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Abstract Currently, serum carcinoembryonic agent (CEA) along with contrast-enhanced imaging and colonoscopy are used for evaluation of recurrence of colorectal cancer. However, CEA is an unreliable and nonspecific biomarker that may fail to rise and signal relapse. Analysis of circulating tumor DNA (ctDNA) in patients offers a minimally invasive method to assess risk of relapse several months ahead of conventional clinical means. Here, we report the case of a colon adenocarcinoma with postoperative liver metastasis diagnosed early by ctDNA measurement, using a personalized NGS-mPCR assay. While ctDNA levels continued to rise, CEA levels tested negative. Metastatic relapse to the liver was promptly confirmed by PET/CT scan. The patient underwent a successful metastasectomy with curative intent. Following surgery, the patient exhibited no evidence of disease and ctDNA levels remained negative. Our case report suggests that the early detection of postoperative molecular residual disease by means of ctDNA measurement can accurately predict mCRC relapse in cases where CEA levels fail to increase. Close monitoring of ctDNA levels during the postoperative period can allow for earlier intervention and more favorable outcomes in relapsing mCRC patients.

In this study, we investigated the therapeutic potential of lentinan in mouse models of inflammatory bowel disease (IBD) and colitis‐associated cancer (CAC). Lentinan decreased the disease activity index and macroscopic and microscopic colon tissue damage in dextran sulphate sodium (DSS)‐induced or TNBS‐induced models of colitis. High‐dose lentinan was more effective than salicylazosulfapyridine in the mouse models of colitis. Lentinan decreased the number of tumours, inflammatory cell infiltration, atypical hyperplasia and nuclear atypia in azoxymethane/DSS‐induced CAC model. It also decreased the expression of pro‐inflammatory cytokines, such as IL‐13 and CD30L, in IBD and CAC model mice possibly by inhibiting Toll‐like receptor 4 (TLR4)/NF‐κB signalling and the expression of colon cancer markers, such as carcinoembryonic antigen, cytokeratin 8, CK18 and p53, in CAC model mice. In addition, lentinan restored the intestinal bacterial microbiotal community structure in IBD model mice. Thus, it shows therapeutic potential in IBD and CAC model mice possibly by inhibiting TLR4/NF‐κB signalling‐mediated inflammatory responses and disruption of the intestinal microbiotal structure.

Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro , which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.

Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1 - PD1 + effector T cells and with more minor increases in TCF1 + PD1 + resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII + macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell – T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

Background CEA, CYFRA21-1 and NSE are tumor markers used for monitoring the response to chemotherapy in advanced adenocarcinoma, squamous cell carcinoma and small-cell lung cancer, respectively. Their role in cancer immunotherapy needs to be elucidated. Methods Patients with advanced non-small cell lung cancer (NSCLC) were treated with nivolumab 3 mg/kg every 2 weeks within the Italian Nivolumab Expanded Access Program. Blood samples were collected at baseline, at each cycle up to cycle 5 and then every two cycles until patient’s withdrawn from the study. All patients underwent a CT-scan after every 4 cycles of treatment and responses were classified according to RECIST 1.1. The biomarkers serum levels were measured with a chemiluminescent microparticle immunoassay for CEA and with an immuno radiometric assay for CYFRA21-1 and NSE. The markers values at baseline and after 4 cycles were used to analyze the relationship between their variation over baseline and the tumor response, evaluated as disease control rate (DCR: CR + PR + SD), and survival (PFS and OS). Results A total of 70 patients were evaluable for the analysis. Overall, a disease control was obtained in 24 patients (35.8%, 4 PR + 20 SD). After 4 cycles of nivolumab a CEA or CYFRA21-1 reduction ≥ 20% over the baseline was significantly associated with DCR (CEA, p = 0.021; CYFRA21-1, p < 0.001), PFS (CEA, p = 0.028; CYFRA21-1, p < 0.001) and OS (CEA, p = 0.026; CYFRA21-1, p = 0.019). Multivariate analysis confirmed the ability of CYFRA21-1 reduction ≥ 20% to predict DCR (p = 0.002) and PFS (p < 0.001). Conclusion The reduction in serum level of CYFRA21-1 or CEA might be a reliable biomarker to predict immunotherapy efficacy in NSCLC patients. NSE was not significant for monitoring the efficacy of nivolumab.

Erlotinib (ER) and gefitinib (GB) are frequently prescribed for patients with advanced lung adenocarcinoma (ADC). Although both ER and GB significantly extend median survival, several patients experience early disease relapse due to treatment resistance. This study aimed to identify common genes associated with acquired resistance to ER or GB. This integrative analysis of transcriptomic data identified differentially expressed genes by comparing sensitive PC-9 cells with ER-resistant (PC-9/ER) or GB-resistant (PC-9/GB) cell lines. A Venn diagram was used to identify common genes. Candidate genes were confirmed in our generated drug-resistant cell lines. After six months, the PC-9/ER and PC-9/GB cell lines exhibited 40.7-fold and 109.2-fold increases in resistance to ER and GB, respectively. Flow cytometric analysis demonstrated that both resistant cell lines were resistant to cell cycle arrest and apoptosis induced by ER or GB. Through integrated analysis, we found that two genes, KRT13 and CEACAM5, were up-regulated in both the PC-9/ER and PC-9/GB cell lines. The expression patterns of these two genes did not align with the transcriptome, with only CEACAM5 exhibiting consistent gene and protein expressions. Our findings indicate that CEACAM5 serves as a biomarker for predicting the response of patients to ER and GB treatments.

Background Adoptive transfer of regulatory T cells (Tregs) has demonstrated safety, feasibility and early signs of efficacy in promoting immunological tolerance in inflammatory conditions such as graft-versus-host disease (GvHD). Chimeric antigen receptor (CAR)-engineered Tregs offer localized activation and suppression compared to polyclonal Tregs, but their clinical translation is limited by high manufacturing costs, lengthy developing times and fixed single-antigen specificity. To address these limitations, we employed the universal adapter Reverse CAR (RevCAR) system, which harbors a peptide epitope lacking intrinsic antigen specificity but provides flexibility in targeting through the use of an antigen-specific RevCAR Target Module (RevTM). As a proof-of-concept, we used a RevTM targeting carcinoembryonic antigen (CEA), which is highly expressed in the gastrointestinal (GI) tract, as a potential strategy to achieve localized immunosuppression in GI acute GvHD. Methods To support clinical translation, we established an automated, GMP-compatible, clinical-scale manufacturing process. Tregs were magnetically enriched from leukapheresis using the CliniMACS ® Plus, followed by high-purity sorting on the MACSQuant ® Tyto ® . The sorted cells were virally transduced and the RevCAR Tregs were expanded on the CliniMACS Prodigy ® to obtain clinically relevant cell numbers. The harvested products were evaluated for phenotype, stability, antigen specificity and suppressive function. Results Across five manufacturing runs, Tregs (CD4 + CD25 high CD127 low FOXP3 + ) with a median initial purity of 94% were expanded to achieve a median therapeutic yield of 602 × 10 6 cells. The final product maintained a high purity (median: 91.9%) and exhibited high RevCAR expression (median: 60% RevCAR + ). Mass cytometry analysis revealed that expanded RevCAR Tregs predominantly exhibited a central memory phenotype with high expression of functional and homing markers. Under experimental pro-inflammatory conditions, the cells maintained stable FOXP3 and Helios expression with minimal pro-inflammatory cytokine production. Importantly, RevCAR Tregs showed antigen-specific activation upon target engagement via the CEA-specific RevTM and robust, dose-dependent suppression. Conclusion The study establishes a scalable, GMP-compatible process for manufacturing pure, stable and functional universal RevCAR Tregs for clinical applications. Furthermore, the RevCAR system offers a promising approach toward an allogenic, off-the-shelf Treg therapy capable of treating diverse immune-mediated diseases with spatial precision.

Background Epithelial–mesenchymal transition (EMT) is implicated in the metastatic process and presents a challenge to epithelial cell adhesion molecule-based detection of circulating tumor cells (CTCs), which have been demonstrated to be a prognostic indicator in metastatic breast cancer. Although evidence has indicated that heterogeneity of CTCs based on EMT markers is associated with disease progression, no standard recommendations have been established for clinical practice. This study aimed to evaluate the prognostic significance of dynamic CTC detection based on EMT for metastatic breast cancer patients. Methods We enrolled 108 human epidermal growth factor receptor 2-negative metastatic breast cancer patients from the prospective phase III CAMELLIA study and applied the CanPatrol CTC enrichment technique to identify CTC phenotypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) in peripheral blood samples. Receiver operating characteristic curve analyses of total CTC count and the proportion of mesenchymal CTCs for predicting the 1-year progression-free survival (PFS) rate were conducted to determine the optimal cut-off values, and Kaplan–Meier analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic value of the cut-off values of both total CTC count and the proportion of mesenchymal CTCs in combination. Results For predicting the 1-year PFS rate, the optimal cut-off value of total CTC count was 9.5 (Area under the curve [AUC] = 0.538, 95% confidence interval [CI] = 0.418–0.657), and that of the proportion of mesenchymal CTCs was 10.7% (AUC = 0.581, 95% CI = 0.463–0.699). We used the two cut-off values in combination to forecast PFS in which the total CTC count was equaled to or exceeded 10/5 mL with the proportion of mesenchymal CTCs surpassed 10.7%. Patients who met the combined criteria had significantly shorter median PFS than did those who did not meet the criteria (6.2 vs. 9.9 months, P  =0.010). A nomogram was constructed based on the criteria and significant clinicopathological characteristics with a C-index of 0.613 ( P  = 0.010). Conclusions The criteria, which combine the total CTC count and the proportion of mesenchymal CTCs, may be used to monitor therapeutic resistance and predict prognosis in patients with metastatic breast cancer. Trial registration ClinicalTrials.gov . NCT01917279. Registered on 19 July 2013, https://clinicaltrials.gov/ct2/show/NCT01917279?term=NCT01917279&rank=1 .

Aggressive variant prostate cancer (AVPC) originates from metastatic prostate cancer (mPCa) following androgen receptor-targeted therapies, leading to diverse pathological subtypes, notably castration-resistant prostate cancer (CRPC). Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), is consistently expressed across AVPC phenotypes, including neuroendocrine prostate carcinoma (NEPC) and double-negative prostate carcinoma (DNPC), which are significant subtypes of CRPC, making it a promising therapeutic target. In this study, A high-affinity nanobody, B12, specific to CEACAM5, was discovered through phage library screening. B12 exhibited robust binding capabilities, enhanced tumor accumulation, and effective tissue penetration, facilitating rapid in vivo imaging of AVPC. The conjugation of B12 with PE38 to create the immunotoxin B12-PE38 showed significant anti-tumor activity in AVPC xenograft models, including one that mimics bone metastasis. When B12-PE38 was combined with docetaxel, it elicited enhanced tumor inhibitory effects, effectively inhibiting tumor progression. This study underscores CEACAM5 as a target for precise imaging and targeted therapy in AVPC, introducing novel diagnostic and therapeutic strategies for a disease that currently faces a dearth of effective treatment options due to the scarcity of well-defined targets. Graphical Abstract

Purpose CA 19-9 is the only established tumor marker in pancreatic cancer (PC); the prognostic role of other serum markers like CEA, CRP, LDH or bilirubin has not yet been defined. Methods We pooled pre-treatment data on CA 19-9, CEA, CRP, LDH and bilirubin levels from two German multicenter randomized phase II trials together with prospective patient data from one high-volume German Cancer Center. Marker levels were assessed locally before the start of palliative first-line therapy for advanced PC and serially during treatment (for CA 19-9 only). Clinical and biomarker data (overall 12 variables) were correlated with the efficacy endpoints time-to-progression (TTP) and overall survival (OS) by using uni- and multivariate Cox models. Results Data from 291 patients were included in this pooled analysis; 253 patients (87 %) received treatment within prospective clinical trials. Median TTP in the study cohort was 5.1 months and median OS 9.0 months. In univariate analysis, pre-treatment CA 19-9 (HR 1.55), LDH (HR 2.04) and CEA (HR 1.89) levels were significantly associated with TTP. Regarding OS, baseline CA 19-9 (HR 1.46), LDH (HR 2.07), CRP (HR 1.69) and bilirubin (HR 1.62) were significant prognostic factors. Within multivariate analyses, pre-treatment log [CA 19-9] (as continuous variable for TTP) and log [bilirubin] as well as log [CRP] (for OS) had an independent prognostic value. A CA 19-9 decline of ≥25 % during the first two chemotherapy cycles was predictive for TTP and OS, independent of the applied CA 19-9 assay. Conclusion Baseline CA 19-9 and CA 19-9 kinetics during first-line chemotherapy are prognostic in advanced PC. Besides that finding other serum markers like CRP, LDH and bilirubin can also provide prognostic information on TTP and OS.