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Background Radioembolization is an established treatment modality in colorectal cancer patients with liver-dominant disease in a salvage setting. Selection of patients who will benefit most is of vital importance. The aim of this study was to assess response (and mode of progression) at 3 months after radioembolization and the impact of baseline characteristics. Methods Three months after radioembolization with either yttrium-90 resin/glass or holmium-166, anatomic response, according to RECIST 1.1, was evaluated in 90 patients. Correlations between baseline characteristics and efficacy were evaluated. For more detailed analysis of progressive disease as a dismal clinical entity, distinction was made between intra- and extrahepatic progression, and between progression of existing metastases and new metastases. Results Forty-two patients (47%) had extrahepatic disease (up to five ≥ 1 cm lung nodules, and ≤ 2 cm lymph nodes) at baseline. No patients showed complete response, 5 (5.5%) patients had partial response, 16 (17.8%) had stable disease, and 69 (76.7%) had progressive disease. Most progressive patients (67/69; 97%) had new metastases (intra-hepatic N = 11, extrahepatic N = 32; or both N = 24). Significantly fewer patients had progressive disease in the group of patients presenting without extrahepatic metastases at baseline (63% versus 93%; p = 0.0016). Median overall survival in patients with extrahepatic disease was 6.5 months, versus 10 months in patients without extrahepatic disease at baseline (hazard ratio 1.79, 95%CI 1.24–2.57). Conclusions Response at 3-month follow-up and survival were heavily influenced by new metastases. Patients with extrahepatic disease at baseline had a worse outcome compared to patients without.

High levels of oxidative stress in disseminated colorectal cancer tumor cells may form a therapeutically exploitable vulnerability. However, it is unclear whether oxidative stress and damage persist in metastases. Therefore, we analyzed markers of oxidative damage in primary colorectal tumors and their corresponding liver metastases. Markers of generic and oxidative DNA damage [phosphorylated histone H2AX (γH2AX) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] were significantly higher in liver metastases compared with their corresponding primary tumors. Chemotherapy and/or radiotherapy before tumor resection was associated with increased persistent oxidative DNA damage, and this effect was more pronounced in metastases. Immunohistochemistry-based molecular classification into epithelial- and mesenchymal-like molecular subtypes revealed that untreated mesenchymal-like tumors contained lower levels of oxidative DNA damage compared with epithelial-like tumors. Treated mesenchymal-like tumors, but not epithelial-like tumors, showed significantly higher levels of γH2AX and 8-OHdG. Mesenchymal-like tumors expressed significantly lower levels of phosphorylated nuclear factor erythroid 2-related factor 2, a master regulator of the antioxidant response, and nuclear factor erythroid 2-related factor 2-controlled genes. Of interest, a positive 8-OHdG status identified a subgroup of mesenchymal-like metastases with a poor overall survival. An increased capacity to tolerate therapy-induced oxidative damage in mesenchymal-like colorectal cancer may explain, at least in part, the poor responsiveness of these tumors to chemotherapy, which could contribute to the poor survival of this patient subgroup.

Reactive oxygen species (ROS) function as second messengers in signal transduction, but high ROS levels can also cause cell death. MTH1 dephosphorylates oxidized nucleotides, thereby preventing their incorporation into DNA and protecting tumour cells from oxidative DNA damage. Inhibitors of MTH1 (TH588 and (S)-crizotinib) were shown to reduce cancer cell viability. However, the MTH1-dependency of the anti-cancer effects of these drugs has recently been questioned. Here, we have assessed anti-tumour effects of TH588 and (S)-crizotinib in patient-derived 3D colorectal cancer cultures. Hypoxia and reoxygenation – conditions that increase intracellular ROS levels – increased sensitivity to (S)-crizotinib, but not to TH588. (S)-crizotinib reduced tyrosine phosphorylation of c-MET and ErbB3 whereas TH588 induced a mitotic cell cycle arrest, which was not affected by adding ROS-modulating compounds. Furthermore, we show that both compounds induced DNA damage that could not be prevented by adding the ROS inhibitor N-acetyl-L-cysteine. Moreover, adding ROS-modulating compounds did not alter the reduction in viability in response to TH588 and (S)-crizotinib. We conclude that TH588 and (S)-crizotinib have very clear and distinct anti-tumour effects in 3D colorectal cancer cultures, but that these effects most likely occur through distinct and ROS-independent mechanisms.

Metastatic colorectal cancer (mCRC) is a devastating disease causing 700.000 deaths annually worldwide. Metastases most frequently develop in the liver. Partial hepatectomy has dramatically improved clinical outcome and is the only curative treatment option for eligible patients with mCRC. Pre-clinical studies have shown that surgical procedures can have tumor-promoting local 'side-effects’ such as hypoxia and inflammation, thereby altering the behaviour of residual tumor cells. In addition, systemically released factors following (colon or liver) surgery can act as a wakeup-call for dormant tumor cells in distant organs and/or help establish a pre-metastatic niche. Tumor handling during resection may also increase the number of circulating tumor cells. Despite the overwhelming amount of pre-clinical data demonstrating the pro-tumorigenic side effects of surgery, clinical evidence is scarce. Indications for hepatic surgery are rapidly increasing due to a rise in the incidence of mCRC and a trend towards more aggressive surgical treatment. Therefore, it is increasingly important to understand the principles of surgery-induced tumor growth, in order to devise perioperative or adjuvant strategies to further enhance long-term tumor control. In the current study we review the evidence for surgery-stimulated tumor growth and suggest strategies to assess the clinical relevance of such findings.

PURPOSE: To assess applicability of metabolic tumor response assessment on 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) after radioembolization (RE) in patients with colorectal liver metastases (CRLM) by comparison with one-dimensional size-based response assessment on MR imaging. MATERIALS AND METHODS: This prospective cohort study comprised 38 patients with CRLM undergoing RE. MR imaging and 18F-FDG PET/CT imaging were performed at baseline, 1 month (n = 38), and 3 months (n = 21). Longest tumor diameter (LTD) reduction on MR imaging at these time points was compared with reduction in total lesion glycolysis (TLG) on 18F-FDG PET/CT. Hepatic response was compared between RECIST and total liver TLG and correlated with overall survival (OS). RESULTS: TLG and LTD were positively correlated in 106 analyzed metastases (38 patients) at 1 month and 58 metastases (22 patients) at 3 months. Agreement was poor, with LTD underestimating TLG response. A significant association with prolonged OS was found in total liver TLG at 1 month (HR 0.64, P < .01) and 3 months (HR 0.43, P < .01). For LTD, a significant association with OS was found at 3 months (HR 0.10, P < .01). Important differences in liver response classification were found, with total liver TLG identifying more patients and situations where there appeared to be treatment benefit compared with RECIST. CONCLUSIONS: TLG response assessment on 18F-FDG PET/CT appears to be more sensitive and accurate, especially at early follow-up, than size-based response assessment on MR imaging in patients with CRLM treated by RE. Semiautomated liver response assessment with total liver TLG is objective, reproducible, rapid, and prognostic.

Surgical removal of colorectal cancer (CRC) liver metastases generates areas of tissue hypoxia. Hypoxia imposes a stem-like phenotype on residual tumor cells and promotes tumor recurrence. Moreover, in primary CRC, gene expression signatures reflecting hypoxia and a stem-like phenotype are highly expressed in the aggressive Consensus Molecular Subtype 4 (CMS4). Therapeutic strategies eliminating hypoxic stem-like cells may limit recurrence following resection of primary tumors or metastases. Here we show that expression of DNA repair genes is strongly suppressed in CMS4 and inversely correlated with hypoxia-inducible factor-1 alpha (HIF1α) and HIF-2α co-expression signatures. Tumors with high expression of HIF signatures and low expression of repair proteins showed the worst survival. In human tumors, expression of the repair proteins RAD51, KU70 and RIF1 was strongly suppressed in hypoxic peri-necrotic tumor areas. Experimentally induced hypoxia in patient derived colonospheres in vitro or in vivo (through vascular clamping) was sufficient to downregulate repair protein expression and caused DNA damage. Hypoxia-induced DNA damage was prevented by expressing the hydroperoxide-scavenging enzyme glutathione peroxidase-2 (GPx2), indicating that reactive oxygen species mediate hypoxia-induced DNA damage. Finally, the hypoxia-activated prodrug Tirapazamine greatly augmented DNA damage and reduced the fraction of stem-like (Aldefluorbright) tumor cells in vitro, and in vivo following vascular clamping. We conclude that decreased expression of DNA repair proteins and increased DNA damage in hypoxic tumor areas may be therapeutically exploited with hypoxia-activated prodrugs, and that such drugs reduce the fraction of Aldefluorbright (stem-like) tumor cells.

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.

Purpose To determine the surgical and oncologic outcomes after major liver surgery for colorectal liver metastases (CRLM) at a Dutch University Hospital. Patients and Methods Consecutive patients with CRLM who had undergone major liver resection, defined as ≥ 4 liver segments, between January 2000 and December 2015 were identified from a prospectively maintained database. Results Major liver surgery was performed in 117 patients. Of these, 26 patients had undergone formal extended left or right hemihepatectomy. Ninety-day postoperative mortality was 8%. Major postoperative complications occurred in 27% of patients; these adverse events were more common in the extended hemihepatectomy group. Median disease-free survival was 11 months and median overall survival 44 months. Conclusion Major liver surgery, including formal extended hemihepatectomy, is associated with significant operative morbidity and mortality but can confer prolonged overall survival for patients with CRLM.