Digital spatial profiling of the microenvironment of muscle invasive bladder cancer.

Muscle invasive bladder cancer (MIBC) is a molecularly diverse disease with varied clinical outcomes. Molecular studies typically employ bulk sequencing analysis, giving a transcriptomic snapshot of a section of the tumour. However, tumour tissues are not homogeneous, but are composed of distinct compartments such as the tumour and stroma. To investigate the molecular profiles of bladder cancer, whilst also maintaining the spatial complexity of the tumours, we employed whole transcriptome Digital Spatial Profiling (DSP). With this method we generated a dataset of transcriptomic profiles of tumour epithelium, stroma, and immune infiltrate. With these data we investigate the spatial relationship of molecular subtype signatures and ligand signalling events. We find that Basal/Squamous and Classical subtypes are mostly restricted to tumour regions, while the stroma-rich subtype signatures are abundant within the stroma itself. Additionally, we identify ligand signalling events occurring between tumour, stroma, and immune infiltrate regions, such as immune infiltrate derived GPNMB, which was highly correlated with VEGFA expression within the tumour. These findings give us new insights into the diversity of MIBC at a molecular level and provide a dataset with detailed spatial information that was not available before in bladder cancer research.

Randomized Phase 3 Trial of the Hypoxia Modifier Nimorazole Added to Radiation Therapy With Benefit Assessed in Hypoxic Head and Neck Cancers Determined Using a Gene Signature (NIMRAD).

PURPOSE: Tumor hypoxia is an adverse prognostic factor in head and neck squamous cell carcinoma (HNSCC). We assessed whether patients with hypoxic HNSCC benefited from the addition of nimorazole to definitive intensity modulated radiation therapy (IMRT). METHODS AND MATERIALS: NIMRAD was a phase 3, multicenter, placebo-controlled, double-anonymized trial of patients with HNSCC unsuitable for concurrent platinum chemotherapy or cetuximab with definitive IMRT (NCT01950689). Patients were randomized 1:1 to receive IMRT (65 Gy in 30 fractions over 6 weeks) plus nimorazole (1.2 g/m2 daily, before IMRT) or placebo. The primary endpoint was freedom from locoregional progression (FFLRP) in patients with hypoxic tumors, defined as greater than or equal to the median tumor hypoxia score of the first 50 patients analyzed (≥0.079), using a validated 26-gene signature. The planned sample size was 340 patients, allowing for signature generation in 85% and an assumed hazard ratio (HR) of 0.50 for nimorazole effectiveness in the hypoxic group and requiring 66 locoregional failures to have 80% power in a 2-tail log-rank test at the 5% significance level. RESULTS: Three hundred thirty-eight patients were randomized by 19 centers in the United Kingdom from May 2014 to May 2019, with a median follow-up of 3.1 years (95% CI, 2.9-3.4). Hypoxia scores were available for 286 (85%). The median patient age was 73 years (range, 44-88; IQR, 70-76). There were 36 (25.9%) locoregional failures in the hypoxic group, in which nimorazole + IMRT did not improve FFLRP (adjusted HR, 0.72; 95% CI, 0.36-1.44; P = .35) or overall survival (adjusted HR, 0.96; 95% CI, 0.53-1.72; P = .88) compared with placebo + IMRT. Similarly, nimorazole + IMRT did not improve FFLRP or overall survival in the whole population. In total (N = 338), 73% of patients allocated nimorazole adhered to the drug for ≥50% of IMRT fractions. Nimorazole + IMRT caused more acute nausea compared with placebo + IMRT (Common Terminology Criteria for Adverse Events version 4.0 G1+2: 56.6% vs 42.4%, G3: 10.1% vs 5.3%, respectively; P < .05). CONCLUSIONS: Addition of the hypoxia modifier nimorazole to IMRT for locally advanced HNSCC in older and less fit patients did not improve locoregional control or survival.

Technical development and validation of a clinically applicable microenvironment classifier as a biomarker of tumour hypoxia for soft tissue sarcoma.

BACKGROUND: Soft tissue sarcomas (STS) are rare, heterogeneous tumours and biomarkers are needed to inform management. We previously derived a prognostic tumour microenvironment classifier (24-gene hypoxia signature). Here, we developed/validated an assay for clinical application. METHODS: Technical performance of targeted assays (Taqman low-density array, nanoString) was compared in 28 prospectively collected formalin-fixed, paraffin-embedded (FFPE) biopsies. The nanoString assay was biologically validated by comparing to HIF-1α/CAIX immunohistochemistry (IHC) in clinical samples. The Manchester (n = 165) and VORTEX Phase III trial (n = 203) cohorts were used for clinical validation. The primary outcome was overall survival (OS). RESULTS: Both assays demonstrated excellent reproducibility. The nanoString assay detected upregulation of the 24-gene signature under hypoxia in vitro, and 16/24 hypoxia genes were upregulated in tumours with high CAIX expression in vivo. Patients with hypoxia-high tumours had worse OS in the Manchester (HR 3.05, 95% CI 1.54-5.19, P = 0.0005) and VORTEX (HR 2.13, 95% CI 1.19-3.77, P = 0.009) cohorts. In the combined cohort, it was independently prognostic for OS (HR 2.24, 95% CI 1.42-3.53, P = 0.00096) and associated with worse local recurrence-free survival (HR 2.17, 95% CI 1.01-4.68, P = 0.04). CONCLUSIONS: This study comprehensively validates a microenvironment classifier befitting FFPE STS biopsies. Future uses include: (1) selecting high-risk patients for perioperative chemotherapy; and (2) biomarker-driven trials of hypoxia-targeted therapies.

Long-Term Outcomes of Radical Radiation Therapy with Hypoxia Modification with Biomarker Discovery for Stratification: 10-Year Update of the BCON (Bladder Carbogen Nicotinamide) Phase 3 Randomized Trial (ISRCTN45938399).

PURPOSE: Many muscle-invasive bladder cancers are hypoxic, which limits the efficacy of radiation therapy. Hypoxia modification using carbogen and nicotinamide has been tested in a phase 3 trial, Bladder Carbogen Nicotinamide. We present mature follow-up data with biomarker predictions of outcomes. METHODS AND MATERIALS: Bladder Carbogen Nicotinamide is a prospective, phase 3, multicenter, randomized, 2-arm, nonblinded clinical trial. Participants were randomized to receive radical radiation therapy (RT; control arm) alone or with the addition of carbogen (98% O2; 2% CO2) and nicotinamide (CON). Patients with muscle-invasive or high-grade non-muscle invasive bladder cancer were included. Tumor tissue was collected at entry and was analyzed for tumor necrosis, hypoxia (24-gene signature), and basal and luminal tumor molecular subtypes. Overall survival (OS) and disease-free survival and relationships with biomarker status outcomes are analyzed using multivariable Cox regression and log-rank analysis. RESULTS: We analyzed 333 patients with a median follow-up of 10.3 years. The 10-year OS rates were 30% (95% confidence interval [CI], 0.23-0.39) in RT + CON patients and 24% (95% CI, 0.18-0.33) in the RT-alone patients (hazard ratio [HR], 0.80; 95% CI, 0.61-1.04; P = .08). The greatest benefit from CON was seen in patients with tumor necrosis (n = 79; 5-year OS, 53% vs. 33% in patients without tumor necrosis; HR, 0.59; 95% CI, 0.36-0.99; P = .04). Cases with a high hypoxia gene score (n = 75) had a 5-year OS rate of 51%, compared to 34% for a low score (HR, 0.64; 95% CI, 0.38-1.08; P = .09); those with the basal molecular subtype (n = 70) had a 5-year OS rate of 58%, compared to 38% for those with the luminal subtype (HR, 0.58; 95% CI, 0.32-1.06; P = .08). CONCLUSIONS: Although the improvement in long-term OS in the whole population is not statistically significant, patients selected by necrosis and high hypoxia gene score benefitted from hypoxia modification.

Correction: Validation of a hypoxia related gene signature in multiple soft tissue sarcoma cohorts.

[This corrects the article DOI: 10.18632/oncotarget.23280.].

Mapping Hypoxia in Renal Carcinoma with Oxygen-enhanced MRI: Comparison with Intrinsic Susceptibility MRI and Pathology.

Purpose To cross-validate T1-weighted oxygen-enhanced (OE) MRI measurements of tumor hypoxia with intrinsic susceptibility MRI measurements and to demonstrate the feasibility of translation of the technique for patients. Materials and Methods Preclinical studies in nine 786-0-R renal cell carcinoma (RCC) xenografts and prospective clinical studies in eight patients with RCC were performed. Longitudinal relaxation rate changes (∆R1) after 100% oxygen inhalation were quantified, reflecting the paramagnetic effect on tissue protons because of the presence of molecular oxygen. Native transverse relaxation rate (R2*) and oxygen-induced R2* change (∆R2*) were measured, reflecting presence of deoxygenated hemoglobin molecules. Median and voxel-wise values of ∆R1 were compared with values of R2* and ∆R2*. Tumor regions with dynamic contrast agent-enhanced MRI perfusion, refractory to signal change at OE MRI (referred to as perfused Oxy-R), were distinguished from perfused oxygen-enhancing (perfused Oxy-E) and nonperfused regions. R2* and ∆R2* values in each tumor subregion were compared by using one-way analysis of variance. Results Tumor-wise and voxel-wise ∆R1 and ∆R2* comparisons did not show correlative relationships. In xenografts, parcellation analysis revealed that perfused Oxy-R regions had faster native R2* (102.4 sec-1 vs 81.7 sec-1) and greater negative ∆R2* (-22.9 sec-1 vs -5.4 sec-1), compared with perfused Oxy-E and nonperfused subregions (all P < .001), respectively. Similar findings were present in human tumors (P < .001). Further, perfused Oxy-R helped identify tumor hypoxia, measured at pathologic analysis, in both xenografts (P = .002) and human tumors (P = .003). Conclusion Intrinsic susceptibility biomarkers provide cross validation of the OE MRI biomarker perfused Oxy-R. Consistent relationship to pathologic analyses was found in xenografts and human tumors, demonstrating biomarker translation. Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Validation of a hypoxia related gene signature in multiple soft tissue sarcoma cohorts.

PURPOSE: There is a need for adjuvant/neo-adjuvant treatment strategies to prevent metastatic relapse in soft tissue sarcoma (STS). Tumor hypoxia is associated with a high-risk of metastasis and is potentially targetable. This study aimed to derive and validate a hypoxia mRNA signature for STS for future biomarker-driven trials of hypoxia targeted therapy. MATERIALS AND METHODS: RNA sequencing was used to identify seed genes induced by hypoxia in seven STS cell lines. Primary tumors in a training cohort (French training) were clustered into two phenotypes by seed gene expression and a de novo hypoxia signature derived. Prognostic significance of the de novo signature was evaluated in the training and two independent validation (French validation and The Cancer Genome Atlas) cohorts. RESULTS: 37 genes were up-regulated by hypoxia in all seven cell lines, and a 24-gene signature was derived. The high-hypoxia phenotype defined by the signature was enriched for well-established hypoxia genes reported in the literature. The signature was prognostic in univariable analysis, and in multivariable analysis in the training (n = 183, HR 2.16, P = 0.0054) and two independent validation (n = 127, HR 3.06, P = 0.0019; n = 258, HR 2.05, P = 0.0098) cohorts. Combining information from the de novo hypoxia signature and a genome instability signature significantly improved prognostication. Transcriptomic analyses showed high-hypoxia tumors had more genome instability and lower immune scores. CONCLUSIONS: A 24-gene STS-specific hypoxia signature may be useful for prognostication and identifying patients for hypoxia-targeted therapy in clinical trials.

The hypoxia marker CAIX is prognostic in the UK phase III VorteX-Biobank cohort: an important resource for translational research in soft tissue sarcoma.

BACKGROUND: Despite high metastasis rates, adjuvant/neoadjuvant systemic therapy for localised soft tissue sarcoma (STS) is not used routinely. Progress requires tailoring therapy to features of tumour biology, which need exploration in well-documented cohorts. Hypoxia has been linked to metastasis in STS and is targetable. This study evaluated hypoxia prognostic markers in the phase III adjuvant radiotherapy VorteX trial. METHODS: Formalin-fixed paraffin-embedded tumour biopsies, fresh tumour/normal tissue and blood were collected before radiotherapy. Immunohistochemistry for HIF-1α, CAIX and GLUT1 was performed on tissue microarrays and assessed by two scorers (one pathologist). Prognostic analysis of disease-free survival (DFS) used Kaplan-Meier and Cox regression. RESULTS: Biobank and outcome data were available for 203 out of 216 randomised patients. High CAIX expression was associated with worse DFS (hazard ratio 2.28, 95% confidence interval: 1.44-3.59, P<0.001). Hypoxia-inducible factor-1α and GLUT1 were not prognostic. Carbonic anhydrase IX remained prognostic in multivariable analysis. CONCLUSIONS: The VorteX-Biobank contains tissue with linked outcome data and is an important resource for research. This study confirms hypoxia is linked to poor prognosis in STS and suggests that CAIX may be the best known marker. However, overlap between single marker positivity was poor and future work will develop an STS hypoxia gene signature to account for tumour heterogeneity.

A Gene Signature for Selecting Benefit from Hypoxia Modification of Radiotherapy for High-Risk Bladder Cancer Patients.

Purpose: Hypoxia modification improves overall survival in muscle-invasive bladder cancer patients who undergo radiotherapy. There is evidence that hypoxic tumors benefit most from hypoxia modification. The study aimed to identify or derive a hypoxia gene signature that predicts benefit from hypoxia-modifying treatment in bladder cancer.Experimental Design: Published hypoxia signatures were tested and a new one derived by analyzing bladder cancer transcriptomic data from public databases. Tumor samples were available from the BCON phase III randomized trial of radiotherapy alone or with carbogen and nicotinamide (CON). Gene expression data were generated for 151 tumors using Affymetrix Human 1.0 Exon ST arrays and used for independent validation.Results: A 24-gene signature was derived, which was prognostic in four of six independent surgical cohorts (n = 679; meta HR, 2.32; 95% CI, 1.73-3.12; P < 0.0001). The signature was also prognostic in BCON patients receiving radiotherapy alone (n = 75; HR for local relapse-free survival, 2.37; 95% CI, 1.26-4.47; P = 0.0076). The signature predicted benefit from CON (n = 76; HR, 0.47; 95% CI, 0.26-0.86; P = 0.015). Prognostic significance (P = 0.017) and predictive significance (P = 0.058) remained after adjusting for clinicopathologic variables. A test for interaction between hypoxia status and treatment arms was significant (P = 0.0094).Conclusions: A 24-gene hypoxia signature has strong and independent prognostic and predictive value for muscle-invasive bladder cancer patients. The signature can aid identification of patients likely to benefit from the addition of carbogen and nicotinamide to radiotherapy. Clin Cancer Res; 23(16); 4761-8. ©2017 AACR.

Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas.

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma.

A 26-gene hypoxia signature predicts benefit from hypoxia-modifying therapy in laryngeal cancer but not bladder cancer.

PURPOSE: Tumor hypoxia is associated with a poor prognosis, hypoxia modification improves outcome, and hypoxic status predicts benefit from treatment. Yet, there is no universal measure of clinical hypoxia. The aim of this study was to investigate whether a 26-gene hypoxia signature predicted benefit from hypoxia-modifying treatment in both cancer types. EXPERIMENTAL DESIGN: Samples were available from 157 T2-T4 laryngeal cancer and 185 T1-T4a bladder cancer patients enrolled on the accelerated radiotherapy with carbogen and nicotinamide (ARCON) and bladder carbogen nicotinamide (BCON) phase III randomized trials of radiotherapy alone or with carbogen and nicotinamide (CON) respectively. Customized TaqMan low density arrays (TLDA) were used to assess expression of the 26-gene signature using quantitative real-time PCR. The median expression of the 26 genes was used to derive a hypoxia score (HS). Patients were categorized as TLDA-HS low (≤median) or TLDA-HS high (>median). The primary outcome measures were regional control (RC; ARCON) and overall survival (BCON). RESULTS: Laryngeal tumors categorized as TLDA-HS high showed greater benefit from ARCON than TLDA-HS low tumors. Five-year RC was 81% (radiotherapy alone) versus 100% (CON) for TLDA-HS high (P=0.009). For TLDA-HS low, 5-year RC was 91% (radiotherapy alone) versus 90% (CON; P=0.90). TLDA-HS did not predict benefit from CON in bladder cancer. CONCLUSION: The 26-gene hypoxia signature predicts benefit from hypoxia-modifying treatment in laryngeal cancer. These findings will be evaluated in a prospective clinical trial.

Necrosis predicts benefit from hypoxia-modifying therapy in patients with high risk bladder cancer enrolled in a phase III randomised trial.

BACKGROUND AND PURPOSE: Addition of carbogen and nicotinamide (hypoxia-modifying agents) to radiotherapy improves the survival of patients with high risk bladder cancer. The study investigated whether histopathological tumour features and putative hypoxia markers predicted benefit from hypoxia modification. MATERIALS AND METHODS: Samples were available from 231 patients with high grade and invasive bladder carcinoma from the BCON phase III trial of radiotherapy (RT) alone or with carbogen and nicotinamide (RT+CON). Histopathological tumour features examined were: necrosis, growth pattern, growing margin, and tumour/stroma ratio. Hypoxia markers carbonic anhydrase-IX and glucose transporter-1 were examined using tissue microarrays. RESULTS: Necrosis was the only independent prognostic indicator (P=0.04). Necrosis also predicted benefit from hypoxia modification. Five-year overall survival was 48% (RT) versus 39% (RT+CON) (P=0.32) in patients without necrosis and 34% (RT) versus 56% (RT+CON) (P=0.004) in patients with necrosis. There was a significant treatment by necrosis strata interaction (P=0.001 adjusted). Necrosis was an independent predictor of benefit from RT+CON versus RT (hazard ratio [HR]: 0.43, 95% CI 0.25-0.73, P=0.002). This trend was not observed when there was no necrosis (HR: 1.64, 95% CI 0.95-2.85, P=0.08). CONCLUSIONS: Necrosis predicts benefit from hypoxia modification in patients with high risk bladder cancer and should be used to select patients; it is simple to identify and easy to incorporate into routine histopathological examination.

Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma.

BACKGROUND AND PURPOSE: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. MATERIALS AND METHODS: Tumour samples (n=201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). RESULTS: The assay was sensitive with linear reaction efficiencies across a 4 log(10) range of inputted cDNA (0.001-10 ng/µl). Intra- (CoV=6.9%) and inter- (CoV=2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO(2). TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p<0.01) and positive pimonidazole scores (p=0.005). CONCLUSIONS: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material.

The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens.

Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.

Gene therapy of patient-derived T lymphocytes to target and eradicate colorectal hepatic metastases.

PURPOSE: The overall aim of this study was to develop a novel treatment for colorectal cancer based on the use of gene therapy. Genetic modification of T lymphocytes has been used to specifically target and kill tumor cell lines directly. To test the efficacy of this method with clinically relevant materials, this study investigated the potential of T lymphocytes derived from patients with advanced colorectal disease to target autologous primary tumor material. METHODS T lymphocytes isolated preoperatively were modified genetically with recombinant retroviruses encoding CD3zeta-based chimeric immune receptors and were tested for functional activity against freshly isolated autologous tumor cells harvested from hepatic colorectal metastases. RESULTS: Patient-derived T cells were successfully transduced, and chimeric immune receptor expression was confirmed. Carcinoembryonic antigen expression on freshly isolated colorectal tumor cells was also demonstrated by molecular and immunohistochemical techniques. T cells expressing the anticarcinoembryonic antigen receptor were specifically activated by coculture with disaggregated or intact, diced tumor, whereas control non-carcinoembryonic antigen-targeted T-cell populations failed to activate. CONCLUSIONS: These results indicate that gene-targeted primary T lymphocytes depict specific functional activity against autologous colorectal tumor cells. This evidence indicates that chimeric immune receptor-expressing T cells may be able to circumvent the mechanisms used by tumor cells to avoid immune cell activity in vivo. This study emphasizes the potential of this approach as a therapy for carcinoembryonic antigen-expressing primary colorectal tumor and its metastases.