Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets

Low Pretreatment CD4⁺:CD8⁺ T Cell Ratios and CD39⁺CD73⁺CD19⁺ B Cell Proportions Are Associated with Improved Relapse-Free Survival in Head and Neck Squamous Cell Carcinoma

The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV+ patients contained increased proportions of CD39−CD73−CD4+ T cells and reduced proportions of CD39−/+CD73+CD4+ T cells compared to the equivalent cells from HPV− patients. Notably, the pretreatment CD4+:CD8+ T cell ratios and CD39+CD73+CD19+ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted

Expression of cancer stem cell markers is prognostic in metastatic gastroesophageal adenocarcinoma

Gastroesophageal adenocarcinoma is a common and highly lethal malignancy. Cancer stem cells (CSCs) have a key role in the development and progression of metastatic disease. While expression of CSC markers CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) in locoregional gastroesophageal cancer is known to be associated with poorer clinical outcomes, the significance of CSC marker expression in distal metastatic disease is unknown. We investigated the clinicopathological and prognostic associations of the CSC markers, CD44, CD133, and ALDH1, on metastatic deposits from gastroesophageal adenocarcinomas, and evaluated the association of CSC expression with urokinase-type plasminogen activator receptor (uPAR) expression. Of the 36 patients included in the study, 16 (44%) were positive for CD44, 13 (36%) were positive for CD133, and 26 (72%) were positive for ALDH1. CD44 expression was significantly associated with poorer overall survival (OS) in univariate [hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.3–6.9, p=0.008] and multivariate analyses (HR 2.5, 95%CI 1.1–6.2, p=0.04). ALDH1 expression was significantly associated with poorer OS in univariate (HR 2.4, 95% CI 1.01–5.7, p=0.04) analysis but was not significant in multivariate analysis. Both CD44 and ALDH1 expression were significantly associated with uPAR expression. We found no association between CD133 expression and OS. CD44 expression on metastatic disease from gastroesophageal adenocarcinomas is an independent prognostic marker associated with poorer OS. These results expand current evidence to support the role of CSCs as biomarkers in metastatic gastroesophageal cancer

Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets

Expression of cancer stem cell markers is prognostic in metastatic gastroesophageal adenocarcinoma

Gastroesophageal adenocarcinoma is a common and highly lethal malignancy. Cancer stem cells (CSCs) have a key role in the development and progression of metastatic disease. While expression of CSC markers CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) in locoregional gastroesophageal cancer is known to be associated with poorer clinical outcomes, the significance of CSC marker expression in distal metastatic disease is unknown. We investigated the clinicopathological and prognostic associations of the CSC markers, CD44, CD133, and ALDH1, on metastatic deposits from gastroesophageal adenocarcinomas, and evaluated the association of CSC expression with urokinase-type plasminogen activator receptor (uPAR) expression. Of the 36 patients included in the study, 16 (44%) were positive for CD44, 13 (36%) were positive for CD133, and 26 (72%) were positive for ALDH1. CD44 expression was significantly associated with poorer overall survival (OS) in univariate [hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.3-6.9, p=0.008] and multivariate analyses (HR 2.5, 95%CI 1.1-6.2, p=0.04). ALDH1 expression was significantly associated with poorer OS in univariate (HR 2.4, 95% CI 1.01-5.7, p=0.04) analysis but was not significant in multivariate analysis. Both CD44 and ALDH1 expression were significantly associated with uPAR expression. We found no association between CD133 expression and OS. CD44 expression on metastatic disease from gastroesophageal adenocarcinomas is an independent prognostic marker associated with poorer OS. These results expand current evidence to support the role of CSCs as biomarkers in metastatic gastroesophageal cancer