLiPSC-derived mesoderm into the MSCs and lin-CD34+CD45+ hematopoietic progenitors, the levels of BCR-ABL protein did not change significantly, indicating that the major epigenetic regulation of BCR-ABL expression occurs during the transition to mesoderm. In spite of the decrease in BCR-ABL expression, the total pTyr levels significantly increased following transition of CMLiPSCs to mesoderm and blood cells, suggesting recovery of BCR-ABL kinase activity during differentiation. Interestingly, we found that imatinib had no effect on CFC potential of the most primitive lin-CD34+CD45+ hematopoietic progenitors derived from CMLiPSCs, while significant inhibition in hematopoietic CFC potential was observed when we used the patient's bone marrow cells. Following expansion of lin-CD34+CD45+ progenitors in serum-free medium with cytokines, we found that more differentiated hematopoietic cells became imatinib sensitive. The differential response of progenitors versus more differentiated cells to imatinib recapitulate the clinical observation that CML stem cells display innate resistance to imatinib but their differentiated progenies become sensitive to this BCR-ABL kinase inhibitor. The iPSC-based models provide several advantages for the study of CML pathogenesis. iPSCs can provide an unlimited supply of hematopoietic cells carrying patient-specific genetic abnormalities. Using well-defined temporal windows and surface markers, distinct cell subsets with tumor-initiating/tumor-propagating potential after transplantation in immunodeficient mice could be identified and used for drug screening. iPSC models make it possible to address CML stem-cell potential at various stages of differentiation for which it may be difficult to obtain samples from the patient, for example, at the hemangioblast stage. They also provide a unique opportunity to explore the interplays between epigenetics and oncogene function, as we have demonstrated using the CMLiPSCs. The major unsolved question is why CML stem cells are naturally resistant to imatinib, and this question can be addressed using the iPS system. Disclosures:Slukvin:CDI: Consultancy, Equity Ownership.

A genomic approach to predict synergistic combinations for breast cancer treatment

We leverage genomic and biochemical data to identify synergistic drug regimens for breast cancer. In order to study the mechanism of the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) in breast cancer, we generated and validated genomic profiles of drug response using a series of breast cancer cell lines sensitive to each drug. These genomic profiles were then used to model drug response in human breast tumors and show significant correlation between VPA and SAHA response profiles in multiple breast tumor datasets, highlighting their similar mechanism of action. The genes deregulated by VPA and SAHA converge on the cell cycle pathway (Bayes Factor 5.21, and 5.94, respectively, p-value 10−8.6 and 10−9, respectively). In particular, VPA and SAHA upregulate key cyclin-dependent kinase (CDK) inhibitors. In two independent datasets, cancer cells treated with CDK inhibitors have similar gene expression profile changes to the cellular response to HDAC inhibitors. Together, these results led us to hypothesize that VPA and SAHA may interact synergistically with CDK inhibitors such as PD-033299. Experiments show that HDAC and CDK inhibitors have statistically significant synergy in both breast cancer cell lines and primary 3-dimensional cultures of cells from pleural effusions of patients. Therefore, synergistic relationships between HDAC and CDK inhibitors may provide an effective combinatorial regimen for breast cancer. Importantly, these studies provide an example of how genomic analysis of drug response profiles can be used to design rational drug combinations for cancer treatment.

Xenotransplantation of surgical specimens of human breast cancer directly into genetically engineered prolactin-humanized immunodeficient mice.

76 Background: Prolactin is an important hormone in mammary gland biology and is indispensible for lobulo-alveolar development during pregnancy and homeostasis during lactation. Prolactin has also been implicated in promoting proliferation, survival, and/ or differentiation of breast cancer. Importantly, murine and bovine prolactin, which are the major lactogens in current laboratory experimental conditions in vivo and in vitro, fail to effectively activate human prolactin receptors because of species incompatibility. Therefore, current established human breast cancer cell lines have been selected in the absence of human prolactin and do not represent the potential subgroup of cancers that depends on prolactin for survival or growth. Methods: In an effort to improve in vivo preclinical modeling of human breast cancer and to establish new human breast cancer lines that are prolactin-dependent or prolactin-sensitive, we used genetic-engineering to generate a human prolactin knock-in mouse line, which expresses physiological levels of human prolactin in place of mouse prolactin. We have crossed the prolactin-humanized mouse line into the immunodeficient NSG strain for effective xenotransplantation studies. Fresh surgical specimens from breast cancer patients were directly transplanted orthotopically into female mice within 2 hours of surgery. We then followed the growth of the xenograft tumors over time and retransplanted the tumors if they engrafted. Results: Our initial data suggested about a take rate of ~35% for the xenografted tumors. Estrogen receptor-positive luminal tumors usually have a very low engraftment rate in immunodeficient mice. However, we observed a relatively high rate of engraftment for luminal tumors. Conclusions: This new prolactin-humanized mouse model is highly relevant for in vivo exploration of tumor biology and drug response testing of breast cancer, especially for luminal subtypes.

Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level.

In vitro chemosensitivity assay for patients with gynecologic sarcoma.

e20513 Background: This study describes the in vitro drug response of tumors from patients with gynecologic sarcoma and explores the clinical implication of in vitro assessment. Methods: Tumors from patients diagnosed with ovarian or uterine sarcoma that were analyzed for response to any of seven drugs (carboplatin [Carb], cisplatin [Cis], paclitaxel [Ptx], docetaxel [Dtx], doxorubicin [Dox], gemcitabine [Gem], ifosfamide [Ifo]) with an in vitro chemosensitivity assay (ChemoFx, Precision Therapeutics, Inc, Pittsburgh PA) from February 1, 2007 to May 3, 2010 were included for analysis. Tumor response was determined from dose response curves. To evaluate combination therapy, tumors were considered sensitive if responsive to at least one of the two drugs. Fisher’s exact test was used to compare group differences. Results: 68 tumors were available for analysis, 21 of which originated in the ovary and 47 in the uterus. 54 of the tumors were primary and 14 recurrent. The most common histologic type was carcinosarcoma (n=53), followed by leiomyosarcoma (n=13), and other (2). 24% of tumors tested were responsive to Carb, 30% to Cis, 33% to Ptx, 9% to Dtx, 18% to Dox, 26% to Gem, 27% to Ifo, 40% to Carb+Ptx, 39% to Cis+Ifo, 48% to Ptx+Ifo, 24% to Gem+Dtx, and 27% to Gem+Dox. The ovarian sarcomas tended to be more responsive in vitro than the uterine sarcomas and the primary more than the recurrent tumors. Carcinosarcomas tended to be more chemoresponsive in general and significantly more responsive than leiomyosarcomas to Ifo (P=0.02). Conclusions: These in vitro chemosensitivity results indicate that ovarian sarcomas tend to be more chemoresponsive than uterine sarcomas and that carcinosarcomas are more chemoresponsive than leiomyosarcomas. Of note, carcinosarcomas were significantly more responsive to Ifo than were leiomyosarcomas. The differential patterns of response observed in these gynecologic sarcomas suggest that in vitro tumor response testing may enable physicians to individualize chemotherapy regimens for their patients.

Studying Therapy Response and Resistance in Mouse Models for BRCA1-Deficient Breast Cancer

Worldwide, more than one million women are diagnosed with breast cancer every year, making it the most common malignancy of females in the developed world. Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 account for 4-6% of all breast cancer cases, and mutation carriers have a lifetime risk of 80% for developing breast cancer and 40% for developing ovarian cancer. Current treatment options are limited and often do not lead to cure. In the 17 years since the discovery of BRCA1, the generation of mouse models for BRCA1 deficiency has greatly aided our understanding of it's role in tumorigenesis. In contrast to human BRCA1 mutation carriers, mice carrying heterozygous mutations in Brca1 did not develop spontaneous tumors. This led to the generation of conditional mouse models in which tissue-specific Brca1 deletion induces formation of mammary tumors that closely resemble human BRCA1-mutated breast tumors. These models have proven useful for studying BRCA1-related tumor development, drug response and resistance. BRCA1-deficient cancer cells are defective in DNA repair mediated by homologous recombination (HR) and therefore highly sensitive to DNA-damaging agents such as platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors. However, BRCA1-mutated tumors can develop resistance to these drugs; hence improved treatment strategies are critical. Existing mouse models have already proven useful for preclinical testing of (combinations of) therapeutic agents that may be beneficial for the treatment of patients with BRCA1-mutated tumors. In this review, we discuss the progress made towards modeling BRCA1-deficient breast cancer in mice and what we have learned from preclinical studies using these models.

Targeted Therapies Development in the Treatment of Advanced Nonsmall Cell Lung Cancer

Nonsmall cell lung cancer (NSCLC) is diagnosed in the majority of patients in advanced stage of disease. In this setting, the prognosis is very poor with median survival of 9–12 months and palliative chemotherapy being the standard of care. However, chemotherapy in advanced disease seems to have reached a plateau with no doublet combination shown to be clinically superior to the others. Advances in the knowledge of tumor biology and mechanisms of oncogenesis has granted the singling out of several molecular targets for NSCLC treatment. Targeted therapies are designed to interfere with specific aberrant biologic pathways involved in tumorigenesis. A large amount of preclinical in vivo and in vitro data have been gathered on the antitumor properties of a number of new biological agents, both as single agents and combined with other conventional treatment modalities such as chemotherapy. Several targeted therapies have been introduced in cancer treatment, and in particular gefitinib and erlotinib—two epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKI) and bevacizumab (an antiangiogen agent) have been introduced in advanced NSCLC, and others are in current clinical development. The present issue includes 5 papers dedicated to targeted therapies of NSCLC, 2 on basic science and 3 focusing on medical treatment. The first paper by K. S. S. Enfield et al. deals with the role of microRNA gene dosage alteration assessment in order to define potential tumor drug resistance and response to chemotherapy. The authors, by instigating a logical stepwise strategy, have identified specific microRNAs that are associated with resistance to several chemotherapeutic agents and provide a proof-of-principle demonstration of how these various databases may be exploited to derive relevant pharmacogenomic results. The second paper is a complete review on targeted therapies in NSCLC. The authors describe the identified tumor target and biomarkers opening the field of personalized medicine. They deal with already registered drugs in current clinical practice and the new agents under current investigation. R. Costanzo et al. in the third paper reviewed the role of gefitinib in the treatment of advanced NSCLC harbouring an activating EGFR mutation. Gefitinib and erlotinib can be considered the standard first-line treatment in this subgroup of patients based on their superiority showed in terms of progression-free survival, response rate, and quality of life as compared to chemotherapy. To date gefitinib has provided data on Asians only and has not been approved in the USA while erlotinib, with data on both Asians and Caucasians, has been approved worldwide. When added to chemotherapy, continuous treatment with tyrosine kinase inhibitors (TKIs) offers no benefit in comparison with chemotherapy alone. This may be due to the antagonistic effect of TKIs and chemotherapy: cells are pushed into the G-0 phase of the cell cycle and are therefore resistant to cytotoxic drugs. In the fourth paper M. Zwitter et al. report the results of a phase II study in patients with advanced NSCLC with EGFR mutation using an intermittent schedule of erlotinib and chemotherapy with cisplatin plus gemcitabine in order to overcome the previously described negative interference of drugs. The authors observed even on a small patients' population interesting results and mild toxicity. In the last, paper Escobar M. et al. make a state-of-the-art review based on the available literature regarding the use of bortezomib as a single agent or in combination with chemotherapy in patients with lung cancer. The ubiquitin-proteasome system like other cellular pathways is critical for the proliferation and survival of cancer cells; thus, proteosome inhibition has become a very attractive anticancer therapy. Cesare Gridelli Enriqueta Felip

Detailed Molecular Fingerprinting of Four New Anaplastic Thyroid Carcinoma Cell Lines and Their Use for Verification of RhoB as a Molecular Therapeutic Target

Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.

QA procedures for multimodality preclinical tumor drug response testing

There are growing expectations that imaging biomarkers for tumor therapeutic drug response assessment will speed up preclinical testing of anticancer drugs in rodent models. The only imaging biomarker presently approved by the U.S. Food and Drug Administration is tumor size measurement based on either World Health Organization (WHO) criteria or Response Evaluation Criteria in Solid Tumors (RECIST). Frequently, preclinical data are accumulated from multiple research centers on multiple continents using scanners from different manufacturers and sometimes even using different imaging modalities. Very expensive cancer drug response studies can be compromised by inadequate controls to assure precision and accuracy of tumor size measurements. This project was undertaken to develop standardized quality assurance (QA) procedures using a multimodality preclinical tumor response phantom to validate the accuracy of tumor size measurements based on WHO criteria, RECIST, or global tumor volume criteria for evaluation of cytostatic drugs. A tumor response phantom containing five low contrast test objects designed to simulate animal tumor models was made of tissue-mimicking materials. Imaging of the phantom was performed using three modalities in two institutions to evaluate size measurement of tumor-simulating test objects. Evaluation of tumor measurements from the three commonly used imaging devices in two different institutions for monitoring tumor size changes showed that a single phantom for multiple modalities was feasible. The tumor response phantom validated precision and accuracy of tumor response data input from ultrasound, computed tomography, and/or magnetic resonance imaging devices. Measurement results show that the standardized QA procedures using the tumor response phantom can provide a rationale check of data that excludes input from poorly maintained instruments, inadequate measurement protocols, or random operator error that frequently introduce unacceptable variability or systematic error in multiple institutions trials.

Tissue-Engineered Three-Dimensional Tumor Models to Study Tumor Angiogenesis

Cell-microenvironment interactions play a critical role in the transformation of normal cells into cancer; however, the underlying mechanisms and effects are far from being well understood. Tissue Engineering provides innovative culture tools and strategies to study tumorigenesis under pathologically relevant culture conditions. Specifically, integration of biomaterials, scaffold fabrication, and micro/nano-fabrication techniques offers great promise to reveal the dynamic role of chemical, cell-cell, cell-extracellular matrix, and mechanical interactions in the pathogenesis of cancer. Due to the central importance of blood vessel formation in tumor growth, progression, and drug response, this review will discuss specific design parameters for the development of culture microenvironments to study tumor angiogenesis. Tumor engineering approaches have the potential to revolutionize our understanding of cancer, provide new platforms for testing of anti-cancer drugs, and may ultimately result in improved treatment strategies.