Jeroen A.C.M. Goos

SATB2 in Combination With Cytokeratin 20 Identifies Over 95% of all Colorectal Carcinomas

The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n=1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.

Maspin is a marker for early recurrence in primary stage III and IV colorectal cancer

Background:Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC.Methods:Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses.Results:Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (<6 months) and prolonged (>24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location.Conclusion:High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.

MGL ligand expression is correlated to BRAF mutation and associated with poor survival of stage III colon cancer patients.

Colorectal cancer (CRC) is the third most prevalent cancer type worldwide with a mortality rate of approximately 50%. Elevated cell-surface expression of truncated carbohydrate structures such as Tn antigen (GalNAcα-Ser/Thr) is frequently observed during tumor progression. We have previously demonstrated that the C-type lectin macrophage galactose-type lectin (MGL), expressed by human antigen presenting cells, can distinguish healthy tissue from CRC through its specific recognition of Tn antigen. Both MGL binding and oncogenic BRAF mutations have been implicated in establishing an immunosuppressive microenvironment. Here we aimed to evaluate whether MGL ligand expression has prognostic value and whether this was correlated to BRAFV600E mutation status. Using a cohort of 386 colon cancer patients we demonstrate that high MGL binding to stage III tumors is associated with poor disease-free survival, independent of microsatellite instability or adjuvant chemotherapy. In vitro studies using CRC cell lines showed an association between MGL ligand expression and the presence of BRAFV600E. Administration of specific BRAFV600E inhibitors resulted in decreased expression of MGL-binding glycans. Moreover, a positive correlation between induction of BRAFV600E and MGL binding to epithelial cells of the gastrointestinal tract was found in vivo using an inducible BRAFV600E mouse model. We conclude that the BRAFV600E mutation induces MGL ligand expression, thereby providing a direct link between oncogenic transformation and aberrant expression of immunosuppressive glycans. The strong prognostic value of MGL ligands in stage III colon cancer patients, i.e. when tumor cells disseminate to lymph nodes, further supports the putative immune evasive role of MGL ligands in metastatic disease.

Glucose Transporter 1 (SLC2A1) and Vascular Endothelial Growth Factor A (VEGFA) Predict Survival After Resection of Colorectal Cancer Liver Metastasis.

OBJECTIVE To investigate the individual and combined prognostic value of HIF1α, SLC2A1, and vascular endothelial growth factor A (VEGFA) in a multi-institutional cohort of patients with resected colorectal cancer liver metastasis (CRCLM). BACKGROUND In the majority of patients with CRCLM, resection seems not to be curative, despite its curative intent. Overexpression of hypoxia-inducible factor 1α (HIF1α), glucose transporter 1 (SLC2A1; also known as GLUT1), and VEGFA has been associated with tumor progression and poor prognosis of patients with colorectal cancer (CRC). METHODS Tissue microarrays were generated using CRCLM and patient-matched primary CRC from patients who underwent CRCLM resection between 1990 and 2010. Prognostic value of HIF1α, SLC2A1, and VEGFA was determined by immunohistochemistry. A 500-fold cross-validated hazard rate ratio (HRRav) for overall survival was calculated. RESULTS HIF1α, SLC2A1, and VEGFA expression could be evaluated in 328, 350, and 335 patients, respectively. High SLC2A1 expression was associated with good prognosis (HRRav, 0.67; P (HRR >1)  < 0.01) and high VEGFA expression to poor prognosis (HRRav, 1.84; P (HRR < 1)  = 0.02), also after multivariate analysis including established clinicopathological prognostic variables (HRRav, 0.67; P (HRR > 1)  < 0.01 and HRRav, 1.50; P (HRR < 1)  = 0.02, respectively). SLC2A1 showed prognostic value particularly in patients treated with systemic therapy (P < 0.01), whereas the prognostic value of VEGFA expression was mainly observed in patients not treated with systemic therapy (P < 0.01). Prognosis was especially poor in patients with both low SLC2A1 and high VEGFA expression (P < 0.01). HIF1α expression was not associated with survival. CONCLUSIONS SLC2A1 and VEGFA expression are prognostic molecular biomarkers for patients with CRCLM with added value to established clinicopathological variables.

Epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prognostic biomarkers for patients with resected colorectal cancer liver metastases.

Background:Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ∼40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM.Methods:Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure.Results:EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance).Conclusions:EGFR and PTGS2 expressions are prognostic molecular biomarkers with added value to standard clinicopathological variables for patients with resectable CRCLM.

Combination of episulfide ring-opening polymerization with ATRP for the preparation of amphiphilic block copolymers.

We report for the first time the combination of ATRP and ring-opening episulfide polymerization as a means to synthesize polysulfide-based low-dispersity amphiphilic block copolymers. The most significant finding is the possibility to perform ATRP under mild conditions using poly(propylene sulfide) macroinitiators, apparently without any significant copper sequestration by the polysulfides. Using glycerol monomethacrylate (GMMA) as a hydrophilic monomer, the polymers self-assembled in colloidal structures with a morphology depending on the PS/GMMA ratio, but also probably on GMMA degree of polymerization. We here also present a new AFM-based method to calculate the average number of amphiphilic macromolecules per micelle.

Molecular imaging of aurora kinase A (AURKA) expression: Synthesis and preclinical evaluation of radiolabeled alisertib (MLN8237).

INTRODUCTION Survival of patients after resection of colorectal cancer liver metastasis (CRCLM) is 36%-58%. Positron emission tomography (PET) tracers, imaging the expression of prognostic biomarkers, may contribute to assign appropriate management to individual patients. Aurora kinase A (AURKA) expression is associated with survival of patients after CRCLM resection. METHODS We synthesized [(3)H]alisertib and [(11)C]alisertib, starting from [(3)H]methyl nosylate and [(11)C]methyl iodide, respectively. We measured in vitro uptake of [(3)H]alisertib in cancer cells with high (Caco2), moderate (A431, HCT116, SW480) and low (MKN45) AURKA expression, before and after siRNA-mediated AURKA downmodulation, as well as after inhibition of P-glycoprotein (P-gp) activity. We measured in vivo uptake and biodistribution of [(11)C]alisertib in nude mice, xenografted with A431, HCT116 or MKN45 cells, or P-gp knockout mice. RESULTS [(3)H]Alisertib was synthesized with an overall yield of 42% and [(11)C]alisertib with an overall yield of 23%±9% (radiochemical purity ≥99%). Uptake of [(3)H]alisertib in Caco2 cells was higher than in A431 cells (P=.02) and higher than in SW480, HCT116 and MKN45 cells (P<.01). Uptake in A431 cells was higher than in SW480, HCT116 and MKN45 cells (P<.01). Downmodulation of AURKA expression reduced [(3)H]alisertib uptake in Caco2 cells (P<.01). P-gp inhibition increased [(3)H]alisertib uptake in Caco2 (P<.01) and MKN45 (P<.01) cells. In vivo stability of [(11)C]alisertib 90min post-injection was 94.7%±1.3% and tumor-to-background ratios were 2.3±0.8 (A431), 1.6±0.5 (HCT116) and 1.9±0.5 (MKN45). In brains of P-gp knockout mice [(11)C]alisertib uptake was increased compared to uptake in wild-type mice (P<.01) CONCLUSIONS: Radiolabeled alisertib can be synthesized and may have potential for the imaging of AURKA, particularly when AURKA expression is high. However, the exact mechanisms underlying alisertib accumulation need further investigation. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE Radiolabeled alisertib may be used for non-invasively measuring AURKA protein expression and to stratify patients for treatment accordingly.

A prognostic classifier for patients with colorectal cancer liver metastasis, based on AURKA, PTGS2 and MMP9

Background Prognosis of patients with colorectal cancer liver metastasis (CRCLM) is estimated based on clinicopathological models. Stratifying patients based on tumor biology may have additional value. Methods Tissue micro-arrays (TMAs), containing resected CRCLM and corresponding primary tumors from a multi-institutional cohort of 507 patients, were immunohistochemically stained for 18 candidate biomarkers. Cross-validated hazard rate ratios (HRRs) for overall survival (OS) and the proportion of HRRs with opposite effect (P(HRR < 1) or P(HRR > 1)) were calculated. A classifier was constructed by classification and regression tree (CART) analysis and its prognostic value determined by permutation analysis. Correlations between protein expression in primary tumor-CRCLM pairs were calculated. Results Based on their putative prognostic value, EGFR (P(HRR < 1) = .02), AURKA (P(HRR < 1) = .02), VEGFA (P(HRR < 1) = .02), PTGS2 (P(HRR < 1) = .01), SLC2A1 (P(HRR > 1) < 01), HIF1α (P(HRR > 1) = .06), KCNQ1 (P(HRR > 1) = .09), CEA (P (HRR > 1) = .05) and MMP9 (P(HRR < 1) = .07) were included in the CART analysis (n = 201). The resulting classifier was based on AURKA, PTGS2 and MMP9 expression and was associated with OS (HRR 2.79, p < .001), also after multivariate analysis (HRR 3.57, p < .001). The prognostic value of the biomarker-based classifier was superior to the clinicopathological model (p = .001). Prognostic value was highest for colon cancer patients (HRR 5.71, p < .001) and patients not treated with systemic therapy (HRR 3.48, p < .01). Classification based on protein expression in primary tumors could be based on AURKA expression only (HRR 2.59, p = .04). Conclusion A classifier was generated for patients with CRCLM with improved prognostic value compared to the standard clinicopathological prognostic parameters, which may aid selection of patients who may benefit from adjuvant systemic therapy.

Abstract 5217: MMP9 is a prognostic biomarker for metastatic colorectal cancer

Background: Worldwide almost 700000 patients are diagnosed with colorectal cancer (CRC) each year. Of these patients more than 45% die as a consequence of metastases, of which the majority is located in the liver. Although patient selection for hepatectomy by 18 FDG PET has significantly reduced futile surgery (Ruers, J Nucl Med 2009), 70% of all operated patients still die of metastases within 5 years after hepatic resection. To improve prediction of patient outcome, novel PET tracers with better prognostic value are needed. Matrix metalloproteinases, frequently associated with reduced survival in several types of cancer, might serve as prognostic biomarkers that can be used in PET imaging. Aim: To investigate the prognostic value of MMP9 expression in colon cancer liver metastases. Methods: Formalin-fixed paraffin-embedded colon cancer liver metastases, collected from 85 patients having had curative hepatectomy between 1990 and 2009, were incorporated into tissue microarrays (TMAs). MMP9 expression in both epithelial and stromal cells was assessed by immunohistochemistry and expression was correlated to patient survival data. Survival analysis was performed using a log rank test. Results: Mean age of the colon cancer patients (62.1% males, 37.9% females) was 62.0 (standard deviation 10.5). Chemotherapy was received by 45.9% (15.3% neoadjuvant, 14.1% adjuvant, 16.5% palliative) and radiofrequency ablation (RFA) was performed in 10.8%. Median overall survival was 35.0 months (standard deviation 5.7). High expression of MMP9 by epithelial colon cancer cells within the liver metastases was associated with increased survival rates (p=0.007 for disease-free survival and p=0.056 for overall survival). MMP9 expression in stromal cells was not associated with patient survival. Discussion and conclusion: Our results suggest that elevated levels of MMP9 in metastatic epithelial colon cancer cells are indicative of a good prognosis. Whether MMP9 qualifies as a target with prognostic relevance for imaging of lesions in patients with metastatic colorectal cancer remains to be determined. This research was supported by the Center for Translational Molecular Medicine (DeCoDe project). 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 5217. doi:10.1158/1538-7445.AM2011-5217

Abstract 3125: KCNQ1 expression is a strong prognostic biomarker for disease recurrence in stage II and III colon cancer

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at high risk of recurrence and stage III patients at low risk of recurrence are key unmet clinical needs. We previously identified KCNQ1 as a tumour suppressor gene of which loss of expression was associated with poor survival in patients with CRC liver metastases. The present study aimed to examine the prognostic value of KCNQ1 in stage II and III colon cancer patients. Methods: KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. KCNQ1 protein expression was evaluated by immuno-histochemistry on tissue microarrays of 386 stage II and III colon cancer patients. Results: Low KCNQ1 mRNA expression in microsatellite stable (MSS) stage II colon cancers was associated with poor disease free survival (DFS) (HR 3.35; 95% CI 1.16-9.66; p Conclusion: We conclude that KCNQ1 is a strong prognostic biomarker for prediction of disease recurrence (HR∼4) and may aid stratification of patients with stage II MSS colon cancer and stage III MSI CRC for adjuvant chemotherapy. Because KCNQ1 protein expression is determined by immuno-histochemistry, this biomarker can be implemented in standard clinical care using existing workflows. Citation Format: Sjoerd H. den Uil, Veerle M.H. Coupe, Janneke F. Linnekamp, Evert van den Broek, Jeroen A.C.M. Goos, Pien M. Delis-van Diemen, Eric J.T. Belt, Nicole C.T. van Grieken, Patricia M. Scott, Louis Vermeulen, Jan Paul Medema, Herman Bril, Hein B.A.C. Stockmann, Robert T. Cormier, Gerrit A. Meijer, Remond J. Fijneman. KCNQ1 expression is a strong prognostic biomarker for disease recurrence in stage II and III colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3125.