Primary Patient Material Research Articles

Abstract 4543: High-throughput combination screening identifies novel drug-drug pairings for a Bruton's tyrosine kinase inhibitor against the ABC subtype of diffuse large B-cell lymphomas.

Abstract The vast majority of cancer treatments currently administered to patients consist of combinations of more than one drug via routine infusions that adhere to specific dosing schedules. It is thought that this multi-arm and time dependent approach will kill not only the tumor cells within the primary site, but also any metastatic lesions, and importantly, any circulating tumor cells (CTCs) which may still exist in the blood. Combination therapies have also been developed as a means to reduce general cytotoxic side effects and prevent resistance and recurrence. Our labs have recently developed a high throughput screening platform to test compounds in pair-wise combinations to rapidly and systematically identify additive, synergistic and antagonistic drug combinations. This HTS capability can easily generate hundreds of dose response matrices in a single study and can increase significantly when applied to multiple cell lines. We are using this combination screening platform with in vitro models from both established cell lines and primary patient material, and we expect it will serve as a very valuable tool and a starting point when designing clinical trials after these combinations show promise within in vivo models. In a proof of concept study, we tested combinations of compounds that effectively kill 2 established lines of the ABC sub-type of diffuse large B-cell lymphoma (DLBCL); TMD8 and HBL1. We will present the infrastructure and methods that we have developed to implement the combination screens, visualize data from the combination dose response comparisons and numerically compare combinations in terms of their response matrices. We will also describe how this approach allows us to investigate putative polypharmacological effects that play a role in compound combination responses. Finally, we will show the results of a combination screen with TMD8 and HBL1 cells, including the identification of a novel drug-drug combination for the BTK inhibitor ibrutinib (PCI-32765) which is of both basic and translational interest for the treatment of DLBCL. Citation Format: Lesley A. Mathews, Rajarshi Guha, Paul Shinn, Ryan M. Young, Kian-Huat Lim, Jonathan Keller, Dongbo Liu, Adam Yasgar, Crystal McKnight, Matthew B. Boxer, Damien Y. Duveau, Jian-kang Jiang, Sam Michael, Bryan T. Mott, Paresma R. Patel, William Leister, David J. Maloney, Christopher A. LeClair, Ganesha Rai, Ajit Jadhav, Brian D. Peyser, Christopher P. Austin, Scott Martin, Anton Simeonov, Marc Ferrer, Louis Staudt, Craig J. Thomas. High-throughput combination screening identifies novel drug-drug pairings for a Bruton's tyrosine kinase inhibitor against the ABC subtype of diffuse large B-cell lymphomas. [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 4543. doi:10.1158/1538-7445.AM2013-4543

Abstract 4170: Mutation and chemosensitivity profiling of 18 human melanoma cell lines

Abstract Malignant melanomas account for a substantial proportion of cancers worldwide and are largely resistant to conventional therapy. The heterogeneity and the invasiveness of melanomas present notable challenges for anti-cancer treatment. Recently, several mutations in RAF, RAS, and PTEN were identified that allow for prognosis of cancer development and progression. Therefore, inhibitors of the RAF-MEK-ERK pathway form attractive candidates for molecular-based targeted treatment of melanoma cancer. Here, we present the molecular characterization and chemosensitivity profiling of 18 human melanoma cell lines. Eight of these were established by Oncotest from human tumor xenografts originally derived from primary patient material. The molecular profiling included mutational analysis for BRAF, PIK3CA, KRAS, NRAS, TP53, and PTEN by sequencing. The chemosensitivity profiles were determined in vitro using a fluorescence-based cytotoxicity assay and included commonly used chemotherapeutic agents for treatment of melanoma, targeted agents (sorafenib and bortezomib) and various BRAF (PLX-4032, PLX-4720, GDC-0879) and MEK (CI-1040, PD0325901) inhibitors. The clinically relevant BRAF V600E substitution was the most prevalent mutation and was found in more than 50% of the melanoma cell lines. The chemosensitivity profiling revealed diverse patterns of selectivity and potency with mean IC50 values between 0.4 nM (vinblastine) and 8.3 µM (carboplatin). Each of the BRAF inhibitors tested was clearly more active in melanoma cell lines carrying a BRAF mutation. On average, IC50 values of BRAF inhibitors were more than 20-fold lower for the BRAF V600E mutated cell lines compared to the wild type cell lines. However, the BRAF mutated cell line MEXF 462NL was resistant towards BRAF inhibitors and it is warranted to compare its gene expression profile and mutational status with BRAF mutated cell lines responding to BRAF inhibitors. By using an ex vivo clonogenic assay, similar activity patterns for BRAF inhibitors were found, with mutated BRAF tumor xenografts showing markedly higher sensitivity. Potential synergistic activity of BRAF inhibitors with other inhibitors of the RAF-MEK-ERK pathway or with standard chemotherapeutic agents will be presented. The mutational status of selected cancer related genes, as well as responsiveness towards many cytotoxic and targeted agents was analyzed for 18 melanoma cell lines. This extensive genotypic and phenotypic characterization makes this melanoma cell line panel a valuable tool for in vitro profiling of novel compounds, in particular inhibitors of the RAF-MEK-ERK pathway. 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 4170. doi:1538-7445.AM2012-4170

A Novel Mouse Model of Multiple Myeloma Representative of Human Disease and Its Use in Preclinical Therapeutic Assessment

Abstract 2907In order to aid the pre-clinical development of novel therapeutics for multiple myeloma, an in vivo model which recapitulates the human condition in particular tumor growth patterns and response to treatment is required. An important feature of such a model is the interaction of the myeloma cells with the bone marrow microenvironment as this is known to modulate tumor activity and protect against drug-induced apoptosis. We have developed a model with myeloma restricted to the bone marrow, which proceeds rapidly from initial inoculation to disease progression, and possesses a range of chemo-sensitive markers with which to monitor anti-tumor response.Female NOD/SCID γcnull mice were injected inta-osseously with luciferase-tagged myeloma cell lines. Disease progression was monitored weekly by bioluminescent imaging (BLI) and measurement of paraprotein levels (ELISA). These methods were compared to histological assessment of tumor infiltration and MRI which provided a quantitative measurement of progression. On T2-weighted images tumor was identified as a hyperintense signal enclosed within cortical bone. Tumor burden was quantified from regions of interest drawn on the periphery of the hyperintense signal.Luciferase-tagged cells engrafted by 3 weeks at the injection site and progressed to the femurs, spine and pelvis from week 4. BLI showed a significant increase in radiance from 5.6×105 to 43.0×105p/s/cm2/sr between weeks 5 and 7 (p<0.05). Quantification of tumor volume by MRI showed a significant increase from 6.4mm3 to 27.6mm3 between weeks 4 and 8 (p<0.05) and μCT demonstrated lytic disease. Serum levels of Igλ increased from 860ng/ml to 4325ng/ml during this period (p<0.05), which mirrored the changes seen with BLI and MRI. Flow cytometry and histology confirmed the confinement of CD138 positive myeloma cells within the bone. These results indicate successful engraftment of human myeloma cell lines with induction of myeloma in a pattern similar to the human condition.We have adapted this model to study primary patient material. 10 mice were implanted with samples from 3 cases of plasma cell leukemia with complex cytogenetics. 5 of these developed myeloma confined to the bone marrow, 2 with additional plasmacytoma localized at the injection site, over a period of 1–5months. We have characterized the original patient cells with gene expression, SNP based gene mapping and have characterized the nature of the engrafted cells using similar technology.We have also shown the model is suitable for preclinical assessment of anti-myeloma agents using bortezomib and a novel aminopeptidase inhibitor, tosedostat (CHR-2797). Non-treated mice displayed a significant increase in radiance from 16.13×105 to 69.00×105p/s/cm2/sr (p<0.01). In comparison, in the bortezomib and tosedostat treated groups no significant increase in radiance was seen (bortezomib: 5.22×105 to 1.12×105 p/s/cm2/sr; tosedostat: 9.92×105 to 13.78×105p/s/cm2/sr). Paraprotein levels mimicked these changes in BLI. At the end of treatment Igλ levels in control, bortezomib and tosedostat treated mice were 2473.7, 132.5 and 923.0ng/ml, respectively. Igλ levels in both treatment groups were significantly different from control (p<0.001). Average tumor volumes derived from MRI were significantly different in bortezomib (14.7mm3) and tosedostat treated (23.4mm3) groups compared to non-treatment (33.0mm3). The volumes for the bortezomib treated group showed no significant difference from control mice. In addition, there was a decrease in CD138 expression by flow cytometry in bone aspirates from treatment groups compared to control which was mirrored in histological samples.In conclusion using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma with secretion of paraprotein, disease confined to the bone marrow, lytic bone lesions and spinal compression. In addition, this model is suitable for assessing the efficacy of novel therapeutics in vivo, using a number of non-invasive tumor markers such as BLI and MRI. Disclosures:Morgan:J&J: Honoraria, Speakers Bureau. Davies:J&J: Honoraria, Speakers Bureau.

Abstract C110: Mutation and chemosensitivity profile of a panel of 25 patient-derived melanoma xenografts.

Abstract Melanomas account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of melanomas present unique therapeutic challenges. Recently a number of somatic mutations were determined, which influence the prognosis and thus therapeutic needs markedly. Here we present a panel of patient-derived melanoma xenografts, established by Oncotest from primary patient material. From 80 patient explants, growth in serial passage was observed in 43% of the cases. Molecular profiling included mutational analysis for BRAF, PI3Kalpha, KRAS, NRAS, TP53, CTNNB1, MET and JAK3 using OncoCarta™ and exon sequencing. The chemosensitivity profile was determined in vitro using 2D and 3D culture assays. Clinically relevant BRAF mutations were the most prevalent mutation and were found in almost two thirds of the melanoma models. The second most common gene mutated was NRAS, with about one quarter of the models harbouring activating mutations of NRAS. PI3Kalpha mutations were rare, and for KRAS no mutations were detected. Most of the melanomas showed multiple mutations of the investigated genes. Chemosensitivity profiling included cytotoxic (e.g. cisplatin, carboplatin, vinblastine, vincristine, ifosfamide, paclitaxel, and docetaxel), as well as targeted drugs (e.g. sorafenib and PLX-4720). The compounds showed diverse patterns of selectivity and potency: Differential activity was determined e.g. for vinblastine, vincristine, paclitaxel, and docetaxel, but also for the BRAF inhibitor PLX-4720. PLX-4720 was most active in melanoma models carrying the BRAF mutation as determined in monolayer and soft agar assays. Activity of vincristine revealed a correlation of sensitivity with a BRAF wild type genotype and resistance with BRAF mutation. The latter result was also described recently for melanoma patients with BRAF mutations, showing a diminished duration of response to treatment with vincristine, dacarbazine, bleomycin, lomustine, and human leukocyte interferon. A collection of 25 patient-derived melanoma models is presented. Their mutational status as well as their responsiveness towards many cytotoxic and targeted agents was analyzed. This extensive genotypic and phenotypic characterization makes them a valuable tool for hypothesis generation and testing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C110.

Abstract 3461: A chemical screen for the identification of novel therapeutics for pediatric brain tumors

Abstract Treatment of brain tumors in children has greatly improved over the past decades, but current treatment protocols lack specificity, killing not only tumor cells, but also “healthy” normal brain cells. Moreover, current therapy still fails in approximately 20% of medulloblastoma patients, 50% of ependymomas and 80% of children suffering from high grade glioma. Treatment mostly consists of surgery, followed by chemotherapy and radiotherapy, and although this kind of therapy is among the most effective treatment options available, it is not sufficient. Therefore, new therapeutic drugs are required that specifically kill brain tumor cells and/or sensitize to irradiation, sparing normal brain tissue. In order to identify such novel compounds, drug screens were set up in a semi-high throughput format, using Gaussia luciferase (Gluc) activity as cell viability readout. As opposed to other forms of luciferase, Gluc is secreted in the medium, allowing the measurement of luciferase activity without lysing the cells. Since the Gluc signal has been shown to be linearly related to cell number, this assay can be used to determine cell viability at different points after drug treatment. In addition, an Acumen eX3 laser scanning cytometer was used in parallel, to determine cell number and cytometric parameters. We screened two small molecule libraries (2960 compounds) for cell growth inhibitory or radiosensitizing properties on pediatric high-grade glioma, ependymoma and medulloblastoma cell lines and primary patient material. Cells were incubated with a concentration of 1µM of the different compounds, either as single agent or in combination with 3 Gray irradiation, and cell viability was determined over a period of 4 days of incubation. A reduction of &amp;gt;75% of cell growth was considered to be significant. In general, 4 different groups of small molecules could be distinguished: 1) compounds that did not affect cell proliferation compared to controls, 2) compounds that block cell cycle progression, 3) compounds that induce cell death independently of irradiation, and 4) radiosensitizers. A total of 114 compounds was identified that inhibited cell growth and/or sensitized towards irradiation. Among the compounds that induced cell death were microtubule inhibitors, alkylating agents, topoisomerase inhibitors and anthracyclins. Besides these known anti-cancer agents we also identified a large number of cardiotonic drugs, benzimidazoles and limonoids that effectively killed brain tumor cells. We are currently investigating these hits and expanding our drug screens with additional primary pediatric brain tumor cells. In addition, we are repeating the screens on non neoplastic primary human cells, including astrocytes, to assess the therapeutic index of these drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3461. doi:10.1158/1538-7445.AM2011-3461

Abstract 3209: Gene expression micro array analysis of diagnosis and matched relapse pediatric AML samples indicates that immune regulatory pathways and epigenetic factors are involved in disease progression

Abstract Due to optimization of induction treatment, approximately 90% of AML pediatric patients achieve a complete remission. However, relapses still occur in 30-40% of patients, with dismal outcome. In order to find new options for targeted treatment, we aim to identify key molecules and signaling pathways involved in the development of relapses. To this end we use gene expression profiling. Total mRNA of initial diagnosis and matched relapse bone marrow samples (&amp;gt;80% blasts) was obtained from 27 pediatric AML patients and used for Affymetrix HGU 133 plus 2.0 microarrays. For statistical analyses we used BioConductor packages, Significance Analysis of Microarrays (SAM) and BRB Arraytools. Pathway analysis was performed using Ingenuity and Gene Set Enrichment Analysis (GSEA). Analysis of paired diagnosis and relapse samples revealed large inter-patient differences in the number of genes that were differentially expressed. Unsupervised cluster analysis showed that for 47% of patients the diagnosis and relapse sample cluster next to each other. The remaining paired samples clustered more distant and interestingly the majority of these pairs also showed changes in mutation status of FLT3 or RAS. The former have been described by us to be associated with shorter time to relapse (Bachas et al, Blood, 2010). Pathway analysis of the differentially expressed genes (&amp;gt; 1.5 fold) revealed molecular pathways implicated in cancer, inflammatory disease, hematopoietic development and genetic disorders. Involved biological processes were related to the immune system, nuclear organization and intracellular trafficking. We performed in silico class prediction and found 31 genes to be differentially expressed and discriminative for diagnosis or relapse samples with an accuracy of more than 80%, as determined by ‘leave-one-out’ cross validation. Of the 31 genes, 29 showed a lowered expression in the relapse sample when compared to the initial diagnosis sample (median intensity ratio diagnosis/relapse = 1.74, range=1.47-2.33, P29th gene=0.032) and 7 of these 29 genes have functions in the maintenance of chromatin structure. Preliminary RT-PCR results confirmed this for an independent set of patient samples. In vitro cytotoxicity experiments on primary patient material using relevant experimental drugs are underway. In conclusion, we identified genes and pathways that were significantly differentially expressed between diagnosis and relapse samples. The majority of discrimative genes are down-regulated at relapse with an important contribution of genes involved in maintenance of chromatin structure. Our efforts are directed to determine if these findings will be instrumental to design therapies aimed at preventing relapses. Financially supported by the Dutch Cancer Society (VU 2005-3666, J.C.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3209. doi:10.1158/1538-7445.AM2011-3209

In Acute Lymphoblastic Leukaemia, Stemness Is Frequent and Ubiquitous

AbstractAbstract 92Research on cancer stem cells, cells that self-renew and reconstitute the full phenotype of the original malignancy, has yielded controversial results regarding their frequency and identity for many cancers. The hierarchical stem cell model has been well established in some malignancies such as acute myeloid leukemia and states that only rare, immunophenotypically immature blasts harbor stem cell activity, resembling a normal physiological hierarchy. The opposing stochastic model proposes that stemness in cancer cells is supported by extrinsic stimuli and that a substantial fraction of malignant cells have this potential. Continued optimization of in vivo xenotransplantation modeling recently caused a paradigm shift for some cancers, for example in malignant melanoma where stem cell activity was found in as many as 1 in 4 cells. For acute lymphoblastic leukemia (ALL) we and others previously challenged the hierarchical model by demonstrating that both immature and more mature leukemic blasts contain self-renewal properties (Cancer Cell 2008, 14(1), p47-58). In this study we address the frequency of leukemic stem cells in the bulk leukemia and also, more specifically, in subpopulations of different blast maturity by using unsorted and highly purified flow sorted cell fractions. Primary patient material as well as leukemic blasts harvested from engrafted mouse bone marrow (secondary and tertiary material) were sorted for their CD10, CD20 or CD34 expression followed by orthotopic intrafemoral transplantation into severely immunocompromised NOD/scid IL2Rγnull (NSG) mice. Engraftment of transplanted CD19+CD10low and CD19+CD10high, CD19+CD20low and CD19+CD20high and CD19+CD34low and CD19+CD34high blast populations was monitored by 5 color flow cytometry using material from consecutive bone marrow punctures, final bone marrow harvests and/or single cell suspensions from spleens. Primary ALL samples from 15 high risk (BCR/ABL positive (n=8), BCR/ABL like ALL (n=2), high hyperdiploid/MRD positive (n=2), MRD positive (n=1), MLL/AF4 (n=2)), 3 intermediate risk (high WBC/MRD negative (n=2), age >10 years (n=1)) and 3 standard risk (n=3) patients were included. Cells sorted into CD19+CD10low and CD19+CD10high fractions were transplanted from primary patient material (n=4, HR; n=1, SR) and from secondary samples (n=4, HR; n=1; IR) with cells from one HR patient used at limiting dilutions. As few as 100 sorted cells of either fraction were sufficient to repopulate the leukemia. CD19+CD20high and CD19+CD20 low fractions from primary (n=7, HR; n=1, IR), secondary (n=5, HR; n=1, IR) and tertiary material (n=2, HR; n=1, IR) engrafted NSG mice. Limiting dilutions were performed on secondary (n=4, HR) and tertiary material (n=2, HR). Cell numbers required for engraftment varied between leukemias with as few as 100 cells being sufficient to cause engraftment. Limiting dilution experiments using CD19+CD34high and CD19+CD34low fractions from secondary (n=1, HR) and tertiary (n=1, HR) material yielded engraftment with as few as 10 CD19+CD34high and 100 CD19+CD34low cells. Similarly, unsorted primary (n=11, HR; n=2, IR), secondary (n=2, HR) and tertiary material (n=1, HR) required as few as 10 cells for leukemic reconstitution. Taken together, both unsorted and sorted blasts of all immunophenotypes and transplanted with low numbers were able to reconstitute the complete original phenotype of the patient leukemia. All limiting dilutions were transplanted down to 10 cells per mouse and those mice not engrafted yet are still under observation. Furthermore, the ability to self-renew was demonstrated by serial transplantation. Finally, we compared expression of self-renewal associated genes (BMI1, EZH2, HMGA2, MEIS1, TERT) in CD19+CD34low and CD19+CD34high fractions of 5 HR and 1 SR samples with that in cord blood. Interestingly, expression of these genes was not dependent on the CD34 status of the leukemic cells, whereas HMGA2, MEIS1 and TERT were upregulated in CD34+ cord blood cells. In summary we provide strong evidence for the stochastic cancer stem cell model in B precursor ALL by demonstrating that (i) a broad spectrum of blast immunophenotypes exhibit stem cell characteristics and (ii) that this stemness is highly frequent among ALL cells. Disclosures:No relevant conflicts of interest to declare.

Targeting Multiple Active Kinase Pathways In 11q23 Translocated Pediatric Acute Myeloid Leukemia Using a VEGFR2 Antibody Together with a MEK Inhibitor.

AbstractAbstract 3626One of the major pathways involved in the angiogenic process is the VEGF/VEGFR signaling pathway. VEGFA was found to be an independent prognostic factor for therapeutic outcome in AML (de Bont et al, 2002;Aguayo et al, 1999). Moreover, it has been shown that after chemotherapy the expression level of VEGFR2 is restored to normal in the bone marrow of AML patients in complete remission (Padro et al, 2002). Both VEGF and its receptors Flt-1(VEGFR1) and KDR (VEGFR2) are expressed by leukemic blasts which results in both a paracrine and an autocrine VEGF/VEGFR2 pathway in AML (Dias et al, 2001). In response to VEGF stimulation, VEGFR2 has been shown to transmit intracellular signals leading to activation of multiple downstream signaling pathways including the mitogen-activated protein (MAP) kinases, JAK)STAT, and Phosphoinositide 3-kinases (PI3 kinases). In AML it has been described that simultaneous activation of these pathways results in a poor prognosis (Kornblau et al, 2006). Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene at locus 11q23 are associated with a poor outcome. These MLL translocations represent 15–20% of pediatric acute myeloid leukemia's. In this study we show that VEGFR2 expression is significantly higher on blasts of 11q23 translocated acute myeloid leukemia's compared to blasts of AML patients with another karyotype. In addition, using peptide arrays and proteome profiler arrays we have found that the VEGF receptor signaling pathway and part of the MAP kinase signaling pathway are active and or phosphorylated in blasts of 11q23 translocated patients (Fig. 1). Proteins from these active pathway are potential targets in the treatment of pediatric 11q23 translocated AML. To further investigate these potential targets we have performed ex vivo drug target assays using an inhibitor and an antibody against two key proteins from the major signal transduction pathways, VEGFR2 antibody (IMC1121b) and MEK inhibitor (U0126). No effect of VEGFR2 inhibition was measured in two AML cell lines (HL-60 and THP-1). Whereas, MEK inhibition had an effect on both cell lines, LC50 8.7 μM and 7.5 μM for HL-60 and THP-1 respectively. Cell survival of the THP-1 cell line (containing a 11q23 translocation) was substantially inhibited after addition of a combination therapy with an inhibitor against MEK and a antibody against VEGFR2 (LC50 2.9 μM). In contrast, the VEGFR2 antibody in combination with the MEK inhibitor had only a marginal effect on the HL-60 cell line (LC50 7.7 μM). Future experiments to investigate the effect of the combination therapy on primary patient material are underway. Overall these data show for the first time that targeting multiple active pathways in pediatric 11q23 AML could result in promising opportunities concerning future therapy options. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Relapsed AML Patients with Mutational Shifts Harbor An Oligoclonal Primitive Cell Compartment at Presentation; Evidence for Clonal Selection towards Relapse

AbstractAbstract 751Previously we observed instability of type I/II mutations between diagnosis and relapse in a large portion (38%) of relapsed AML patients and this was associated with outcome (Bachas et al, Blood, 2010). Such instabilities may be explained by clonal selection during disease progression from oligoclonal initial AML populations. This hypothesis was tested in 6 AML patients with recurrent disease and mutational shifts between diagnosis and relapse. We determined the mutation profiles of mature and of immature subfractions of AML cells in initial diagnosis samples and related these to the corresponding relapse samples. Small amounts of cells (<50 cells) from mature and immature AML subfractions were sorted, followed by direct PCR assays for the detection of the frequently instable FMS like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD), Wilms Tumor 1 (WT1) and RAS mutations and the relatively stable Nucleophosmin 1 (NPM1) mutation. AML blasts were cell sorted based on CD45dim expression and primitive cell populations were defined by CD34/CD38 co-expression and refined based on the expression of CD133 and CD117. A lineage infidelity (Lininf) marker was used for designation of aberrant cells (Van Rhenen et al, Leukemia, 2007). Lymphocytes were sorted based on scatter properties and CD45bright expression and were regarded as wild type controls of primary patient material in mutation analyses. Immunophenotypes, cytogenetics and minimal residual disease (MRD) status were routinely assessed in our laboratory. Our results are indicative of an oligoclonal immature AML cell compartment at presentation: the immature CD45dimCD34+CD38dim/- compartments of initial samples were more heterogeneous according to their mutation profiles, when compared to the bulk of mature CD45dimCD34+ blast cells. Mutation profiles differed for at least one molecular marker among immature subfractions of the initial samples within 5 out of 6 patients. In the remaining patient, the mutation profiles were identical among sorted fractions of the initial sample. When comparing these data with the mutation profile of the bulk of AML cells of the relapse samples, in the latter patient and 2 out of the 5 above referred heterogeneous patients, mutational shifts could not be retraced to any subfraction of the initial sample. Strikingly, in the 3 other patients we found evidence for the clonal selection of a minor immature subclone of the initial sample, with a specific mutation profile that was predominantly present at relapse. In two of these patients we detected FLT3/ITD mutations of specific lengths, which were predominantly present at relapse, in a CD34+CD38−Lininf or CD34+CD38dimLininf subfraction of the initial sample. This ITD was undetectable in the bulk of AML cells by standard diagnostics. Similarly, in the other patient with clonal selection, the CD34+CD38−Lininf compartment of the initial sample was enriched for a WT1 exon 7 mutation that predominated at relapse, whereas no WT1 mutations were detected in the bulk of AML cells at presentation. In concordance with these observations, immunophenotype and MRD data also suggested a clonal selection in these patients. In the 3 patients that did show a mutational shift, but lacked direct indications for clonal selection, a de novo mutation may have been induced during therapy. Alternatively, the use of additional immunophenotypical and molecular markers may be required for these samples to identify the relevant subclones. The key finding of our study is that we could detect in the immature fraction of initial samples of 3 AML patients a rare and otherwise undetectable subclone with a distinctive mutational profile that was predominant in the bulk of AML cells at relapse. This has several clinical implications: risk group stratification based on the presence of mutations that are associated with prognosis can be refined by genetic characterization of oligoclonal immature AML subpopulations that may play a role in the development of relapse. In addition, early and accurate detection of these subclones will be crucial in the prevention of relapse by accurate timing and application of targeted treatment. In conclusion, our results underline the concept of oligoclonality in AML and the involvement of clonal selection of immature chemoresistant subclones in the development of relapse. This study was supported by the Dutch Cancer Society (VU2005-3666). Disclosures:No relevant conflicts of interest to declare.

Regenerative Therapy and Cancer: In Vitro and In Vivo Studies of the Interaction Between Adipose-Derived Stem Cells and Breast Cancer Cells from Clinical Isolates

Adipose-derived stem cells (ASCs) have been proposed to stabilize autologous fat grafts for regenerative therapy, but their safety is unknown in the setting of reconstructive surgery after mastectomy. Both bone marrow mesenchymal stem cells (MSCs) and ASC have been shown to enhance tumorigenesis of established breast cancer cell lines, but primary patient material has not been tested. Here, we ask whether ASC promote the in vitro growth and in vivo tumorigenesis of metastatic breast cancer clinical isolates. Metastatic pleural effusion (MPE) cells were used for coculture experiments. ASC enhanced the proliferation of MPE cells in vitro (5.1-fold). For xenograft experiments (100 sorted cells/injection site), nonhematopoietic MPE cells were sorted into resting and active populations: CD90+ resting (low scatter, 2.1%≥2N DNA), CD90+ active (high scatter, 10.6%≥2N DNA), and CD90-. Resting CD90+ MPE cells were tumorigenic in 4/40 sites but growth was not augmented by ASC. Active CD90+ MPE cells were tumorigenic (17/40 sites) only when coinjected with ASC (p=0.0005, χ2 test). The multilineage potentiality and MSC-like immunophenotype of ASC were confirmed by flow cytometry, differentiation cultures, and immunostaining. The secretome profile of ASC resembled that reported for MSC, but included adipose-associated adipsin and the hormone leptin, shown to promote breast cancer growth. Our data indicate that ASC enhance the growth of active, but not resting tumor cells. Thus, reconstructive therapy utilizing ASC-augmented whole fat should be postponed until there is no evidence of active disease.

Abstract 3282: Molecular characterization of a panel of patient-derived breast cancer xenografts

Abstract Breast cancer is a heterogeneous disease for which several subtypes have been described according to histological and molecular determinants. Therapeutic decisions are dependent on tumor staging which includes molecular markers like estrogen receptor (treatment with anti-estrogens or aromatase inhibitors) or HER2 (treatment with HER2 targeting agents Trastuzumab or Lapatinib). Here we present a panel of 12 patient-derived breast cancer xenografts, eleven of which have been established by Oncotest from primary patient material. Molecular profiling included expression analysis of receptors for estrogens (ER), androgens (AR) and progesterone (PR), HER2 and the E2F transcription factor 1 by quantitative RT-PCR and Affymetrix HG U133 plus2.0 array analysis. HER2 protein and phosphorylation were determined by ELISA and - for selected models - FISH analysis was performed to determine gene amplification. MAXF 713 and MAXF 1398 are ER positive, luminal B subtype tumors, whereas MAXF 1162 is a HER2-overexpressing, Lapatinib sensitive tumor. The high HER2 levels in MAXF 1162 are based on a gene amplification. MAXF 1322 is borderline HER2-positive, but insensitive towards Lapatinib and Herceptin. The majority of tumor models (8 out of 12) belong to the basal-like, triple negative breast cancer subtype. In addition, activation of downstream signaling molecules of the Akt and MAPK pathways (Akt, Erk1/2 and p70S6K) were analysed. Akt signaling is important in many breast cancers and 8 out of 12 tumor models depicted activated Akt, some of which also had highly activated p70S6K. Erk1/2 phosphorylation was less prominent and slightly higher compared to the Oncotest lysate pool in MAXF 1322. The Oncotest lysate pool incorporates 106 different tumor lysates and proved to be a valuable control lysate to estimate a mean expression and/or activation level. Finally, the mutational status of PI3Kα and PTEN was assessed with PTEN protein expression also evaluated by Western Blot. Modifications in the PI3Kα activity and its negative effector PTEN modulate Akt kinase activity and downstream signaling. Finally, the estrogen-dependency of the ER-positive models MAXF 713 and MAXF 1398 and their growth properties in dependence of subcutaneous versus orthotopic implantation were assessed. The xenograft models are routinely implanted subcutaneously, which sustains tumor growth. Nevertheless we found that of the 12 MAXF models as studied, 4 grew faster when implanted orthotopically into the mammary fat pad. In summary, a unique collection of patient-derived mammary xenograft models has been characterised comprehensively in order to enable a pre-study selection of suitable tumors for in-vivo efficacy testing. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3282.

High fidelity of phenotype and genotype in serial NOD/SCID pediatric acute lymphoblastic leukemia

Primary ALL cells are very difficult to culture in vitro and currently available cell lines only poorly reflect the heterogeneous nature of the disease. Many groups therefore use murine xenotransplantation models for primary pediatric ALL. But, to date it remains unclear as to what extent the characteristics of the original patient leukemia are retained over consecutive xenograft passages or whether there is selection for a clone with characteristics distinct from the bulk leukemia at diagnosis. We here address this question for 7 consecutive leukemias diagnosed in our clinic and directly transplanted onto unconditioned NOD/SCID-mice. After primary passage, clinical presentation, immunophenotype and gene expression profiles were highly similar to the diagnostic leukemia, with minor differences arising from the presence of normal human hematopoietic cells in primary patient material. Moreover, all characteristics analyzed remained stable after secondary and tertiary passages. The model precisely recapitulates the primary human leukemia in the mouse and thereby reflects the heterogeneity and inherent variability of different primary patient leukemia cells.

C-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis

Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.

Disease Propagating Blasts in Standard and High Risk Acute Lymphoblastic Leukemia Are Frequent and of Diverse Immunophenotype.

Abstract 1421Poster Board I-444Conflicting results in the field of cancer stem cells have reignited debate regarding the frequency and identity of cells with the ability to self renew and to propagate the complete phenotype of the malignancy. Initially it was suggested by different studies that cancer stem cells represent only a small minority of the malignant population and that the immunophenotypes of these cells resemble a rather immature type in the cell hierarchy. More recent data from our own and other groups have challenged these findings by demonstrating that cells at different maturity levels within the leukemic hierarchy have cancer stem cell abilities and that the frequency of the leukemia maintaining cell is higher than previously thought (Cancer Cell 2008, 14(1), p47-58). We use an in vivo NOD/scid IL2Rγnull (NSG) mouse intra-femoral transplant model to determine the clonogenicity of sorted candidate leukemic stem cell populations, characterized by specific immunophenotypes. We selected the surface markers CD10 and CD20, in order to differentiate between rather immature and more mature cells. Furthermore we carried out limiting dilution experiments on sorted (CD20) and unsorted leukemic blasts to investigate the frequency of the proposed leukemic stem cells. Flow sorted ALL blasts of CD19+CD20low and CD19+CD20high as well as of CD19+CD10low and CD19+CD10high immunophenotype were transplanted into NSG mice. Sorts were performed on primary patient material and on leukemic blasts that had been harvested following prior passage in mice. Different subtypes of ALL were included (high risk: BCR/ABL (t9;22) positive (patients L4967, L4951, L49101, L8849, L2510), high hyperdiploid/MRD positive high risk (L754, L835), intermediate risk: high WBC/MRD negative (L736, L784), age >10 years (L803)). CD20 sorts were performed on primary patient material (L4951, L49101, L754, L835 and L776), on secondary samples harvested from engrafted primary mice (L4967, L4951, L2510, L736 and L754) and on tertiary samples harvested from engrafted secondary mice (L4967 and L736). In total 151 mice were transplanted, with 122 showing engraftment in consecutive bone marrow punctures or in bone marrow harvests. CD10 sorts were performed on primary patient material (L784 and L49101) and on secondary samples harvested from engrafted primary mice (L4951, L8849, L2510 and L803) with 31 out of 52 mice transplanted with sorted material showing engraftment as seen with CD20 sorted cells. Blasts of all selected immunophenotypes were able to engraft the leukemia in unconditioned NSG mice as determined by 5 color flow cytometry. In particular, sorted cells of both fractions were able to reconstitute the complete phenotype of the leukemia. Harvested cells from engrafted mice could then be re-sorted into high and low antigen expressing fractions and successfully re-engrafted on secondary and tertiary mice. Cell purities of transplanted cells were usually higher than 90% (range 67-100%). The ability of all populations to serially engraft mice demonstrates long-term self-renewal capacity. Two additional patients were used in the limiting dilution assays (high WBC/t(4;11) high risk (L826); low WBC/MRD negative low risk (L792)) and experiments were performed on primary unsorted and secondary sorted material. Cell numbers necessary for ALL engraftment differed between individual leukemias but as little as 100 cells proved to be sufficient in one unsorted and in both the CD19+CD20low and CD19+CD20high fractions (Table 1). Mice transplanted with 10 cells only are still under observation.Table 1PatientTransplantPopulationCell doseMice engrafted/transplantedL4951SecondaryCD20 high5003/3CD20 low3/3CD20 high1003/3CD20 low3/3L2510SecondaryCD20 high3,0002/4CD20 low4/4CD20 high3000/4CD20 low1/4L49101PrimaryUnsorted5003/41000/4L792PrimaryUnsorted1,0005/51001/5L826PrimaryUnsorted1,0003/41000/4In conclusion we present strong evidence that leukemia-propagating cells are much more prevalent than previously thought and that blasts of diverse immunophenotype are able to serially reconstitute the complete leukemia in immune-deficient mice. Disclosures:No relevant conflicts of interest to declare.

Hypermethylation of CD44 Is Characteristic of Specific Lymphoma Subtypes.

Abstract 3469Poster Board III-357Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a common hallmark of cancer. It is generally agreed, that CpG island hypermethylation profiles are specific for different tumor types [Costello et al., 2000, Nat Genet 25:132-138]. Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification), methylation of 24 different TSG was analyzed in 40 lymphoma cell lines representing Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). On average 8 ± 2.8 TSG out of the 24 analyzed were methylated per lymphoma cell line, whereas 0/24 TSG were methylated in healthy donor tonsils. Methylation frequencies decreased from HL, ALCL, BL, FL, DLBCL to MCL cell lines. The TSG methylation status of the most relevant genes was verified by methylation-specific PCR. Moreover, TSG hypermethylation correlated with transcriptional silencing as assessed by quantitative real time PCR. While our studies on the methylation status of TSG in lymphoma cell lines support previous methylation analyses performed on primary lymphoma patient material for many of the genes analyzed here, MS-MLPA screening also revealed a new interesting candidate: CD44. Hypermethylation of CD44 was characteristic of ALCL, BL, FL and DLBCL cell lines and allowed their discrimination from MCL and HL cell lines. In CD44 hypermethylated cell lines expression of CD44 was re-inducible at mRNA and protein levels by treatment with the demethylating agent 5-Aza-2'-deoxycytidine, confirming its epigenetic regulation. Furthermore, CD44 ligation with an anti-CD44 antibody induced apoptosis in CD44+ (CD44 unmethylated) lymphoma cell lines whereas CD44 hypermethylated cell lines showed no response. Thus, CD44 might be an interesting new epigenetic marker and a potential molecular target for the diagnosis and treatment of specific lymphoma subtypes. DisclosuresNo relevant conflicts of interest to declare.

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.

Identification of Novel Biomarkers for Prediction of Response to Tyrosine Kinase Inhibitors (TKIs) by Proteomic Profiling of Imatinib as Well as 2nd and 3rd Generation TKIs in Vitro

The tyrosine kinase inhibitor (TKI) Imatinib (IM) represents the gold standard firstline treatment for patients with newly diagnosed chronic myeloid leukemia (CML). For patients developing resistance or intolerance to Imatinib, the 2nd generation TKIs Dasatinib (DASA) and Nilotinib (NILO) which are approved, and Bosutinib (BOSU) which is in clinical development, possess activity against almost all mutant forms of BCR-ABL which confer Imatinib-resistance, except the gatekeeper mutation, T315I. This latter mutation occurs in approximately 15% of clinically observed mutations in chronic phase CML and confers resistance to all currently approved agents. Therefore compounds have been evaluated for activity against T315I mutant BCR-ABL, among which combined Aurora kinase and Abl inhibitors such as PHA-739358 (PHA) have been identified as showing some promise.In the current study, we used a classical proteomics approach to generate drug profiles of the TKIs (IM, DASA, NILO and PHA), in order to identify biomarkers that are either compound or drug group (i.e. 1st, 2nd or 3rd generation TKI) specific and could potentially be used as biomarkers for response prediction in vivo. For in vitro screening, we used murine Ba/F3 cells expressing wild-type (p210, wt) BCRABL or mutants, which are either low grade (M351T) or absolutely resistant (T315I) to Imatinib. Using 2D-gel electropheresis and mass spectrometry, we could identify a total of 68 individual protein spots which were differentially regulated in cells when treated with equieffective concentrations (IC50) of TKIs. Using in silico overlay of the different 2D-gels (Delta2D, Decodon GmbH Greifswald), 42, 38, 41 and 15 spots were found to be specifically differentially regulated in Ba/F3 cells expressing wt BCR-ABL under either IM, NILO, DASA and PHA, respectively. Interestingly, hierarchical cluster analysis based on these candidate proteins identified similar protein expression patterns for IM, NILO and DASA in comparison to PHA. Using genontology analysis (Panther software), the majority of the proteins belonged to the group of nucleic acid binding proteins (25%), cytoskeletal proteins (13%) and chaperones (12%). In contrast to the broad response of the different TKIs on wt Bcr-Abl cells, changes in protein expression patterns induced in cells carrying the M315T BCR-Abl mutation were substantially less pronounced (IM: 9, NILO: 12, DASA: 28, PHA: 17) with the strongest response seen in Dasatinibtreated cells, consistant with the compound being a powerful inhibitor of both wt and M351T BCR-ABL signaling. With the exception of the combined BCR-ABL and Aurorakinase inhibitor PHA (7 proteins), cells expressing T315I BCR-ABL exhibited no altered protein expression in response to treatment with either IM or the other 2nd generation TKIs. The protein expression patterns identified were used for systems biology network analysis using Metacore software (GeneGo), which enabled the elucidation of signaling pathways and identification of transcription factors involved in TKI response. Besides known regulators of BCR-ABL signaling, such as c-Myc and p53, we were able to identify novel TKI-dependent candidate proteins (e.g. eIF5a) and post-translational modifications (PTM) that, pending validation in primary patient material might effectively be used as biomarkers for response prediction in the near future.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material. We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis. By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor. Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Human Leukemia and Lymphoma Cell Lines as Models and Resources

Tumor cell lines are widely used as oncologic models and resources, forming, along with primary patient material and animal models, one of three major subjects for cancer investigation. With the advent of the Human Genome Project (HGP) and the ensuing provision of sequencing data and mapped clones, human cancer cell lines, notably those derived from leukemia-lymphoma (LL) have become increasingly productive tools for cancer gene ascertainment and characterization. Hence, the roles of putative novel cancer genes may be investigated using diverse panels of LL cell lines, both individually by PCR-based methods, and globally by transcriptional chip-profiling. Similar studies have also enabled the faithfulness with which cancer cell lines model their supposed in vivo counterparts to be quantified at last. Several recent transcriptional profiling studies indicate that of all tumor types well characterized human LL cell lines most accurately model the gene expression patterns of their corresponding primary tumors. Analysis using genomic arrays tells a similar story for the stability of chromosome rearrangements in LL cell lines. Well characterized LL cell lines also provide ideal tools for investigating the druggability of individual gene products, e.g. by measuring their transcript levels using q(uantitative)-PCR methods in cells subjected to treatments with small interfering (si)-RNAs. We provide a list of authentic, well characterized examples for prospective investigators, since many circulating cell lines have been cross-contaminated and describe DNA profiling methods which, together with classic and molecular cytogenetic analyses, inform authentication. We also review the problem of mycoplasma contamination and means for its eradication.

Deeper Insights into Hematological Oncology Disorders via Single-Cell Phospho-Signaling Analysis

An understanding of hematological cancer cell signaling processes poses one of the most complex and intractable problems in modern biomedical inquiry. While we understand some of the fundamental players that contribute to oncogenic processes, significant effort is focused upon determining how these individual players relay information to each other to create the composite functions of a cancer cell. Efforts designed to understand these processes at the single cell level will undoubtedly allow for understanding of the heterogeneity of hematological tumors as well as, simultaneously, the function of the 'responding' immune system. I will relate some of the insights our laboratory has developed over the last several years applying single-cell phospho-flow cytometry to the study of signaling in primary patient material and murine models. While it is clear that this analysis now allows us to accomplish phospho-signaling biochemistry at the single cell level with primary cell material, we are only beginning to develop some of the bioinformatics tools to appropriately display the vast amount of information collected by such approaches. These approaches, however, have already allowed us to develop approaches that prognosticate patient outcomes based on signaling status, prior to any treatment, as well as subgroup patient subtypes according to signaling states. The modest efforts to date presage a time where it should be possible to provide far more tailored therapies specific to the varied diseases represented by the hematological malignancies.