149 Quantitative mutational assessment of circulating tumor DNA using massively parallel deep sequencing in plasma and urine from advanced colorectal cancer patients

Poster Session – Preclinical Models 148 POSTER RAS synthetic lethal interactions from yeast to human cells S. van Wageningen1 , A. Prahallad1 , G. Heynen1 , R. Rothstein2 , R. Bernards1 . 1 Netherlands Cancer Institute Antoni van Leeuwenhoek hospital, Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Columbia University, Genetics Department, New York, USA Background: Synthetic lethal (SL) interactions are used to develop targeted cancer therapy. However, novel SL interactions discovered in mammalian cell cultures are often cell type specific and are therefore only relevant to a small, or difficult to define, subset of patients. We developed a strategy in which we prioritize potential SL drug targets using the genetically tractable model system Saccharomyces cerevisiae. Material and Methods: Weperformed a SL screen by expressing a constitutively active RAS allele, RAS2(V19), in ~4800 S. cerevisiae strains in which each individual gene is deleted. Next we tested if SL interactions were conserved in human cancer cell lines. Results: The yeast screen yielded a hit list highly enriched for mutants with a defect in ‘endoplasmic reticulum (ER)-to-Golgi-to-vacuole’ transport. Moreover, we found that this list had a significant overlap with strains sensitive to b-mercaptoethanol, DTT and tunicamycin. We hypothesized that ER homeostasis was disturbed in these cells. The two gene deletion mutants most sensitive to ER stress are IRE1 and HAC1. These genes make up the unfolded protein response (UPR) in yeast; the signaling pathway that restores ER homeostasis. Both UPR genes were SL with RAS2(V19). Next we asked if we could detect a SL interaction between oncogenic RAS and the UPR in human cells. We find that a SL interaction between oncogenic RAS and the UPR is dependent on specific RAS effector pathways in human cell cultures. Conclusions: The UPR is conserved in evolution. However, signaling pathways downstream of RAS have diverged over time. We will present how the interaction between oncogenic RAS and the UPR has evolved in human cells and how this interaction can be exploited for therapeutic intervention. 149 POSTER Quantitative mutational assessment of circulating tumor DNA using massively parallel deep sequencing in plasma and urine from advanced colorectal cancer patients J.C. Poole1 , C.R.T. Vibat1 , L. Benesova2 , B. Belsanova2 , S. Hancock1 , T.L. Lu1 , M.G. Erlander1 , M. Minarik2 . 1 Trovagene Inc., R&D, San Diego, USA; 2 Genomac Research Institute, Center for Applied Genomics of Solid Tumors, Prague, Czech Republic Background: Technologies enabling the assessment of circulating tumor DNA (ctDNA) in biofluids expand the clinical utility to detect and monitor cancer patient oncogenic mutations by minimally invasive and non-invasive liquid biopsy methods. Mutational tumor load quantification with high clinical sensitivity is vital for robust individualized assessment of systemic therapeutic responsiveness and resistance. A quantitative ctDNA assay using a massively parallel deep sequencing approach was developed to determine patient ctDNA mutational status. Material and Methods: Initial assay development was for the simultaneous detection of 13 known (7 reported) oncogenic mutations in KRAS codons 12/13. An ultrashort 31bp region encompassing KRAS codons 12/13 was PCR amplified; G12A/C/D/R/S/V, and G13D mutations were enriched by suppressing wild-type (WT) sequence amplification with a WT blocking oligo. Barcoded adaptor primers were added for compatibility with massively parallel deep sequencing. Limits of detection (LOD) were independently determined for each of the 7 KRAS mutations by spiking 5–500 copies of each mutant into 60 ng of a WT genomic background. Limits of quantitation (LOQ) were confirmed with 7 copies of each mutation in an increasing WT genomic DNA background of 60–360 ng. Archived, matched plasma and urine samples (stored between 3−5 years prior to ctDNA extraction) from 20 treatment na¨ıve, advanced cancer patients with known tumor tissue KRAS mutations determined by an accredited clinical laboratory, were used in a retrospective setting for a blinded pilot study. These samples were used to compare KRAS status in urine and plasma to tumor tissue, and assess clinical sensitivity of the ctDNA assay. Results: LOD data for 5–500 KRAS G12A/C/D/R/S/V, and G13D mutant copies in 60 ng WT DNA showed a highly correlative response with an average R2 of 0.90 for the 7 mutations evaluated. LOQ assessed for each mutation in an increasing WT DNA background, revealed a robust signal for each mutation versus WT alone; an estimated analytical LOD of 7 copies per ~100,000 genome equivalents (0.007%) was observed. Of 20 blinded retrospective plasma ctDNA samples evaluated, 19 (95%) displayed the KRAS mutation concordant with tumor tissue. Of 20 matched urine samples tested, 16 were deemed evaluable; 15 (94%) had a significant sequence call consistent to tumor and to plasma.

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Conclusion: The developed method for quantitative massively parallel deep sequencing of ctDNA for KRAS mutational assessment has reported high clinical sensitivity in plasma and urine. This technical approach is scalable and has the potential for detecting and quantifying a multitude of genomic alterations indicative of therapeutic responsiveness and resistance. Expansion of retrospective colorectal patient cohort described herein will be reported. Partially supported by the Czech Ministry of Health grant NT 13660. 150 POSTER Pirin downregulates E-cadherin gene expression and contributes to EMT K. Komai1 , Y. Niwa1 , Y. Sasazawa1 , S. Simizu1 . 1 Keio University, Faculty of Science and Technology, Yokohama, Japan Background: Downregulation of E-cadherin, a cell–cell adhesion protein, induces epithelial–mesenchymal transition (EMT), which plays crucial roles in metastatic progression. A nuclear protein pirin enhances NF-úB related transcription by binding to Bcl3-p50 complex, which is important for the SLUG expression and melanoma migration. Additionally, proteomics analysis indicated that pirin expression was decreased in metastatic adenoid cystic carcinoma cells. Although these reports suggest that pirin may be involved in tumor metastasis, there are no reports which directly demonstrate the contribution of pirin to metastasis. Here, we investigated the effects of pirin on EMT which associates with metastasis, and its mechanisms. Material and Methods: Pirin was overexpressed or silenced in HeLa cells, and then EMT-related genes and protein expressions were detected. The morphological changes of pirin stably-expressing HeLa cells were measured by employing parameter of ‘circularity’, [4p(area)/(perimeter)2 ]×100, which decreases by morphological changes from cobblestone-like epithelial cells to spindle-like mesenchymal cells. The effect of pirin on cell migration and anticancer drug resistance was measured by wound healing assay and MTT assay, respectively. The binding of wild-type pirin or its mutant to Bcl3 was confirmed by GST pull-down assay using recombinant GST-Bcl3. Results: Knockdown of pirin increased E-cadherin gene expression whereas overexpression of pirin decreased its level. Pirin stably-expressing HeLa cells exhibited spindle-like morphology and loss of cell–cell adhesion, which are reminiscent of EMT. From the result of MTT assay, we demonstrated that pirin contributed to acquire anticancer drug resistance. Furthermore, RNAi experiment revealed that pirin positively regulated A549 cell migration. Next, we examined whether Bcl3, a binding partner of pirin, is involved in EMT induction by pirin overexpression. GST pull-down analysis indicated that Pirin/E103A mutant was decreased its binding ability to Bcl3; however, as with wild-type pirin, this mutant also downregulated E-cadherin gene expression, suggesting that pirin decreases E-cadherin expression in Bcl3-SLUG-axis-independent manner. Conclusions: Pirin downregulates E-cadherin gene expression in Bcl3 independent manner, and contributes to EMT and cancer malignancy. These data provide evidence that pirin may be a potent target toward cancer therapy. 151 POSTER Impact of EGFR amplification pattern on the expression of miRNA-200c in primary glioblastoma multiforme L. Munoz ˜ Hidalgo1 , C. Lopez ´ Gines2 , E. Serna3 , D. Monleon1 , R. Callaghan2 , R. Gil Benso2 , H. Martinetto4 , A. Gregori Romero2 , J. Gonzalez Darder5 , M. Cerda Nicolas2 . 1 Fundation HCU-INCLIVA, Pathology, Valencia, Spain; 2 University of Valencia, Pathology, Valencia, Spain; 3 University of Valencia, UCIM, Valencia, Spain; 4 Institute FLENI, Neurological, Buenos Aires, Argentina; 5 Clinical Hospital, Neurosurgery, Valencia, Spain Glioblastoma Multiforme (GBM) is the most common tumor in the primary tumors of the central nervous system, accounting for 60% of neoplasms in this location. It is a highly aggressive tumor with a median survival of twelve months. Heterogeneity in the biological behavior of this neoplasm of astrocytic glial origin is expressed in the infiltrative nature, this is a critical feature in glioblastoma. Several miRNAs have been related with different types of cancer, some of them related with ability of modulation behavior neoplastic cells expressed epithelial/mesenchymal changes. In this regard, miRNA expression is deregulated in most, if not all, types of cancer. Based on the literature the most common dysregulation of miRNAs in GBM is over-expression; more than two hundred miRNAs have been found to be significantly overexpressed. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns in primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have