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Recent years have seen improvements in strategies to treat pancreatic cancer. Pancreatic cancer is a leading cause of cancer-related death in the world, and is characterised by rapid disease progression, highly invasive tumour phenotype and resistance to chemotherapy. Patient prognosis is extremely grim, with a one-year survival rate of just 10% and only a 5% chance of surviving beyond five years. There has been little change in the treatment regimen, with 5-FU-based therapies being the usual route. Gemcitabine has also offered some relief over the past two decades, with modest improvements in median survival. This lack of choice has increased the call for new treatments, and indeed, novel drugs are now being investigated for their use in pancreatic cancer. These include those agents classically applied to other indications such as doxycycline and doxorubicin, as well as dietary components such as curcumin and genistein. Each of these drugs possesses different levels of activity both as single agents and in combinatorial approaches with gemcitabine. This review will discuss the difficulties in treating pancreatic cancer, and will summarise the progress and latest development in drugs used within this field of cancer.

Although intratumor heterogeneity has been inferred in multiple myeloma (MM), little is known about its subclonal phylogeny. To describe such phylogenetic trees in a series of patients with MM, we perform whole-exome sequencing and single-cell genetic analysis. Our results demonstrate that at presentation myeloma is composed of two to six different major clones, which are related by linear and branching phylogenies. Remarkably, the earliest myeloma-initiating clones, some of which only had the initiating t(11;14), were still present at low frequencies at the time of diagnosis. For the first time in myeloma, we demonstrate parallel evolution whereby two independent clones activate the RAS/MAPK pathway through RAS mutations and give rise subsequently to distinct subclonal lineages. We also report the co-occurrence of RAS and interferon regulatory factor 4 (IRF4) p.K123R mutations in 4% of myeloma patients. Lastly, we describe the fluctuations of myeloma subclonal architecture in a patient analyzed at presentation and relapse and in NOD/SCID-IL2Rγ(null) xenografts, revealing clonal extinction and the emergence of new clones that acquire additional mutations. This study confirms that myeloma subclones exhibit different survival properties during treatment or mouse engraftment. We conclude that clonal diversity combined with varying selective pressures is the essential foundation for tumor progression and treatment resistance in myeloma.

Gemcitabine is currently the leading therapeutic for pancreatic cancer treatment, despite growing resistance. Studying the mechanisms that underlie gemcitabine resistance and discovery of agents that increase the tumour sensitivity to gemcitabine, is therefore desirable. The thalidomide analogue lenalidomide has been approved for use in multiple myeloma in combination with dexamethasone. Although it is primarily immunomodulatory, it also has direct effects on tumours. We investigated the sensitivity of three pancreatic cell lines PANC-1, MIA-PaCa-2 and BxPC-3 to gemcitabine. We observed that PANC-1 cells display most resistance to gemcitabine and MIA-PaCa-2 are most sensitive. Western blot analysis revealed that PANC-1 exhibits high phosphorylated extracellular signal-regulated kinase (pERK) expression, whereas MIA-PaCa-2 displays low expression. Combining gemcitabine and lenalidomide reduced the IC(50) of gemcitabine up to 40% (p0.05). Western blot analysis showed lenalidomide significantly reduced pERK expression in all cell lines (p0.05). It was hypothesised that gemcitabine sensitivity could be increased through combination with a pERK-reducing agent. The mitogen-activated kinase (MEK) specific inhibitor U0126 was used on PANC-1 cells to restore gemcitabine sensitivity. U0126 significantly increased cell killing by gemcitabine from 30% to 60% (p0.001). Sensitive MIA-PaCa-2 cells were transfected with a constitutively active MEK mutant to reduce gemcitabine sensitivity. Transfection resulted in a significant reduction in cell killing by gemcitabine from 54-16% (p0.05). These results provide evidence that ERK activity underlies sensitivity to gemcitabine and that addition of an agent that reduces this activity, such as lenalidomide, enhances gemcitabine efficacy. In conclusion, these results provide an understanding of gemcitabine resistance and could be used to predict successful combination therapies.

Multiple Myeloma (MM) is a proliferation of aberrant plasma cells in the bone marrow. Ig translocations or hyperdiploidy are MM initiating events present in all clonal cells in contrast to other secondary lesions involved in progression (e.g. RASmutations), which may be subclonal. We and others have recently described the presence of different tumour clonal populations—a phenomenon called intra-tumour heterogeneity—in MM, yet the phylogenetic relationships of the tumour subclones remains to be fully elucidated. To describe the intra-tumour heterogeneity and tumour phylogenies in a series of t(11;14) MM patients characterised by KRAS/NRAS/BRAF mutations, we combined whole-exome sequencing and single-cell genetic analysis. A novel approach for single-cell multiplex-qPCR analysis using nano-fluidic arrays (Fluidigm) was followed in 100-250 single-cells per sample. The t(11;14) breakpoint was defined using Ig regions-targeted massively-parallel sequencing. Taqman assays specific for detection of the t(11;14) breakpoints and for mutations in selected genes were custom-made designed. Copy number assays for chromosomal regions of interest were also used. This strategy allowed us: first, to report the presence of the t(11;14), mutations and copy number aberrations (gains/losses) at the single-cell level; second, to define subclonal populations; and third, to delineate the most plausible sequence of events for each case. An additional series of 14 MM patients with paired-samples at presentation/relapse was included for the study of the effect of treatment in clonal architecture. Lastly, to analyse the engraftment-ability of subclonal populations, we injected 1x106 CD138+ cells from one of the patient-samples at diagnosis into the tibia of NOD/SCIDyc(null)-mice and compared the engrafted-myeloma with the paired presentation-relapse samples. We demonstrate that MM is comprised of 2-6 clones, related through a linear (3/7 cases) or a branching (4/7) phylogeny. The t(11;14) was seen in 91-100% of tumour cells, supporting its aetiological role as a MM initiating event. For the first time in MM, we describe a parallel evolutionary pattern in two samples that carried double hits in KRAS or KRAS/NRAS. These mutations were acquired separately in two divergent clonal lineages, which were derived from the same ancestor but evolved independently. We suggest that RAS is a true driver mutation in MM as such alterations seem to provide clonal advantages for myeloma subclones in the bone marrow environment. We also report the concomitant acquisition of RAS mutations and IRF4p.K123R mutation in 2% (9/453) screened MM patients. This finding suggests a collaborative effect provided by the mutations in both pathways to trigger myeloma development. We examine the ability of subclonal populations to survive patient treatment by the analysis of paired presentation-relapse samples. Not only did the intra-tumour heterogeneity shift in the transition to relapse meaning that subclonal populations fluctuated during treatment, but also new clones emerged from early and late subclones formerly described at diagnosis. To note, some of these new subclones acquired mutations in KRAS/BRAF during transition, likely leading to relapse. In parallel, we tested the ability to recapitulate the disease in NOD/SCIDyc(null)-mice. Engrafted-myelomas also had clonal fluctuations with 1/3 clones shown at patient-diagnosis neither found at relapse, nor in the engrafted-MM. This missing clone was similarly outcompeted by the other clones during patient treatment and xeno-transplantation. Altogether, these results suggest that subclonal populations have different abilities to survive treatment or xeno-transplantation and hence, to lead to relapse or reconstitution of myeloma by the generation of new clonal subpopulations. We confirm the existence of distinct MM subclones that are related through a linear or a branching phylogeny, with examples of parallel evolution for alteration of the RAS/MAPK pathway. Myeloma subclones are subject of a selection process involving clonal extinction and clonal tides, similar to the theory of Natural Selection by Charles Darwin. In conclusion, intra-tumour heterogeneity is an elementary foundation for Darwinian selection underlying both disease progression and the development of treatment resistance in MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 2385 The t(4;14) group in multiple myeloma (MM) is associated with a significantly impaired prognosis based on the overexpression of MMSET, a histone methyltransferase and epigenetic modifier that is thought to play a major role in myeloma genesis. We have previously shown that t(4;14) myelomas are associated with a specific pattern of DNA hypermethylation using Illumina HumanMethylation27 BeadChip arrays. These results were interpreted as being consistent with that MMSET overexpression leads to specific changes in the epigenetic architecture of MM. However, the detailed mechanism underlying this remains unclear partly due to the low resolution of methylation array technology used. In order to address this we have performed high-resolution genome-wide analyses of DNA methylation for t(4;14) and t(11;14) samples from patients with myeloma, plasma cell leukemia (PCL) and myeloma cell lines (HMCL) using methyl binding domain next generation sequencing (MBD-seq) in order to define DNA methylation patterns specific for t(4;14) MM, as well as to analyse methylation changes accompanying the progression from MM to PCL at a high-resolution, genome-wide scale. DNA from 6 myeloma patients at diagnosis [3 MM with t(4;14), 3 MM with t(11;14)], 6 patients with PCL [3 PCL with t(4;14), 3 PCL with t(11;14)] and 6 HMCL [3 with t(4;14), 3 non-t(4;14)] was fragmented. Methylated DNA fractions were captured using biotin-labelled methyl-binding domain 2 (MBD2) protein. Captured sequences bound to MBD were washed and eluted using elution buffers with increasing salt concentrations. Eluted fragments were purified and sequenced on an Illumina GAIIx, using 1.5 lanes per patient sample, generating 36 bp single-end reads. On average, 1.4 Gbases of reads were generated per sample. Sequences were aligned and de-duplicated using stampy and bwa algorithms. The reference genome (build hg19) was divided into overlapping bins of 200 bp (termed probes) and short read coverage per bin was normalised to per million reads aligned. Differentially methylated regions were defined by comparing normalised reads per probe between the t(4;14) and the t(11;14) groups for MM, PCL and HMCL groups. We first compared probe values that were higher in all three t(4;14) MM samples compared to the three t(11;14) samples. About 16500 probe values were higher in t(4,14) cases compared the t(11;14) group, whereas only 470 probes values were higher in all t(11;14) cases compared to t(4;14) cases. This confirms our previous observation that t(4;14) MM cases are characterised by pronounced hypermethylation. Of the 16500 probes values higher in t(4;14), about 9500 probes mapped to gene bodies and 600 to gene promoters, affecting in total about 1600 genes, indicating that gene or gene regulatory sequence hypermethylation is a common feature in t(4;14). Gene set enrichment analyses of these genes demonstrated highly significant enrichment of KEGG pathways ‘pathways in cancer’, ‘cell adhesion molecules’, the GO term ‘cell development’, among others, and an overrepresentation of probes mapping to chromosomal regions on chromosome 1q. When comparing the progression from MM to PCL, about 2600 genomic probe values were higher in all 3 t(11;14) PCL vs all 3 t(11;14) MM and 1600 probes in all 3 t(4;14) PCL vs MM, indicating that hypermethylation from MM to PCL is more pronounced in t(11;14) than in t(4;14). Very few differences in probe values were present when comparing all 6 MM (both t(4;14) and t(11;14)) with the 6 PCL samples, indicating that the epigenetic mechanisms involved in progression from MM to PCL might be different between the cytogenetic subgroups. Enrichment of methylated sequences was strong for both translocation groups when comparing PCLs with HMCLs, demonstrating that the epigenetic architecture of HMCLs differs significantly even from late-stage patient tumour material. This genome-wide methylation analysis provides us with candidate genes that are likely to be directly or indirectly epigenetically modified by MMSET. We are integrating this methylation data with gene expression data to identify expression-methylation correlations. Furthermore, additional experiments using MMSET knockout models will be used to further filter MMSET-specific effects on genome wide methylation. Finally, we go on to define epigenetic markers that could serve as biomarkers for future epigenetic therapies targeting epigenetic modifiers in t(4;14) myeloma. Disclosures: No relevant conflicts of interest to declare.

Abstract 2907 In 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 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 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.

Multiple myeloma (MM) cells rely on protein homeostatic mechanisms for survival. These mechanisms could be therapeutically targeted via modulation of the heat shock response. We studied the roles of Hsp72 and Hsc70, and show that the two major cytoplasmic Hsp70s play a key role in regulating protein homeostasis and controlling multiple oncogenic pathways in MM, and their inhibition can lead to myeloma cell death. Our study provides further evidence that targeting Hsp70 represents a novel therapeutic approach which may be effective in the treatment of MM.