Extrahepatic perfusion and incomplete hepatic perfusion after hepatic arterial infusion pump implantation: incidence and clinical implications.

INTRODUCTION: This study investigates the incidence of extrahepatic perfusion and incomplete hepatic perfusion at intraoperative methylene blue testing and on postoperative nuclear imaging in patients undergoing hepatic arterial infusion pump (HAIP) chemotherapy. METHODS: The first 150 consecutive patients who underwent pump implantation in the Netherlands were included. All patients underwent surgical pump implantation with the catheter in the gastroduodenal artery. All patients underwent intraoperative methylene blue testing and postoperative nuclear imaging (99mTc-Macroaggregated albumin SPECT/CT) to determine perfusion via the pump. RESULTS: Patients were included between January-2018 and December-2021 across eight centers. During methylene blue testing, 29.3% had extrahepatic perfusion, all successfully managed intraoperatively. On nuclear imaging, no clinically relevant extrahepatic perfusion was detected (0%, 95%CI: 0.0-2.5%). During methylene blue testing, 2.0% had unresolved incomplete hepatic perfusion. On postoperative nuclear imaging, 8.1% had incomplete hepatic perfusion, leading to embolization in only 1.3%. CONCLUSION: Methylene blue testing during pump placement for intra-arterial chemotherapy identified extrahepatic perfusion in 29.3% of patients, but could be resolved intraoperatively in all patients. Postoperative nuclear imaging found no clinically relevant extrahepatic perfusion and led to embolization in only 1.3% of patients. The role of routine nuclear imaging after HAIP implantation should be studied in a larger cohort.

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Trastuzumab plus pertuzumab for HER2-amplified advanced colorectal cancer: Results from the drug rediscovery protocol (DRUP).

BACKGROUND: In 2-5% of patients with colorectal cancer (CRC), human epidermal growth factor 2 (HER2) is amplified or overexpressed. Despite prior evidence that anti-HER2 therapy confers clinical benefit (CB) in one-third of these patients, it is not approved for this indication in Europe. In the Drug Rediscovery Protocol (DRUP), patients are treated with off-label drugs based on their molecular profile. Here, we present the results of the cohort 'trastuzumab/pertuzumab for treatment-refractory patients with RAS/BRAF-wild-type HER2amplified metastatic CRC (HER2+mCRC)'. METHODS: Patients with progressive treatment-refractory RAS/BRAF-wild-type HER2+mCRC with measurable disease were included for trastuzumab plus pertuzumab treatment. Primary endpoints of DRUP are CB (defined as confirmed objective response (OR) or stable disease (SD) ≥ 16 weeks) and safety. Patients were enrolled using a Simon-like 2-stage model, with 8 patients in stage 1 and 24 patients in stage 2 if at least 1/8 patients had CB. To identify biomarkers for response, whole genome sequencing (WGS) was performed on pre-treatment biopsies. RESULTS: CB was observed in 11/24 evaluable patients (46%) with HER2+mCRC, seven patients achieved an OR (29%). Median duration of response was 8.4 months. Patients had undergone a median of 3 prior treatment lines. Median progression-free survival and overall survival were 4.3 months (95% CI 1.9-10.3) and 8.2 months (95% CI 7.2-14.7), respectively. No unexpected toxicities were observed. WGS provided potential explanations for resistance in 3/10 patients without CB, for whom WGS was available. CONCLUSIONS: The results of this study confirm a clinically significant benefit of trastuzumab plus pertuzumab treatment in patients with HER2+mCRC.

Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response.

BACKGROUND: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies. METHODS: We optimized drug screen methods on 5-11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient's response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness. RESULTS: Drug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003). CONCLUSIONS: Our study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.

Harnessing the Potential of Real-World Evidence in the Treatment of Colorectal Cancer: Where Do We Stand?

OPINION STATEMENT: Treatment guidelines for colorectal cancer (CRC) are primarily based on the results of randomized clinical trials (RCTs), the gold standard methodology to evaluate safety and efficacy of oncological treatments. However, generalizability of trial results is often limited due to stringent eligibility criteria, underrepresentation of specific populations, and more heterogeneity in clinical practice. This may result in an efficacy-effectiveness gap and uncertainty regarding meaningful benefit versus treatment harm. Meanwhile, conduct of traditional RCTs has become increasingly challenging due to identification of a growing number of (small) molecular subtypes. These challenges-combined with the digitalization of health records-have led to growing interest in use of real-world data (RWD) to complement evidence from RCTs. RWD is used to evaluate epidemiological trends, quality of care, treatment effectiveness, long-term (rare) safety, and quality of life (QoL) measures. In addition, RWD is increasingly considered in decision-making by clinicians, regulators, and payers. In this narrative review, we elaborate on these applications in CRC, and provide illustrative examples. As long as the quality of RWD is safeguarded, ongoing developments, such as common data models, federated learning, and predictive modelling, will further unfold its potential. First, whenever possible, we recommend conducting pragmatic trials, such as registry-based RCTs, to optimize generalizability and answer clinical questions that are not addressed in registrational trials. Second, we argue that marketing approval should be conditional for patients who would have been ineligible for the registrational trial, awaiting planned (non) randomized evaluation of outcomes in the real world. Third, high-quality effectiveness results should be incorporated in treatment guidelines to aid in patient counseling. We believe that a coordinated effort from all stakeholders is essential to improve the quality of RWD, create a learning healthcare system with optimal use of trials and real-world evidence (RWE), and ultimately ensure personalized care for every CRC patient.

Dual targeting of cancer metabolome and stress antigens affects transcriptomic heterogeneity and efficacy of engineered T cells.

Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αß T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BBCD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BBCD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8+ TEGs, the NKG2D-4-1BBCD28TM chimera mainly skewed CD4+ TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8+ subset toward a proliferative state.

Stromal localization of inactive CD8+ T cells in metastatic mismatch repair deficient colorectal cancer.

BACKGROUND: The determinants of metastasis in mismatch repair deficiency with high levels of microsatellite instability (MSI-H) in colorectal cancer (CRC) are poorly understood. Here, we hypothesized that distinct immune and stromal microenvironments in primary tumors may discriminate between non-metastatic MSI-H CRC and metastatic MSI-H CRC. METHODS: We profiled 46,727 single cells using high-plex imaging mass cytometry and analyzed both differential cell type abundance, and spatial distribution of fibroblasts and immune cells in primary CRC tumors with or without metastatic capacity. We validated our findings in a second independent cohort using immunohistochemistry. RESULTS: High-plex imaging mass cytometry and hierarchical clustering based on microenvironmental markers separated primary MSI-H CRC tumors with and without metastatic capacity. Primary tumors with metastatic capacity displayed a high stromal content and low influx of CD8+ T cells, which expressed significantly lower levels of markers reflecting proliferation (Ki67) and antigen-experience (CD45RO) compared to CD8+ T cells in non-metastatic tumors. CD8+ T cells showed intra-epithelial localization in non-metastatic tumors, but stromal localization in metastatic tumors, which was validated in a second cohort. CONCLUSION: We conclude that localization of phenotypically distinct CD8+ T cells within stroma may predict metastasis formation in MSI-H CRC.

Consensus molecular subtype transition during progression of colorectal cancer.

The consensus molecular subtype (CMS) classification divides colorectal cancer (CRC) into four distinct subtypes based on RNA expression profiles. The biological differences between CMSs are already present in CRC precursor lesions, but not all CMSs pose the same risk of malignant transformation. To fully understand the path to malignant transformation and to determine whether CMS is a fixed entity during progression, genomic and transcriptomic data from two regions of the same CRC lesion were compared: the precursor region and the carcinoma region. In total, 24 patients who underwent endoscopic removal of T1-2 CRC were included. Regions were subtyped for CMS and DNA mutation analysis was performed. Additionally, a set of 85 benign adenomas was CMS-subtyped. This analysis revealed that almost all benign adenomas were classified as CMS3 (91.8%). In contrast, CMS2 was the most prevalent subtype in precursor regions (66.7%), followed by CMS3 (29.2%). CMS4 was absent in precursor lesions and originated at the carcinoma stage. Importantly, CMS switching occurred in a substantial number of cases and almost all (six out of seven) CMS3 precursor regions showed a shift to a different subtype in the carcinoma part of the lesion, which in four cases was classified as CMS4. In conclusion, our data indicate that CMS3 is related to a more indolent type of precursor lesion that less likely progresses to CRC and when this occurs, it is often associated with a subtype change that includes the more aggressive mesenchymal CMS4. In contrast, an acquired CMS2 signature appeared to be rather fixed during early CRC development. Combined, our data show that subtype changes occur during progression and that CMS3 switching is related to changes in the genomic background through acquisition of a novel driver mutation (TP53) or selective expansion of a clone, but also occurred independently of such genetic changes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Survival of Patients with Deficient Mismatch Repair Versus Proficient Mismatch Repair Metastatic Colorectal Cancer Receiving Curative-Intent Local Treatment of Metastases in a Nationwide Cohort.

BACKGROUND: It is unclear whether curative-intent local therapy of metastases is of similar benefit for the biological distinct subgroup of patients with deficient mismatch repair (dMMR) metastatic colorectal cancer (mCRC) compared with proficient mismatch repair (pMMR) mCRC. PATIENTS AND METHODS: In this nationwide study, recurrence-free (RFS) and overall survival (OS) were analyzed in patients with dMMR versus pMMR mCRC who underwent curative-intent local treatment of metastases between 2015 and 2018. Subgroup analyses were performed for resection of colorectal liver metastases (CRLM) and cytoreductive surgery ± hyperthermic intraperitoneal chemotherapy (CRS ± HIPEC). Multivariable regression was conducted. RESULTS: Median RFS was 11.1 months [95% confidence interval (CI) 8.5-41.1 months] for patients with dMMR tumors compared with 8.9 months (95% CI 8.1-9.8 months) for pMMR tumors. Two-year RFS was higher in patients with dMMR versus pMMR (43% vs. 21%). Results were similar within subgroups of local treatment (CRLM and CRS ± HIPEC). Characteristics differed significantly between patients with dMMR and pMMR mCRC; however, multivariable analysis continued to demonstrate dMMR as independent factor for improved RFS [hazard ratio (HR): 0.57, 95% CI 0.38-0.87]. Median OS was 33.3 months for dMMR mCRC compared with 43.5 months for pMMR mCRC, mainly due to poor survival of patients with dMMR in cases of recurrence in the preimmunotherapy era. CONCLUSION: Patients with dMMR eligible for curative-intent local treatment of metastases showed a comparable to more favorable RFS compared with patients with pMMR, with a clinically relevant proportion of patients remaining free of recurrence. This supports local treatment as a valuable treatment option in patients with dMMR mCRC and can aid in shared decision-making regarding upfront local therapy versus immunotherapy.

Prognostic value of Lynch syndrome, BRAFV600E , and RAS mutational status in dMMR/MSI-H metastatic colorectal cancer in a pooled analysis of Dutch and French cohorts.

BACKGROUND: Current knowledge on prognostic biomarkers (especially BRAFV600E /RAS mutations) in metastatic colorectal cancer (mCRC) is mainly based on mCRC patients with proficient mismatch repair (pMMR) tumors. It is uncertain whether these biomarkers have the same prognostic value in mCRC patients with deficient mismatch repair (dMMR) tumors. METHODS: This observational cohort study combined a population-based Dutch cohort (2014-2019) and a large French multicenter cohort (2007-2017). All mCRC patients with a histologically proven dMMR tumor were included. RESULTS: In our real-world data cohort of 707 dMMR mCRC patients, 438 patients were treated with first-line palliative systemic chemotherapy. Mean age of first-line treated patients was 61.9 years, 49% were male, and 40% had Lynch syndrome. BRAFV600E mutation was present in 47% of tumors and 30% harbored a RAS mutation. Multivariable regression analysis on OS showed significant hazard rates (HR) for known prognostic factors as age and performance status, however showed no significance for Lynch syndrome (HR: 1.07, 95% CI: 0.66-1.72), BRAFV600E mutational status (HR: 1.02, 95% CI: 0.67-1.54), and RAS mutational status (HR: 1.01, 95% CI: 0.64-1.59), with similar results for PFS. CONCLUSION: BRAFV600E and RAS mutational status are not associated with prognosis in dMMR mCRC patients, in contrast to pMMR mCRC patients. Lynch syndrome is also not an independent prognostic factor for survival. These findings underline that prognostic factors of patients with dMMR mCRC are different of those with pMMR, which could be taken into consideration when prognosis is used for clinical decision-making in dMMR mCRC patients and underline the complex heterogeneity of mCRC.

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer.

Background: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation. Methods: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation. Results: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFß1, VEGFA and lactate, and potently inhibited T cell proliferation. Conclusion: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer.

BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.

Drug-repurposing screen on patient-derived organoids identifies therapy-induced vulnerability in KRAS-mutant colon cancer.

Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.

Predicting early extrahepatic recurrence after local treatment of colorectal liver metastases.

BACKGROUND: Patients who develop early extrahepatic recurrence (EHR) may not benefit from local treatment of colorectal liver metastases (CRLMs). This study aimed to develop a prediction model for early EHR after local treatment of CRLMs using a national data set. METHODS: A Cox regression prediction model for EHR was developed and validated internally using data on patients who had local treatment for CRLMs with curative intent. Performance assessment included calibration, discrimination, net benefit, and generalizability by internal-external cross-validation. The prognostic relevance of early EHR (within 6 months) was evaluated by landmark analysis. RESULTS: During a median follow-up of 35 months, 557 of the 1077 patients had EHR and 249 died. Median overall survival was 19.5 (95 per cent c.i. 15.6 to 23.0) months in patients with early EHR after CRLM treatment, compared with not reached (45.3 months to not reached) in patients without an early EHR. The EHR prediction model included side and stage of the primary tumour, RAS/BRAFV600E mutational status, and number and size of CRLMs. The range of 6-month EHR predictions was 5.9-56.0 (i.q.r. 12.9-22.0) per cent. The model demonstrated good calibration and discrimination. The C-index through 6 and 12 months was 0.663 (95 per cent c.i. 0.624 to 0.702) and 0.661 (0.632 to 0.689) respectively. The observed 6-month EHR risk was 6.5 per cent for patients in the lowest quartile of predicted risk compared with 32.0 per cent in the highest quartile. CONCLUSION: Early EHR after local treatment of CRLMs can be predicted.

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice.

BACKGROUND: The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. METHODS: We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. RESULTS: PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. CONCLUSIONS: By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.

Pre-Diagnostic Circulating Resistin Concentrations Are Not Associated with Colorectal Cancer Risk in the European Prospective Investigation into Cancer and Nutrition Study.

Resistin is a polypeptide implicated in inflammatory processes, and as such could be linked to colorectal carcinogenesis. In case-control studies, higher resistin levels have been found in colorectal cancer (CRC) patients compared to healthy individuals. However, evidence for the association between pre-diagnostic resistin and CRC risk is scarce. We investigated pre-diagnostic resistin concentrations and CRC risk within the European Prospective Investigation into Cancer and Nutrition using a nested case-control study among 1293 incident CRC-diagnosed cases and 1293 incidence density-matched controls. Conditional logistic regression models controlled for matching factors (age, sex, study center, fasting status, and women-related factors in women) and potential confounders (education, dietary and lifestyle factors, body mass index (BMI), BMI-adjusted waist circumference residuals) were used to estimate relative risks (RRs) and 95% confidence intervals (CIs) for CRC. Higher circulating resistin concentrations were not associated with CRC (RR per doubling resistin, 1.11; 95% CI 0.94-1.30; p = 0.22). There were also no associations with CRC subgroups defined by tumor subsite or sex. However, resistin was marginally associated with a higher CRC risk among participants followed-up maximally two years, but not among those followed-up after more than two years. We observed no substantial correlation between baseline circulating resistin concentrations and adiposity measures (BMI, waist circumference), adipokines (adiponectin, leptin), or metabolic and inflammatory biomarkers (C-reactive protein, C-peptide, high-density lipoprotein cholesterol, reactive oxygen metabolites) among controls. In this large-scale prospective cohort, there was little evidence of an association between baseline circulating resistin concentrations and CRC risk in European men and women.

External Validation of the Colon Life Nomogram for Predicting 12-Week Mortality in Dutch Metastatic Colorectal Cancer Patients Treated with Trifluridine/Tipiracil in Daily Practice.

BACKGROUND: Predicting prognosis in refractory metastatic colorectal cancer (mCRC) patients is needed to guide decision making. The Colon Life nomogram was developed to predict 12-week mortality in refractory mCRC patients. The aim of this study is to validate the Colon Life nomogram in last line/refractory patients receiving trifluridine/tipiracil (FTD/TPI) in daily practice. METHODS: The validation cohort consists of 150 QUALITAS study patients, an observational substudy of the Prospective Dutch CRC cohort, who were treated with FTD/TPI between 2016 and 2019. Model performance was assessed on discrimination, calibration, and clinical usefulness. The additional prognostic value of baseline quality of life (QoL) and thymidine kinase (TK1) expression in tissue was explored. RESULTS: Of the 150 patients, 25 (16.7%) died within 12 weeks of starting FTD/TPI treatment. The C-statistic was 0.63 (95% C.I. 0.56-0.70). The observed/expected ratio was 0.52 (0.37-0.73). The calibration intercept and slope were -1.06 (-1.53 to -0.58) and 0.41 (0.01-0.81), respectively, which indicated overestimation of 12-week mortality by the nomogram. Decision curve analysis showed the nomogram did not yield a positive net benefit at clinically meaningful thresholds for predicted 12-week mortality. Addition of QoL to the nomogram improved the C-statistic to 0.85 (0.81-0.89). TK1 expression was associated with progression-free survival but not with overall survival. CONCLUSION: We demonstrated evident miscalibration of the Colon Life nomogram upon external validation, which hampers its use in clinical practice. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model including QoL.