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Purpose: Risk stratification after surgery for colorectal cancer liver metastases (CRLM) is achieved using clinicopathologic variables, however, is of limited accuracy. We sought to derive and externally validate a multigene expression assay prognostic of overall survival (OS) that is superior to clinicopathologic variables in patients with surgically resected CRLM. Experimental Design: We measured mRNA expression in prospectively collected frozen tumor from 96 patients with surgically resected CRLM at Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY). We retrospectively generated a 20-gene molecular risk score (MRS) and compared its prognostic utility for OS and recurrence-free survival (RFS) with three common clinical risk scores (CRS). We then tested the prognostic ability of the MRS in an external validation cohort (European) of 119 patients with surgically resected CRLM at the University Medical Center Utrecht (Utrecht, the Netherlands) and Paul Brousse Hospital (Villejuif, France). Results: For OS in the MSKCC cohort, MRS was the strongest independent prognosticator (HR, 3.7–4.9; P < 0.001) followed by adjuvant chemotherapy (HR, 0.3; P ≤ 0.001). For OS in the European cohort, MRS was the only independent prognosticator (HR, 3.5; P = 0.007). For RFS, MRS was also independently prognostic in the MSKCC cohort (HR, 2.4–2.6; P ≤ 0.001) and the European cohort (HR, 1.6–2.5; P ≤ 0.05). Conclusions: Compared with CRSs, the MRS is more accurate, broadly applicable, and an independent prognostic biomarker of OS in resected CRLM. This MRS is the first externally validated prognostic multigene expression assay after metastasectomy for CRLM and warrants prospective validation. Clin Cancer Res; 22(10); 2575–82. ©2016 AACR.

Purpose: Mutational activation of the KRAS oncogene and overexpression of cyclooxygenase-2 (COX-2) contribute to colorectal carcinoma (CRC) development, but the relationship between these two events is unclear. This study was designed to clarify that relationship and to assess the contribution of KRAS-dependent COX-2 to the seeding of CRC cells in the liver and to their outgrowth as liver metastases in an experimental mouse model. Experimental Design: The effect of RNA interference–mediated KRAS knockdown on COX-2 expression and activity was tested in murine C26 CRC cells. The contribution of KRAS-dependent COX-2 to early metastatic tumor cell seeding (by intravital microscopy) and outgrowth of metastases in the liver (by bioluminescence imaging) was studied by using parecoxib, a novel and highly selective liver-activated COX-2 inhibitor. Intratumoral cell proliferation, apoptosis, and tumor-associated angiogenesis were assessed by immunohistochemistry on liver tissue sections. Results: Stable knockdown of mutant KRASD12 in murine C26 CRC cells by RNA interference lead to a dramatic reduction of COX-2 synthesis and prostaglandin E2 production. Inhibition of host or tumor cell COX-2 activity had no effect on early metastatic cell seeding in the liver but greatly reduced intrahepatic tumor cell proliferation and the rate of liver metastasis outgrowth. COX-2 inhibition had no effect on early tumor vascularization or on tumor cell apoptosis. Conclusions: The high levels of COX-2 enzyme and prostaglandin production in C26 CRC cells are primarily caused by the presence of endogenous mutant KRASD12. Furthermore, COX-2 inhibition affects the tumoral rather than the vascular compartment during the early stages of C26 liver metastasis outgrowth.

Background: Tumors are addicted to pathways neutralizing oxidative stress as a result of their high levels of reactive oxygen species (ROS). These ROS can oxidize nucleotides (NTs) within the precursor pool, which can cause cell death when incorporated into DNA. This tumor specific vulnerability might be exploited therapeutically. The mutT-homologue (MTH1) enzyme is recently identified as a non-oncogene target, which prevents incorporation of oxidized NTs into DNA (Gad et al, 2014; Huber et al, 2014). MTH1 inhibition by TH588 and (S)-Crizotinib caused tumor-specific oxidative DNA damage and cell death (Gad et al, 2014; Huber et al, 2014). Interestingly, hypoxia might enhance sensitivity to MTH1 inhibition (Qiu et al, 2015; Bräutigam et al, 2016). Thus, exploiting tumor oxidative stress via inhibition of MTH1 might be an exciting new approach to fight colorectal cancer (CRC) even in the presence of hypoxia. Aims and Hypotheses: In the present study we aim to compare the ability of TH588 HCl and (S)-Crizotinib to inhibit CRC growth in vitro and in vivo and to assess their mechanism(s) of action, including MTH1-dependent and -independent effects. We hypothesize that both compounds have the potency to inhibit CRC growth, conceivably via different mechanisms of action, which might not dependent on MTH1 inhibition (Kettle et al, 2016). Methods: All data were obtained using a human CRC liver metastasis-derived 3D spheroid line untreated or treated for 3 days with TH588 HCl or (S)-Crizotinib. Spheroids were pre-cultured and treated under normoxia (21% O2), hypoxia (0.1% O2) or only pre-cultured under hypoxia to mimic hypoxia and reoxygenation respectively. Results: Using a CellTiter-Glo cell viability assay we showed that increasing concentrations of (S)-Crizotinib under normoxia reduced cell viability with 23%, 64%, and 98% for 5, 10 and 20 μM respectively. By contrast, the maximal reduction of cell viability after treatment with TH588 HCl was only 32%. Interestingly, the dose response curve of (S)-Crizotinib was steeper when spheroids were reoxygenated just before treatment compared to normoxia and hypoxia, which implies that (S)-Crizotinib is more efficient. Importantly, hypoxia completely abolished the TH588-induced reduction in cell viability seen during normoxia. While the immediate effects on cell viability were different, both drugs (10 μM) severely reduced clone-forming efficiency both under normoxia and after hypoxia-reoxygenation prior to treatment. These results suggest different mechanisms of action of both drugs. As expected, both drugs caused an increase in DNA double-strand breaks, as measured by γH2AX staining. While chemotherapy treatment usually enriches the content of cancer stem cells, so far we did not observe this with either MTH1 inhibitor. Discussion and Conclusions: Until now, most research involving MTH1 inhibitors has been performed using cancer cell lines. In the present study we use patient-derived CRC spheroids and organoids. We previously found that organoids derived from patients with a poor-prognosis type of CRC are generally characterized by high expression of a H2O2 stress signature (Emmink et al, 2014). Exploiting this oxidative stress phenotype via inhibition of MTH1 might be an attractive new therapeutic avenue. Our results suggest that TH588 HCl and (S)-Crizotinib induce DNA damage and effectively reduce clone-forming potential of a CRC spheroid line, which was not limited by hypoxia pre-culturing. Interestingly, both compounds appear to act via different mechanisms. Further research should unravel these mechanisms of action. Citation Format: Lizet M. van der Waals, Danielle A.E. Raats, Jennifer M.J. Jongen, Tobias B. Dansen, Inne H.M. Borel Rinkes, Onno W. Kranenburg. Exploiting oxidative stress using MTH1 inhibitors in colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A29.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable model systems to interrogate pathways involved in pancreatic tumorigenesis and to probe individual responses to novel therapies are urgently needed. To that end, we established methods to culture normal and neoplastic pancreatic duct cells as three-dimensional organoid cultures. Pancreatic organoids can be rapidly generated from resected tumors or fine needle biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Following orthotopic transplant, neoplastic organoids recapitulated the full spectrum of tumor development by forming early-grade neoplasms that progressed to locally invasive and metastatic carcinomas, demonstrating the utility of organoids to model the stages of PDA tumorigenesis. Monolayer cell lines were generated from organoid cultures with high efficiency, creating a diverse collection of new PDA cell lines. To better understand pathways involved in PDA progression, we performed transcriptomic and proteomic analyses of murine organoids derived from normal pancreatic ducts, pancreatic intraepithelial neoplasias (PanINs), and PDAs. These datasets revealed expression changes associated with early and late pancreatic tumorigenesis. To identify genes dysregulated during pancreatic tumorigenesis whose depletion impaired human PDA cells, a CRISPR-Cas competition assay was employed. Taken together, pancreatic organoids offer a novel model system for studying pancreatic cancer biology and can be used to screen for genetic dependencies in PDA. Citation Format: Lindsey A. Baker, Hervé Tiriac, Vincenzo Corbo, Sylvia F. Boj, Chang-il Hwang, Iok In Christine Chio, Danielle D. Engle, Myrthe Jager, Mariano Ponz-Sarvise, Mona S. Spector, Ana Gracanin, Tobiloba Oni, Kenneth H. Yu, Ruben van Boxtel, Meritxell Huch, Keith D. Rivera, John P. Wilson, Michael E. Feigin, Daniel Öhlund, Abram Handly-Santana, Christine M. Ardito-Abraham, Michael Ludwig, Ela Elyada, Brinda Alagesan, Giulia Biffi, Georgi N. Yordanov, Bethany Delcuze, Brianna Creighton, Kevin Wright, Youngkyu Park, Folkert H.M. Morsink, I. Quintus Molenaar, Inne H. Borel Rinkes, Edwin Cuppen, Yuan Hao, Ying Jin, Isaac J. Nijman, Christine Iacobuzio-Donahue, Steven D. Leach, Darryl J. Pappin, Molly Hammell, David S. Klimstra, Olca Basturk, Ralph H. Hruban, George Johan Offerhaus, Robert G.J. Vries, Hans Clevers, David A. Tuveson. Using human patient-derived organoids to identify genetic dependencies in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B16.