In Patients Undergoing CRS/HIPEC for Colorectal Adenocarcinoma with Peritoneal Metastases, Presence of Ascites on Computed Tomography Imaging is not a Prognostic Marker for Survival.

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is a potentially curative treatment for patients with colorectal peritoneal metastases (CRPM). Patient selection is key to optimizing outcomes after CRS/HIPEC. The aim of this study was to determine the prognostic value of ascites diagnosed on preoperative imaging. METHODS: A prospective database of patients eligible for CRS/HIPEC between 2010 and 2020 was retrospectively analyzed. The presence of ascites, postoperative complications, overall survival (OS), disease-free survival (DFS), and completeness of cytoreduction were assessed. Univariable and multivariable logistic regression was performed to identify independent predictors for outcome. RESULTS: Of the 235 included patients, 177 (75%) underwent CRS/HIPEC while 58 (25%) were not eligible for CRS/HIPEC. In 42 of the 177 patients (24%) who underwent CRS/HIPEC, ascites was present on preoperative computed tomography (CT) imaging. Peritoneal Cancer Index (PCI) score was significantly higher in patients with preoperative ascites compared with patients without (11 [range 2-30] vs. 9 [range 0-28], respectively; p = 0.011) and complete cytoreduction was more often achieved in patients without ascites (96.3% vs. 85.7%; p = 0.007). There was no significant difference in median DFS and OS after CRS/HIPEC between patients with and without ascites {10 months (95% confidence interval [CI] 7.1-12.9) vs. 9 months (95% CI 7.2-10.8), and 25 months (95% 9.4-40.6) vs. 27 months (95% CI 22.4-31.6), respectively}. CONCLUSIONS: Ascites on preoperative imaging was not associated with worse survival in CRS/HIPEC patients with CRPM. Therefore, excluding patients from CRS/HIPEC based merely on the presence of ascites is not advisable.

Tumor Seeding During Colonoscopy as a Possible Cause for Metachronous Colorectal Cancer.

BACKGROUND AND AIMS: In patients who have undergone surgery for colorectal cancer (CRC), 3% have recurrence of (metachronous) CRC. We investigated whether tumor seeding during colonoscopy (iatrogenic implantation of tumor cells in damaged mucosa) increases risk for metachronous CRC. METHODS: In a proof of principle study, we collected data from the Dutch National Pathology Registry for patients with a diagnosis of CRC from 2013 through 2015, with a second diagnosis of CRC within 6 months to 3.5 years after surgery. We reviewed pathology reports to identify likely metachronous CRC (histologically proven adenocarcinoma located elsewhere in the colon or rectum from the surgical anastomosis). For 22 patients fulfilling the inclusion criteria, we ascribed the most likely etiology to tumor seeding when endoscopic manipulations, such as biopsies or polypectomy, occurred at the location where the metachronous tumor was subsequently detected, after endoscopic manipulation of the primary tumor. We collected clinical data from patients and compared molecular profiles of the primary and metachronous colorectal tumors using next-generation sequencing. We then examined the source of seeded tumor. We tested whether tumor cells stay behind in the working channel of the endoscope after biopsies of colorectal tumors, and whether these cells maintain viability in organoid cultures. RESULTS: In total, tumor seeding was suspected as the most likely etiology of metachronous CRC in 5 patients. Tumor tissues were available from 3 patients. An identical molecular signature was observed in the primary and metachronous colorectal tumors from all 3 patients. In 5 control cases with a different etiology of metachronous CRC, the molecular signature of the primary and metachronous tumor were completely different. Based on review of 2147 patient records, we estimated the risk of tumor seeding during colonoscopy to be 0.3%-0.6%. We demonstrated that the working channel of the colonoscope becomes contaminated with viable tumor cells during biopsy collection. Subsequent instruments introduced through this working channel also became contaminated. These cells were shown to maintain their proliferative potential. CONCLUSIONS: In an analysis of primary and secondary tumors from patients with metachronous CRC, we found that primary tumor cells might be seeded in a new location after biopsy of the primary tumor. Although our study does not eliminate other possibilities of transmission, our findings and experiments support the hypothesis that tumor seeding can occur during colonoscopy via the working channel of the endoscope. The possibility of iatrogenic seeding seems low. However, our findings compel awareness on this potentially preventable cause of metachronous CRC.

Associations of non-pedunculated T1 colorectal adenocarcinoma outcome with consensus molecular subtypes, immunoscore, and microsatellite status: a multicenter case-cohort study.

Advanced colorectal cancer (CRC) consensus molecular subtype 4 (CMS4) or CRC with a low immunoscore is associated with shorter survival times. Non-metastatic CRC with microsatellite instability (MSI) is associated with a lower risk of recurrence. We evaluated outcome (lymph node metastases [LNM] or cancer recurrence) in these tumor subtypes in patients with surgically-removed non-pedunculated T1 CRC by performing a multicenter case-cohort study. We included all patients in 13 hospitals in the Netherlands from 2000-2014 (n = 651). We randomly selected a subgroup of patients (n = 223) and all patients with LNM or recurrence (n = 63), and median follow-up of 44 months. We centrally reviewed tumor-slides, and constructed and immunostained tissue microarrays determining MSI, CMS (MSI/CMS1, CMS2/3, or CMS4), and immunoscore (I-low/I-high). We used weighted Cox proportional hazard models to evaluate the association of MSI, CMS, and immunoscore with LNM or recurrence, adjusting for conventional histologic risk factors. In the randomly selected subgroup of patients, 7.1% of tumors were MSI/CMS1, 91.0% CMS2/3, 1.8% CMS4, and 25% I-low. In the case-cohort, patients with CMS4 tumors had an increased risk for LNM or recurrence compared with patients with tumors of other CMSs (adjusted hazard ratio [HR], 3.97; 95% CI, 1.12-14.06; P = 0.03). Albeit not significant, tumors with MSI had a lower risk for LNM or recurrence than other tumor subtypes (adjusted HR, 0.52; 95% CI, 0.12-2.30; P = 0.39), whereas tumors with a low immunoscore had an increased risk for LNM or recurrence (adjusted HR, 1.30; 95% CI, 0.68-2.48; P = 0.43). In conclusion, in a case-cohort study of patients with non-pedunculated T1 CRC, MSI, and immunoscore were not significantly associated with adverse outcome after surgery. CMS4 substantially increased the risk of adverse outcome. However, CMS4 is rare in T1 CRCs, limiting its value for determining the risk in patients.

Increased Levels of Oxidative Damage in Liver Metastases Compared with Corresponding Primary Colorectal Tumors: Association with Molecular Subtype and Prior Treatment.

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.

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer.

BACKGROUND AND AIM: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target. METHODS: Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence. RESULTS: KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo, without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture. CONCLUSIONS: RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.

Lymphangiogenic Gene Expression Is Associated With Lymph Node Recurrence and Poor Prognosis After Partial Hepatectomy for Colorectal Liver Metastasis.

OBJECTIVES: To investigate the relevance of lymphangiogenic gene expression in primary and liver metastasis of colorectal cancer (CRC) and identify determinants of lymphatic invasion. BACKGROUND: Lymphatic development promoting vascular endothelial growth factor C (VEGFC) is associated with poor outcome in primary CRC. For colorectal liver metastasis (CRLM), intrahepatic lymph invasion and lymph node metastasis are poor prognostic factors. Exact biological factors promoting lymphatic involvement remain elusive, just as the association with molecular subtypes of CRC. METHODS: We designed a lymphangiogenic gene set (VEGFC, Nrp-2, PDPN, LYVE-1, MRC1, CCL-21) and applied it to large datasets of CRC. Gene expression of the lymphangiogenic signature was assessed in resected CRLM specimens by Rt-QPCR. In vitro experiments were performed with colon cancer cell line Colo320 (high Nrp-2 expression) and human dermal microvascular lymphatic endothelial cells (LECs). RESULTS: Lymphangiogenic gene expression was associated with poor prognosis in both primary and liver metastasis of CRC. CRLM with high expression of consensus molecular subtype-4 identifier genes also exhibited high lymphangiogenic gene expression. Lymph node recurrence following CRLM resection was associated with high expression of VEGFC and Nrp-2. Blocking Nrp-2 significantly reduced invasion of Colo320 cells through an LEC monolayer. CONCLUSIONS: Lymphangiogenic gene expression is correlated with worse prognosis and consensus molecular subtype-4 in both primary and liver metastatic CRC. VEGFC and Nrp-2 expression may be predictive of lymph node involvement in recurrence after resection of CRLM. Nrp-2, expressed on both tumor and LECs, may have a mechanistic role in lymphatic invasion and is a potential novel target in CRC.

Maintenance of Clonogenic KIT(+) Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells.

BACKGROUND & AIMS: Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. METHODS: We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. RESULTS: Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. CONCLUSIONS: Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.

Adding preoperative radiotherapy plus cetuximab to perioperative chemotherapy for resectable esophageal adenocarcinoma: a single-center prospective phase II trial.

BACKGROUND: Local and systemic recurrence are important sources of treatment failure following surgical resection of esophageal adenocarcinoma. We hypothesized that adding preoperative cetuximab and radiotherapy (cetux-RT) to perioperative chemotherapy would increase treatment efficacy with acceptable toxicity. METHODS: In this prospective phase II trial, patients were treated with three cycles of epirubicin, cisplatin, and capecitabine (ECX), followed by cetux-RT. After surgery with curative intent, patients received three more cycles of ECX. Primary endpoints were efficacy, determined by histopathological complete response (pCR) rate, and safety, which was assessed with resectability rate. RESULTS: Of the 12 patients enrolled in this trial, six received at least one dose of cetux-RT. In five patients, cetux-RT was not started because of adverse events (AEs) related to preoperative chemotherapy; one patient had progressive disease. Addition of cetux-RT was well tolerated and did not interfere with the resectability rate (100%). However, the pCR rate was 0, and 50% of patients experienced serious adverse events (SAEs) postoperatively. CONCLUSION: With 12 patients enrolled, the lack of initial signs of efficacy and a high incidence of postoperative SAEs prompted us to end this study prematurely. Perioperative ECX was associated with considerable toxicity, and further treatment intensification is problematic.

KIT promotes tumor stroma formation and counteracts tumor-suppressive TGFß signaling in colorectal cancer.

Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFß1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFß signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFß signaling, while KIT deletion sensitized PDOs to TGFß-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFß signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFß expression, and protects the tumor cells from tumor-suppressive TGFß signaling by inhibiting SMAD2 expression.

Consensus molecular subtype 4 (CMS4)-targeted therapy in primary colon cancer: A proof-of-concept study.

Background: Mesenchymal Consensus Molecular Subtype 4 (CMS4) colon cancer is associated with poor prognosis and therapy resistance. In this proof-of-concept study, we assessed whether a rationally chosen drug could mitigate the distinguishing molecular features of primary CMS4 colon cancer. Methods: In the ImPACCT trial, informed consent was obtained for molecular subtyping at initial diagnosis of colon cancer using a validated RT-qPCR CMS4-test on three biopsies per tumor (Phase-1, n=69 patients), and for neoadjuvant CMS4-targeting therapy with imatinib (Phase-2, n=5). Pre- and post-treatment tumor biopsies were analyzed by RNA-sequencing and immunohistochemistry. Imatinib-induced gene expression changes were associated with molecular subtypes and survival in an independent cohort of 3232 primary colon cancer. Results: The CMS4-test classified 52/172 biopsies as CMS4 (30%). Five patients consented to imatinib treatment prior to surgery, yielding 15 pre- and 15 post-treatment samples for molecular analysis. Imatinib treatment caused significant suppression of mesenchymal genes and upregulation of genes encoding epithelial junctions. The gene expression changes induced by imatinib were associated with improved survival and a shift from CMS4 to CMS2. Conclusion: Imatinib may have value as a CMS-switching drug in primary colon cancer and induces a gene expression program that is associated with improved survival.

Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer.

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/ß-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and ß-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.

A Novel Diagnostic Tool for Selecting Patients With Mesenchymal-Type Colon Cancer Reveals Intratumor Subtype Heterogeneity.

Background: Consensus molecular subtype 4 (CMS4) is a recently identified aggressive colon cancer subtype for which platelet-derived growth factor receptors (PDGFRs) and KIT are potential therapeutic targets. We aimed to develop a clinically applicable CMS4 reverse transcription polymerase chain reaction (RT-qPCR) test to select patients for PDGFR/KIT-targeted therapy. Methods: We used logistic regression to develop a CMS4 prediction rule based on microarray expression values of PDGFRA , PDGFRB , PDGFC , and KIT (566 training and 1259 test samples, using the 273-gene random forest classifier as CMS4 reference standard). We next translated the prediction rule into a single-sample RT-qPCR test, which we independently validated in 29 fresh tumor samples. To study intratumor CMS4 heterogeneity, we used the RT-qPCR test to analyze five random regions of 20 colon tumors. Results: The microarray-based prediction rule diagnosed CMS4-type tumors extremely well in both training and independent test samples (training: area under the curve [AUC] = 0.95, 95% confidence interval [CI] = 0.94 to 0.97; test: AUC = 0.95, 95% CI = 0.94 to 0.96), with excellent calibration and approximately 80% overall net benefit over a large threshold range. Translation into an RT-qPCR test did not affect discrimination (AUC = 0.97, 95% CI = 0.93 to 1.00, independent validation). RT-qPCR analysis of five random tumor regions revealed extensive intratumor CMS4 heterogeneity in nine out of 20 tumors. At least two regions likely have to be analyzed to identify patients that are predominantly CMS4 positive (>50% average CMS4 chance). Conclusion: The CMS4 RT-qPCR test is a promising clinical tool for selecting individual patients for CMS4-subtype-targeted therapy.

A potential role for CCN2/CTGF in aggressive colorectal cancer.

CCN2, also known as connective tissue growth factor (CTGF) is a transcriptional target of TGF-ß signaling. Unlike its original name ("CTGF") suggested, CCN2 is not an actual growth factor but a matricellular protein that plays an important role in fibrosis, inflammation and connective tissue remodeling in a variety of diseases, including cancer. In pancreatic ductal adenocarcinoma, CCN2 signaling induces stromal infiltration and facilitates a strong tumor-stromal interaction. In many types of cancer, CCN2 overexpression has been associated with poor outcome. CMS4 (Consensus Molecular Subtype 4) is a recently identified aggressive colorectal cancer subtype, that is characterized by up-regulation of genes involved in epithelial-to-mesenchymal transition, TGF-ß signaling, angiogenesis, complement activation, and extracellular matrix remodeling. In addition, a high influx of stromal fibroblasts contributes to the mesenchymal-like gene expression profile of this subtype. Furthermore, compared with the other three CMS groups, CMS4 tumors have the worst prognosis. Based on these observations, we postulated that CCN2 might contribute to colorectal cancer progression, especially in the CMS4 subtype. This review discusses the available literature on the role of CCN2 in colorectal cancer, with a focus on the 'fibrotic subtype' CMS4.

Surgical and Oncologic Outcomes After Major Liver Surgery and Extended Hemihepatectomy for Colorectal Liver Metastases.

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.

Detailed imaging and genetic analysis reveal a secondary BRAF(L505H) resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib.

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three-dimensional imaging. In all patients, only a subset of lesions progressed. Next-generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib-resistant patient-derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.