Identification of a gene expression signature associated with brain metastasis in colorectal cancer.

PURPOSE: Brain metastasis (BM) in colorectal cancer (CRC) is a rare event with poor prognosis. Apart from (K)RAS status and lung and bone metastasis no biomarkers exist to identify patients at risk. This study aimed to identify a gene expression signature associated with colorectal BM. METHODS: Three patient groups were formed: 1. CRC with brain metastasis (BRA), 2. exclusive liver metastasis (HEP) and, 3. non-metastatic disease (M0). RNA was extracted from primary tumors and mRNA expression was measured using a NanoString Panel (770 genes). Expression was confirmed by qPCR in a validation cohort. Statistical analyses including multivariate logistic regression followed by receiver operating characteristic (ROC) analysis were performed. RESULTS: EMILIN3, MTA1, SV2B, TMPRSS6, ACVR1C, NFAT5 and SMC3 were differentially expressed in BRA and HEP/M0 groups. In the validation cohort, differential NFAT5, ACVR1C and SMC3 expressions were confirmed. BRA patients showed highest NFAT5 levels compared to HEP/M0 groups (global p = 0.02). High ACVR1C expression was observed more frequently in the BRA group (42.9%) than in HEP (0%) and M0 (7.1%) groups (global p = 0.01). High SMC3 expressions were only detectable in the BRA group (global p = 0.003). Only patients with BM showed a combined high expression of NFAT5, ACVR1C or SMC3 as well as of all three genes. ROC analysis revealed a good prediction of brain metastasis by the three genes (area under the curve (AUC) = 0.78). CONCLUSIONS: The NFAT5, ACVR1C and SMC3 gene expression signature is associated with colorectal BM. Future studies should further investigate the importance of this biomarker signature.

TGFß signalling pathway impacts brain metastases profiles in locally advanced colorectal cancer.

RATIONALE: Colorectal Cancer (CRC) represents the third most common type of cancer in Germany and the second most common cancer-related cause of death worldwide. Distant metastases are still the main limit for patient survival. While liver metastases as well as peritoneal carcinomatosis can often either be resected or treated with systemic therapy, little options remain for brain metastases. Additionally, a number of studies has already investigated hepatic, peritoneal, pulmonary as well as continuing distant metastases in colorectal cancer. Yet, with respect to tumor biology and brain metastases, little is known so far. MATERIAL AND METHODS: Two cohorts, M0 without distant spread and BRA with brain metastases were build. RNA was isolated from paraffin embedded specimen. Gene expression was performed by an RNA NanoString-Analysis using the nCounter® PanCancer Progression Panel by NanoString-Technologies (Hamburg, Germany). Results were analysed by principal component analysis, gene expression and pathway analysis using commonly available databases such as KEGG as benchmark for comparison. RESULTS: We were able to determine a gene signature that provides a sophisticated group separation between M0 and BRA using principal component analysis. All genes with strong loading characteristics on principal component 1 were cross-referenced with the subsequently performed accurate gene set enrichment analysis (GSEA). The GSEA revealed a clear dysregulation of the TGFß pathway in compared cohorts M0 and BRA. Interestingly, the targeted pathways analysis of the identified genes confirmed that in fact almost all strong loading genes of PC1 play a role in the TGFß pathway. CONCLUSION: Our results suggest the TGFß pathway as a crucial player in the development of brain metastases in primary CRC. In some types of colorectal cancer, downregulation of the TGFß pathway might hinder primary colorectal cancer to metastasize to the nervous system. While the paradoxical functioning of the TGFß pathway is still not fully understood, these shed light on yet another clinical implication of this complex pathway.

PPARG activation promotes the proliferation of colorectal cancer cell lines and enhances the antiproliferative effect of 5-fluorouracil.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARG) is a member of the nuclear receptor family. It is involved in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, vascular homeostasis and inflammation. In addition, PPARG agonists, known as thiazolidinediones, are well established in the treatment of type 2 diabetes mellitus. PPARGs role in cancer is a matter of debate, as pro- and anti-tumour properties have been described in various tumour entities. Currently, the specific role of PPARG in patients with colorectal cancer (CRC) is not fully understood. MATERIAL AND METHODS: The prognostic impact of PPARG expression was investigated by immunohistochemistry in a case-control study using a matched pair selection of CRC tumours (n = 246) with either distant metastases to the liver (n = 82), lung (n = 82) or without distant metastases (n = 82). Its effect on proliferation as well as the sensitivity to the chemotherapeutic drug 5-fluorouracil (5-FU) was examined after activation, inhibition, and transient gene knockdown of PPARG in the CRC cell lines SW403 and HT29. RESULTS: High PPARG expression was significantly associated with pulmonary metastasis (p = 0.019). Patients without distant metastases had a significantly longer overall survival with low PPARG expression in their tumours compared to patients with high PPARG expression (p = 0.045). In the pulmonary metastasis cohort instead, a trend towards longer survival was observed for patients with high PPARG expression in their tumour (p = 0.059). Activation of PPARG by pioglitazone and rosiglitazone resulted in a significant dose-dependent increase in proliferation of CRC cell lines. Inhibition of PPARG by its specific inhibitor GW9662 and siRNA-mediated knockdown of PPARG significantly decreased proliferation. Activating PPARG significantly increased the CRC cell lines sensitivity to 5-FU while its inhibition decreased it. CONCLUSION: The prognostic effect of PPARG expression depends on the metastasis localization in advanced CRC patients. Activation of PPARG increased malignancy associated traits such as proliferation in CRC cell lines but also increases sensitivity towards the chemotherapeutic agent 5-FU. Based on this finding, a combination therapy of PPARG agonists and 5-FU-based chemotherapy constitutes a promising strategy which should be further investigated.

Long-Term Progression-Free Survival of a Pre-Treated Patient with Metastatic Colorectal Cancer Receiving Trifluridine/Tipiracil.

Trifluridine/tipiracil is approved for the use in later or last-line setting in previously treated metastatic colorectal cancer (mCRC) patients who progressed on standard anti-tumor drugs including 5-fluorouracil (5-FU), irinotecan, oxaliplatin, anti-VEGF and anti-EGFR antibodies, or who are not considered candidates for those standard therapies. In this report, we describe a 67-year-old male patient with KRAS-mutated mCRC and metachronous liver and lung metastasis who failed prior 5-FU- and irinotecan-containing regimens, but then showed long-term disease control for 31 months on single-agent trifluridine/tipiracil given as second-line treatment. According to our experience, trifluridine/tipiracil is a feasible and effective treatment option in earlier but not necessarily last-line therapy in mCRC patients who are not considered candidates for doublet or triplet chemotherapy. Besides its efficacy, it is associated with maintained quality of life and a manageable toxicity profile. Considering increasing age of mCRC patients and their wish for maintaining an independent lifestyle, further research on the use of trifluridine/tipiracil in earlier lines of systemic mCRC therapy is warranted.

[Molecular pathology of colorectal cancer]. / Molekularpathologie kolorektaler Karzinome.

In recent years, the treatment of colorectal carcinoma has experienced increasing individualization. In addition to RAS and BRAF mutational status that is firmly established in routine diagnostics, new therapeutic options evolved based on MSI and HER2 status as well as primary tumour localization. Offering the best targeted options in therapy requires new evidence-based decision-making algorithms regarding timing and scope of molecular pathological diagnostics in order for patients to receive an optimized therapy according to current treatment guidelines. New targeted therapies, some of which are about to be approved and for which pathology has to provide new molecular pathological biomarkers, will also play an increasingly important role in the future.

Serial Analysis of Gene Mutations and Gene Expression during First-Line Chemotherapy against Metastatic Colorectal Cancer: Identification of Potentially Actionable Targets within the Multicenter Prospective Biomarker Study REVEAL.

Most metastatic colorectal cancer (mCRC) patients succumb to refractory disease due to secondary chemotherapy resistance. To elucidate the molecular changes associated with secondary resistance, we recruited 64 patients with mCRC and hepatic metastases before standard first-line chemotherapy between 2014 and 2018. We subjected DNA from primary tumor specimens (P), hepatic metastasis specimens after treatment (M), and liquid biopsies (L) taken prior to (pre), during (intra), and after (post) treatment to next generation sequencing. We performed Nanostring expression analysis in P and M specimens. Comparative bioinformatics and statistical analysis revealed typical mutational patterns with frequent alterations in TP53, APC, and KRAS in P specimens (n = 48). P and pre-L (n = 42), as well as matched P and M (n = 30), displayed a similar mutation spectrum. In contrast, gene expression profiles classified P (n = 31) and M (n = 23), distinguishable by up-regulation of immune/cytokine receptor and autophagy programs. Switching of consensus molecular subtypes from P to M occurred in 58.3% of cases. M signature genes SFRP2 and SPP1 associated with inferior survival, as validated in an independent cohort. Molecular changes during first-line treatment were detectable by expression profiling rather than by mutational tumor and liquid biopsy analyses. SFRP2 and SPP1 may serve as biomarkers and/or actionable targets.

Disease Modeling on Tumor Organoids Implicates AURKA as a Therapeutic Target in Liver Metastatic Colorectal Cancer.

BACKGROUND & AIMS: Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. METHODS: We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. RESULTS: Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α-related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS-wild-type organoids. In dual epidermal growth factor receptor (EGFR)- pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. CONCLUSIONS: Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression.

Early weight loss is an independent risk factor for shorter survival and increased side effects in patients with metastatic colorectal cancer undergoing first-line treatment within the randomized Phase III trial FIRE-3 (AIO KRK-0306).

Body weight loss is frequently regarded as negatively related to outcomes in patients with malignancies. This retrospective analysis of the FIRE-3 study evaluated the evolution of body weight in patients with metastatic colorectal cancer (mCRC). FIRE-3 evaluated first-line FOLFIRI (folinic acid, fluorouracil and irinotecan) plus cetuximab or bevacizumab in mCRC patients with RAS-WT tumors (ie, wild-type in KRAS and NRAS exons 2-4). The prognostic and predictive relevance of early weight loss (EWL) regarding patient outcomes and treatment side effects were evaluated. Retrospective data on body weight during first 6 months of treatment were evaluated (N = 326). To correlate with efficacy endpoints and treatment side effects, patients were grouped according to clinically significant EWL ≥5% and <5% at Month 3. Age constituted the only significant predictor of EWL following a linear relationship with the corresponding log odds ratio (P = .016). EWL was significantly associated with the incident frequencies of diarrhea, edema, fatigue, nausea and vomiting. Further, a multivariate analysis revealed EWL to be an independent negative prognostic factor for overall survival (32.4 vs 21.1 months; hazard ratio [HR]: 1.64; 95% confidence interval [CI] = 1.13-2.38; P = .0098) and progression-free survival (11.8 vs 9.0 months; HR: 1.72; 95% CI = 1.18-2.5; P = .0048). In conclusion, EWL during systemic treatment against mCRC is significantly associated with patient age. Patients exhibiting EWL had worse survival and higher frequencies of adverse events. Early preventative measures targeted at weight maintenance should be evaluated, especially in elderly patients being at highest risk of EWL.

Expression of CIB1 correlates with colorectal liver metastases but not with peritoneal carcinomatosis.

BACKGROUND: Molecular differences in colorectal cancer (CRC) are associated with the metastatic route. Patient survival is mainly driven by metastatic spread thus it is imperative to understand its key drivers to develop biomarkers for risk stratification, follow-up protocols and personalized therapy. Thus, this study aimed to identify genes associated with the metastatic route in CRC. MATERIAL AND METHODS: CRC patients resected at our clinic from 2005 to 2014 and with a minimum 5-year follow-up were included in this analysis and grouped into CRC with hepatic (HEP), peritoneal (PER) or without distant metastases (M0), and HEP/PER. Firstly, tumor RNA of 6 patients each was isolated by microdissection from formalin-fixed paraffin-embedded specimens and analyzed by a NanoString analysis. Subsequently, these results were validated with immunohistochemistry and correlated to clinicopathological parameters in a larger collective of CRC patients (HEP n = 51, PER n = 44, M0 n = 47, HEP/PER n = 28). RESULTS: Compared to M0, HEP tumors showed 20 differentially expressed genes associated with epithelial-mesenchymal transition (EMT) and angiogenesis. Compared to M0, PER tumors had 18 differentially expressed genes. The finding of different gene signatures was supported by the multidimensional principal component clustering analysis. Tumor perforation did not influence the metastatic route. CIB1 was homogenously and significantly overexpressed in HEP compared to M0 (p < 0.001), but not in PER. Furthermore, immunohistochemical validation demonstrated that the mean CIB1 expression in HEP was 80% higher than in M0 (p < 0.001). CONCLUSION: Gene expression analysis revealed that CIB1 is significantly overexpressed in CRC leading to liver metastases compared to M0 and PER. Thus, the present results suggest that CIB1 may play a crucial role for hematogenous spread to the liver but not for peritoneal carcinomatosis. Consequently, CIB1 seems to be a promising prognostic marker and a potential tool for future targeted therapies as well as early diagnostics and follow-up.

Outcome and Safety after 103 Radioembolizations with Yttrium-90 Resin Microspheres in 73 Patients with Unresectable Intrahepatic Cholangiocarcinoma-An Evaluation of Predictors.

Trans-arterial radioembolization (TARE) is increasingly evaluated for unresectable intrahepatic cholangiocarcinoma (ICC). Not all ICC patients benefit equally well from TARE. Therefore, we sought to evaluate variables predicting progression-free survival (PFS) and overall survival (OS). Patients with non-resectable ICC underwent TARE and were treated with 90Y resin microspheres. Baseline characteristics, biochemical/clinical toxicities, and response were examined for impact on PFS and OS. A total of 103 treatments were administered to 73 patients without major complications or toxicity. Mean OS was 18.9 months (95% confidence intervals (CI); 13.9-23.9 months). Mean and median PFS were 10.1 months (95% CI; 7.9-12.2) and 6.4 months (95% CI; 5.20-7.61), respectively. Median OS and PFS were significantly prolonged in patients with baseline cholinesterase (CHE) ≥ 4.62 kU/L (OS: 14.0 vs. 5.5 months; PFS: 6.9 vs. 3.2 months; p < 0.001). Patients with a tumor burden ≤ 25% had a significantly longer OS (15.2 vs. 6.6 months; p = 0.036). Median PFS was significantly longer for patients with multiple TARE cycles (24.4 vs. 5.8 months; p = 0.04). TARE is a considerable and safe option for unresectable ICC. CA-19-9, CHE, and tumor burden have predictive value for survival in patients treated with TARE. Multiple TARE treatments might further improve survival; this has to be confirmed by further studies.

Gender-dependent survival benefit from first-line irinotecan in metastatic colorectal cancer. Subgroup analysis of a phase III trial (XELAVIRI-study, AIO-KRK-0110).

BACKGROUND: XELAVIRI compared sequential (Arm A) versus initial (Arm B) irinotecan in combination with fluoropyrimidine plus bevacizumab in patients with metastatic colorectal cancer, trial identification: NCT01249638. In the full analysis set of the study, non-inferiority of time to failure of strategy (TFS) was not shown. The present analysis was performed to evaluate the effect of gender on treatment outcome and tolerability. METHODS: The study end-points overall response rate (ORR), progression-free survival (PFS), TFS and overall survival (OS) were evaluated in female versus male patients and in molecular subgroups (i.e. RAS mutational status). Interaction of treatment and gender was tested by likelihood ratio tests. RESULTS: In total, 281 male and 140 female patients (n = 421) were evaluated. Among the male patients, the ORR was 33.6% without and 58.3% with initial irinotecan (P < 0.001). PFS (hazard ratio [HR] 0.54; 95% confidence interval [CI] 0.42-0.69; P < 0.001) and OS (HR 0.63; 95% CI 0.47-0.85; P = 0.002) were also significantly better with initial irinotecan. Among the female patients, the ORR was 42.7% in Arm A and 43.1% in Arm B, PFS was similar (HR 1.09; 95% CI 0.76-1.55; P = 0.649) without and with initial irinotecan. A strong trend for inferior outcome with regard to OS with initial irinotecan was observed (HR 1.46; 95% CI 0.95-2.24; P = 0.081) and the trend reached significance in the multivariate analysis (HR 1.78; 95% CI 1.08-2.95; P = 0.02). Formal interaction of treatment and gender was observed for ORR (P = 0.018), PFS (P = 0.002) and OS (P = 0.001). Treatment-related adverse events were not significantly different between male and female patients. CONCLUSIONS: The present analysis suggests that gender interacts with efficacy of initial irinotecan when used in combination with fluoropyrimidines and bevacizumab. Although male patients derived a significant and clinically meaningful benefit from initial combination chemotherapy, this was not observed in female patients.

Patients with colorectal cancer and brain metastasis: The relevance of extracranial metastatic patterns predicting time intervals to first occurrence of intracranial metastasis and survival.

The aim of the study was to investigate the predictive impact of extracranial metastatic patterns on course of disease and survival in patients with colorectal cancer (CRC) and brain metastasis (BM). A total of 228 patients (134 male [59%], 94 female [41%]) with histologically proven CRC and BM were classified into different groups according to extracranial metastatic patterns. Time intervals to metastatic events and survival times from initial CRC diagnosis, extracranial and intracranial metastasis were analyzed. Extracranial organs mostly affected were liver (102 of 228 [44.7%]) and lung (96 of 228 [42.1%]). Liver and lung metastases were detected in 31 patients (13.6%). Calculated over the entire course of disease, patients with lung metastasis showed longer overall survival (OS) than patients with liver metastasis or patients without lung metastasis (43.9 vs 34.6 [P = .002] vs 35.0 months [P = .002]). From the date of initial CRC diagnosis, lung metastasis occurred later in CRC history than liver metastasis (24.3 vs 7.5 months). Once lung metastasis was diagnosed, BM occurred faster than in patients with liver metastasis (15.8 vs 26.0 months; Δ 10.2 months). Accordingly, OS from the diagnosis of liver metastasis was longer than from lung metastasis (27.1 vs 19.6 months [P = .08]). Once BM was present, patients with lung metastasis lived longer than patients with liver metastasis (3.8 vs 1.1 months [P = .028]). Shortest survival times in all survival categories analyzed revealed patients with concurrent liver and lung metastasis. Patients with CRC and BM form a heterogeneous cohort where extracranial metastasis to liver or lungs predicts survival.

Single-nucleotide variants, tumour mutational burden and microsatellite instability in patients with metastatic colorectal cancer: Next-generation sequencing results of the FIRE-3 trial.

BACKGROUND: Molecular biomarkers and primary tumour sidedness guide treatment decisions in metastatic colorectal cancer. Comprehensive molecular profiling aims to identify targetable alterations and measure tumour mutational burden (TMB) to enable precision oncology. MATERIAL AND METHODS: FoundationOne® next-generation sequencing identified single-nucleotide variants (SNVs), copy number alterations, high TMB (TMB-H) and high-grade microsatellite instability (MSI-H) in patients treated in the FIRE-3 trial. Data were correlated with objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). RESULTS: Three hundred seventy-three (49.6%) of 752 patients provided material for this analysis. Frequent SNVs included TP53, APC, KRAS, PIK3CA, BRAF, SMAD4 and FBXW7. KRAS, BRAF V600E and SMAD4 mutations were confirmed as prognostic biomarkers by logistic penalised regression for ORR. OS was significantly longer in patients with SMAD4 wild-type (WT) tumours than in those with SMAD4-mutated tumours (hazard ratio = 0.59 [95% confidence interval {CI} = 0.34-1.01], p = 0.05), with a higher probability of ORR [odds ratio, SMAD4 SNV versus WT = 0.32 [95% CI = 0.10-0.98], p = 0.05] when treated with cetuximab. MSI-H (30.0%, p = 0.03) and TMB-H (17.3%, p = 0.003) tumours were enriched by FBXW7 mutations. Numerically lower ORR, OS and PFS were observed in MSI-H tumours. CONCLUSIONS: RAS, BRAF V600E and SMAD4 mutations were identified as poor prognostic biomarkers in patients of the FIRE-3 trial, whereas improved outcome was observed for BRAF non-V600E mutation. SMAD4 mutation might provide predictive relevance for cetuximab efficacy. MSI-H tumours showed numerically lower ORR, OS and PFS.

Current treatment options in RAS mutant metastatic colorectal cancer patients: a meta-analysis of 14 randomized phase III trials.

PURPOSE: Although biomarkers for patients with metastatic colorectal cancer exist, the benefit patients with RAS mutated tumors derive from established regimens is unclear. METHODS: Efficacy of therapeutic strategies available for RAS mutated patients (addition of chemotherapeutic agents and/or anti angiogenic agents) were investigated in fourteen randomized controlled phase III trials at trial level by meta-analysing individual study hazard ratios and 95% confidence intervals (95% CI) for overall survival (OS) and progression free survival (PFS). RESULTS: 6810 of 10,748 patients (63.3%) were available (48.5% RAS wildtype, 51.5% RAS mutated). Across all treatment lines, additional treatment efficacy (chemotherapy and/or anti angiogenic agents) was significantly smaller in RAS mutated compared to wildtype tumors for OS and PFS. In detail, patients with RAS mutated metastatic colorectal cancer derived significant benefit in PFS but not in OS by the addition of either chemotherapy or anti angiogenic agents to the respective comparator. In patients with RAS wildtype metastatic colorectal cancer, PFS and OS were improved by the addition of chemotherapy or anti angiogenic agent. CONCLUSION: The therapeutic benefit of additional substances is less distinct in patients with RAS mutated as compared to RAS wildtype metastatic colorectal cancer, especially with regard to OS.

Wnt-driven LARGE2 mediates laminin-adhesive O-glycosylation in human colonic epithelial cells and colorectal cancer.

BACKGROUND: Wnt signaling drives epithelial self-renewal and disease progression in human colonic epithelium and colorectal cancer (CRC). Characterization of Wnt effector pathways is key for our understanding of these processes and for developing therapeutic strategies that aim to preserve tissue homeostasis. O-glycosylated cell surface proteins, such as α-dystroglycan (α-DG), mediate cellular adhesion to extracellular matrix components. We revealed a Wnt/LARGE2/α-DG signaling pathway which triggers this mode of colonic epithelial cell-to-matrix interaction in health and disease. METHODS: Next generation sequencing upon shRNA-mediated silencing of adenomatous polyposis coli (APC), and quantitative chromatin immunoprecipitation (qChIP) combined with CRISPR/Cas9-mediated transcription factor binding site targeting characterized LARGE2 as a Wnt target gene. Quantitative mass spectrometry analysis on size-fractionated, glycoprotein-enriched samples revealed functional O-glycosylation of α-DG by LARGE2 in CRC. The biology of Wnt/LARGE2/α-DG signaling was assessed by affinity-based glycoprotein enrichment, laminin overlay, CRC-to-endothelial cell adhesion, and transwell migration assays. Experiments on primary tissue, human colonic (tumor) organoids, and bioinformatic analysis of CRC cohort data confirmed the biological relevance of our findings. RESULTS: Next generation sequencing identified the LARGE2 O-glycosyltransferase encoding gene as differentially expressed upon Wnt activation in CRC. Silencing of APC, conditional expression of oncogenic ß-catenin and endogenous ß-catenin-sequestration affected LARGE2 expression. The first intron of LARGE2 contained a CTTTGATC motif essential for Wnt-driven LARGE2 expression, showed occupation by the Wnt transcription factor TCF7L2, and Wnt activation triggered LARGE2-dependent α-DG O-glycosylation and laminin-adhesion in CRC cells. Colonic crypts and organoids expressed LARGE2 mainly in stem cell-enriched subpopulations. In human adenoma organoids, activity of the LARGE2/α-DG axis was Wnt-dose dependent. LARGE2 expression was elevated in CRC and correlated with the Wnt-driven molecular subtype and intestinal stem cell features. O-glycosylated α-DG represented a Wnt/LARGE2-dependent feature in CRC cell lines and patient-derived tumor organoids. Modulation of LARGE2/α-DG signaling affected CRC cell migration through laminin-coated membranes and adhesion to endothelial cells. CONCLUSIONS: We conclude that the LARGE2 O-glycosyltransferase-encoding gene represents a direct target of canonical Wnt signaling and mediates functional O-glycosylation of α-dystroglycan (α-DG) in human colonic stem/progenitor cells and Wnt-driven CRC. Our work implies that aberrant Wnt activation augments CRC cell-matrix adhesion by increasing LARGE/α-DG-mediated laminin-adhesiveness. Video abstract.

Treatment of advanced gastrointestinal cancer with genetically modified autologous mesenchymal stem cells: Results from the phase 1/2 TREAT-ME-1 trial.

TREAT-ME-1, a Phase 1/2 open-label multicenter, first-in-human, first-in-class trial, evaluated the safety, tolerability and efficacy of treatment with genetically modified autologous mesenchymal stromal cells (MSC), MSC_ apceth_101, in combination with ganciclovir in patients with advanced gastrointestinal adenocarcinoma. Immunological and inflammatory markers were also assessed. All patients (3 in Phase 1; 7 in Phase 2) received three treatment cycles of MSC_apceth_101 at one dose level on Day 0, 7, and 14 followed by ganciclovir administration according to the manufacturer's instructions for 48─72 h after MSC_apceth_101 injection. Ten patients were treated with a total dose of 3.0 x 106 cells/kg MSC_apceth_101. 36 adverse events and six serious adverse events were reported. Five patients achieved stable disease (change in target lesions of -2 to +28%). For all patients, the median time to progression was 1.8 months (95% CI: 0.5, 3.9 months). Median overall survival could not be estimated as 8/10 patients were still alive at the end of the study (1 year) and therefore censored. Post-study observation of patients showed a median overall survival of 15.6 months (ranging from 2.2─27.0 months). Treatment with MSC_apceth_101 and ganciclovir did not induce a consistent increase or decrease in levels of any of the tumor markers analyzed. No clear trends in the immunological markers assessed were observed. MSC_apceth_101 in combination with ganciclovir was safe and tolerable in patients with advanced gastrointestinal adenocarcinoma, with preliminary signs of efficacy in terms of clinical stabilization of disease.

Prediction of 90Y Radioembolization Outcome from Pretherapeutic Factors with Random Survival Forests.

Our objective was to predict the outcome of 90Y radioembolization in patients with intrahepatic tumors from pretherapeutic baseline parameters and to identify predictive variables using a machine-learning approach based on random survival forests. Methods: In this retrospective study, 366 patients with primary (n = 92) or secondary (n = 274) liver tumors who had received 90Y radioembolization were analyzed. A random survival forest was trained to predict individual risk from baseline values of cholinesterase, bilirubin, type of primary tumor, age at radioembolization, hepatic tumor burden, presence of extrahepatic disease, and sex. The predictive importance of each baseline parameter was determined using the minimal-depth concept, and the partial dependency of predicted risk on the continuous variables bilirubin level and cholinesterase level was determined. Results: Median overall survival was 11.4 mo (95% confidence interval, 9.7-14.2 mo), with 228 deaths occurring during the observation period. The random-survival-forest analysis identified baseline cholinesterase and bilirubin as the most important variables (forest-averaged lowest minimal depth, 1.2 and 1.5, respectively), followed by the type of primary tumor (1.7), age (2.4), tumor burden (2.8), and presence of extrahepatic disease (3.5). Sex had the highest forest-averaged minimal depth (5.5), indicating little predictive value. Baseline bilirubin levels above 1.5 mg/dL were associated with a steep increase in predicted mortality. Similarly, cholinesterase levels below 7.5 U predicted a strong increase in mortality. The trained random survival forest achieved a concordance index of 0.657, with an SE of 0.02, comparable to the concordance index of 0.652 and SE of 0.02 for a previously published Cox proportional hazards model. Conclusion: Random survival forests are a simple and straightforward machine-learning approach for prediction of overall survival. The predictive performance of the trained model was similar to a previously published Cox regression model. The model has revealed a strong predictive value for baseline cholinesterase and bilirubin levels with a highly nonlinear influence for each parameter.

Treatment of advanced gastrointestinal cancer with genetically modified autologous mesenchymal stem cells - TREAT-ME-1 - a phase I, first in human, first in class trial.

PURPOSE: This phase I, first in human, first in class clinical study aimed at evaluating the safety, tolerability and efficacy of treatment with genetically modified mesenchymal stromal cells (MSC) in combination with ganciclovir (GCV). MSC_apceth_101 are genetically modified autologous MSCs used as vehicles for a cell-based gene therapy in patients with advanced gastrointestinal adenocarcinoma. EXPERIMENTAL DESIGN: The study design consisted of a dose-escalation 3 + 3 design. All patients (n = 6) were treated with up to three applications of MSC_apceth_101, followed by GCV infusions given on three consecutive days starting 48 hours after injection of MSC_apceth_101. Three of six patients received a total dose of 1.5 × 106 cells/kg. Two patients received three doses of 1 × 106 cells/kg, while one patient received only two doses of 1 × 106 cells/kg due to a SADR. RESULTS: Six patients received MSC_apceth_101. No IMP-related serious adverse events occurred. Adverse-events related to IMP-injection were increased creatinine, cough, fever, and night sweat. TNF, IL-6, IL-8, IL-10 and sE-Selectin, showed that repeated application is immunologically safe, but induces a switch of the functional properties of monocytes to an inflammatory phenotype. Treatment induced stable disease in 4/6 patients, and progressive disease in 2/6 patients. CONCLUSION: Treatment with MSC_apceth_101 in combination with GCV demonstrated acceptable safety and tolerability in patients with advanced gastrointestinal adenocarcinoma.

Evaluation of Visualization Using a 50/50 (Contrast Media/Glucose 5% Solution) Technique for Radioembolization as an Alternative to a Standard Sandwich Technique.

BACKGROUND: Radioembolization (RE) with 90yttrium (90Y) resin microspheres generally employs a sandwich technique with separate sequential administration of contrast medium (CM), followed by vehicle (e.g., glucose 5% [G5] solution), then 90Y resin microspheres (in G5), then G5, and then CM again to avoid contact of CM and microspheres under fluoroscopic guidance. This study evaluates the visualization quality and safety of a modified sandwich technique with a 50/50-mixture of CM (Imeron 300) and G5 for administration of 90Y resin microspheres. MATERIALS AND METHODS: A retrospective analysis of 81 RE procedures in patients with primary or secondary liver tumors was performed. The quality of angiographic visualization of the hepatic vessels was assessed before the first injection and immediately before the whole dose has been injected. Visualization and flow rate were graded on a 5-point scale: 1 = very good to 5 = not visible/no antegrade flow. Univariate logistic regression models and multiple linear regression models were used to evaluate the prognostic variables associated with visualization and flow scores. RESULTS: Visualization quality was inversely related to flow rate, the lower the flow rate the better the grade of the visualization. Visualization quality was also inversely related to body-mass-index (BMI). Performing RE with the 50/50-CM/G5 mixture resulted in a mean injection time for 1 GBq of 15 min. No clinically significant adverse events, including radiation-induced liver disease were reported. CONCLUSION: RE with a 50/50-mixture of CM and G5 for administration of 90Y resin microspheres in a modified sandwich technique is a safe administration alternative and provides good visualization of hepatic vessels, which is inversely dependent on flow rate and BMI. Injection time was reduced compared with our experience with the standard sandwich technique.