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.

Mice lacking functional CD95-ligand display reduced proliferation of the intestinal epithelium without gross homeostatic alterations.

Homeostasis of the continuously self-renewing intestinal tract involves cell proliferation, migration, differentiation along the crypt-villus-axis and shedding of cells into the gut lumen. CD95-ligand (FAS-ligand, CD95L) is a cytokine that is known for its capacity to induce apoptosis by binding its cognate receptor, CD95 (Fas). More recently, it was discovered that CD95L can also induce other cellular responses, such as proliferation, differentiation and cell migration. CD95L is highly expressed in Paneth cells of the small intestine which are in close contact with intestinal stem cells. This suggests a potential role for CD95L in controlling stem cell function and, possibly, intestinal homeostasis. We analyzed the intestines of mice deficient for functional CD95L (gld) for potential alterations in the diversity of stem-cell-lineages and parameters of intestinal homeostasis. Stem cell diversity was assessed by analyzing methylation patterns of the non-transcribed mMYOD gene. Proliferation was analyzed by BrdU labeling and differentiation was assessed by immunohistochemistry. Of all parameters analyzed, only epithelial cell proliferation was significantly reduced in the small intestines of gld-mice, but not in their colons which lack CD95L expression. We conclude that CD95L has a proliferation-stimulating role during normal turnover of the small intestine, but has a marginal effect on overall intestinal homeostasis.

Diagnostic pitfalls after COVID-19 vaccination in melanoma and breast cancer patients: A case series.

INTRODUCTION: During the current Coronavirus Disease 2019 (COVID-19) pandemic, significant COVID-19 disease-reducing developments have been made, culminating in the COVID-19 vaccines. However, COVID-19 vaccines may complicate oncological staging and follow-up oncological disease course since they may induce the enlargement of lymph nodes. Consequently, this uncertainty may lead to increased distress. PRESENTATION OF CASES: This case series describes seven patients diagnosed with melanoma or breast cancer in whom lymphadenopathy was observed on oncology imaging after COVID-19 vaccination. Four of these patients underwent additional diagnostic testing, all without malignant cells on pathological examination or suspected metastasis on imaging. The remaining patients were re-evaluated, and the lymphadenopathy was interpreted as an adverse outcome of the recent COVID-19 vaccination. In addition, four out of seven patients were vaccinated in the ipsilateral arm relative to the tumor. Abnormal lymph nodes could be observed up to sixty-nine days after COVID-19 vaccination. DISCUSSION AND CONCLUSION: These findings indicate that a COVID-19 vaccination may result in possible false-positive oncological imaging findings in melanoma and breast cancer patients. Moreover, it is advised to administer the vaccine in the contralateral arm of the primary tumor, suspected breast abnormalities, or after the oncologic imaging in melanoma and breast cancer patients.

Macrophages induce "budding" in aggressive human colon cancer subtypes by protease-mediated disruption of tight junctions.

Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the contribution of other stromal cell types is unclear. Here we searched for additional stromal cell types that contribute to aggressive tumor cell behavior. By making use of the 'immunome compendium'-a collection of gene signatures reflecting the presence of specific immune cell-types-we show that macrophage signatures are most strongly associated with a high CAF content and with poor prognosis in multiple large cohorts of primary tumors and liver metastases. Co-culturing macrophages with patient-derived colonospheres promoted 'budding' of small clusters of tumor cells from the bulk. Immunohistochemistry showed that budding tumor clusters in stroma-rich areas of T1 colorectal carcinomas were surrounded by macrophages. In vitro budding was accompanied by reduced levels of the tight junction protein occludin, but OCLN mRNA levels did not change, nor did markers of epithelial mesenchymal transition. Budding was accompanied by nuclear accumulation of ß-catenin, which was also observed in budding tumor cell clusters in situ. The NFκB inhibitor Sanguinarine resulted in a decrease in MMP7 protein expression and both NFκB inhibitor Sanguinarine and MMP inhibitor Batimastat prevented occludin degradation and budding. We conclude that macrophages contribute to the aggressive nature of stroma-rich colon tumors by promoting an MMP-dependent pathway that operates in parallel to classical EMT and leads to tight junction disruption.

Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability.

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.

Practical and Robust Identification of Molecular Subtypes in Colorectal Cancer by Immunohistochemistry.

PURPOSE: Recent transcriptomic analyses have identified four distinct molecular subtypes of colorectal cancer with evident clinical relevance. However, the requirement for sufficient quantities of bulk tumor and difficulties in obtaining high-quality genome-wide transcriptome data from formalin-fixed paraffin-embedded tissue are obstacles toward widespread adoption of this taxonomy. Here, we develop an immunohistochemistry-based classifier to validate the prognostic and predictive value of molecular colorectal cancer subtyping in a multicenter study. EXPERIMENTAL DESIGN: Tissue microarrays from 1,076 patients with colorectal cancer from four different cohorts were stained for five markers (CDX2, FRMD6, HTR2B, ZEB1, and KER) by immunohistochemistry and assessed for microsatellite instability. An automated classification system was trained on one cohort using quantitative image analysis or semiquantitative pathologist scoring of the cores as input and applied to three independent clinical cohorts. RESULTS: This classifier demonstrated 87% concordance with the gold-standard transcriptome-based classification. Application to three validation datasets confirmed the poor prognosis of the mesenchymal-like molecular colorectal cancer subtype. In addition, retrospective analysis demonstrated the benefit of adding cetuximab to bevacizumab and chemotherapy in patients with RAS wild-type metastatic cancers of the canonical epithelial-like subtypes. CONCLUSIONS: This study shows that a practical and robust immunohistochemical assay can be employed to identify molecular colorectal cancer subtypes and uncover subtype-specific therapeutic benefit. Finally, the described tool is available online for rapid classification of colorectal cancer samples, both in the format of an automated image analysis pipeline to score tumor core staining, and as a classifier based on semiquantitative pathology scoring. Clin Cancer Res; 23(2); 387-98. ©2016 AACR.

Paired image- and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures.

Novel spheroid-type tumor cell cultures directly isolated from patients' tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer.

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer. We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear ß-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer. RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 µM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1. Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.

SIRT1/PGC1α-Dependent Increase in Oxidative Phosphorylation Supports Chemotherapy Resistance of Colon Cancer.

PURPOSE: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms. EXPERIMENTAL DESIGN: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy. RESULTS: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy. CONCLUSIONS: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable.