• Cancer stem cell mobilization and therapeutic targeting of the 5T4 oncofetal antigen.

  Harrop, R; O'Neill, E; Stern, Peter L; Oxford BioMedica plc, Windrush Court, Transport Way, Oxford, OX4 6LT, UK (2019)

  Cancer stem cells (CSCs) can act as the cellular drivers of tumors harnessing stem cell properties that contribute to tumorigenesis either as founder elements or by the gain of stem cell traits by the malignant cells. Thus, CSCs can self-renew and generate the cellular heterogeneity of tumors including a hierarchical organization similar to the normal tissue. While the principle tumor growth contribution is often from the non-CSC components, it is the ability of small numbers of CSCs to avoid the effects of therapeutic strategies that can contribute to recurrence after treatment. However, identifying and characterizing CSCs for therapeutic targeting is made more challenging by their cellular potency being influenced by a particular tissue niche or by the capacity of more committed cells to regain stem cell functions. This review discusses the properties of CSCs including the limitations of the available cell surface markers, the assays that document tumor initiation and clonogenicity, the roles of epithelial mesenchymal transition and molecular pathways such as Notch, Wnt, Hippo and Hedgehog. The ability to target and eliminate CSCs is thought to be critical in the search for curative cancer treatments. The oncofetal tumor-associated antigen 5T4 (TBGP) has been linked with CSC properties in several different malignancies. 5T4 has functional attributes that are relevant to the spread of tumors including through EMT, CXCR4/CXCL12, Wnt, and Hippo pathways which may all contribute through the mobilization of CSCs. There are several different immunotherapies targeting 5T4 in development including antibody-drug conjugates, antibody-targeted bacterial super-antigens, a Modified Vaccinia Ankara-basedvaccine and 5T4-directed chimeric antigen receptor T-cells. These immune therapies would have the advantage of targeting both the bulk tumor as well as mobilized CSC populations.
• DNA methylation of immune checkpoints in the peripheral blood of breast and colorectal cancer patients.

  Elashi, AA; Sasidharan, N; Taha, RZ; Shaath, H; Elkord, Eyad; Cancer Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar (2019)

  Aberrant expression of immune checkpoints (ICs) in cancer creates an immunosuppressive microenvironment, which supports immune evasion of tumor cells. We have recently reported that epigenetic modifications are critical for ICs expression in the tumor microenvironment (TME) of primary breast cancer (PBC) and colorectal cancer (CRC). Herein, we investigated transcriptomic expression of ICs (PD-1, CTLA-4, LAG-3, TIM-3, TIGIT) and PD-L1 in peripheral blood of PBC and CRC patients, compared to healthy donors (HD). We found that expressions of TIM-3, TIGIT, PD-L1 were significantly upregulated, while LAG-3 expression was downregulated in peripheral blood of PBC and CRC patients. Demethylation enzymes TET2 and TET3 were also upregulated. In addition, promoter DNA methylation status of PD-1 was significantly hypermethylated, while PD-L1 was hypomethylated in PBC and CRC patients. Furthermore, TIGIT was significantly hypomethylated only in CRC patients. Remarkably, promoter methylation status of LAG-3, TIGIT and PD-L1 was in concordance with transcriptomic expression in CRC: the more the hypomethylation, the higher the expression. In comparison, we found that CTLA-4, TIM-3, TIGIT and PD-L1 in PBC, and CTLA-4 in CRC patients were significantly upregulated in peripheral blood, compared with tumor tissues of the same patients. However, demethylation status of all ICs was higher in TT, except for TIGIT in PBC, and CTLA-4 in CRC patients. These data indicate that the underlying mechanisms behind peripheral upregulation of PD-L1 and TIGIT in cancer patients could be due to aberrant promoter methylation profile. Moreover, demethylation inhibitors together with anti-PD-L1/anti-TIGIT could be a more efficient therapeutic strategy in cancer patients.
• Clinical intensity-modulated proton therapy for Hodgkin lymphoma: which patients benefit the most?

  Ntentas, G; Dedeckova, K; Andrlik, M; Aznar, Marianne Camille; George, B; Kubes, J; Darby, SC; Cutter, DJ; Clinical Trial Service Unit, Nuffield Department of Population Health, University of Oxford, Oxford (2019)

  PURPOSE: Radiotherapy (RT) improves control of Hodgkin lymphoma (HL), but patients undergoing RT are at risk of late effects, including cardiovascular disease and second cancers, due to radiation dose to organs at risk (OAR). Proton therapy (PT) can reduce OAR dose compared to conventional photon RT. However, access to PT is currently limited so referral must be appropriately selective. We aim to identify subgroups of HL patients who could benefit most dosimetrically from RT with PT based on their pre-chemotherapy disease characteristics. METHODS AND MATERIALS: Normal tissue radiation doses were calculated for 21 HL patients treated with deep inspiration breath-hold pencil-beam scanning (PBS) PT and compared to doses from 3D-conformal and partial-arc volumetric modulated (PartArc) photon RT. Pre-chemotherapy disease characteristics associated with significant dosimetric benefits from PBS compared to photon RT were identified. RESULTS: Treatment with PBS was well tolerated and provided with good local control. PBS provided dosimetric advantages for patients whose clinical treatment volume extended below the 7th thoracic level and for female patients with axillary disease. Additionally, an increasing dosimetric benefit for some OAR was observed for increasing target volume. PBS significantly reduced the mean dose to the heart, breast, lungs, spinal cord and esophagus. Dose homogeneity and conformity within the target volume were also superior with PBS but some high dose measures and hot spots were increased with PBS compared to PartArc. CONCLUSIONS: PBS gives good target coverage and local control whilst providing reductions in radiation dose to OAR for individuals receiving RT for HL compared to advanced photon RT. Our findings highlight certain groups of patients who would be expected to gain more dosimetric benefit from PBS. These findings facilitate the selection of patients who should be considered a priority for PT.
• Effect of pembrolizumab on CD4(+) CD25(+) , CD4(+) LAP(+) and CD4(+) TIM-3(+) T cell subsets.

  Toor, SM; Sasidharan, N; Pfister, G; Elkord, Eyad; Cancer Research Center, Qatar, Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar (2019)

  Tumor immune evasion involves the expansion of avidly proliferating immunosuppressive cells and inhibition of effector T cell proliferation. Immune checkpoints (IC) block the activation pathways of tumor-reactive T cells. IC pathways are often exploited by tumor cells to evade immune destruction, and blocking these pathways through IC inhibitors (ICI) has shown promising results in multiple malignancies. In this study, we investigated the effects of an ICI, pembrolizumab, on various T cell subsets in vitro. We compared the suppressive activity of CD4+ CD25+ regulatory T cells (conventional Treg ) with T cells expressing T cell immunoglobulin-3+ (TIM-3+ ) and latency-associated peptide (LAP)+ T cells. We found that LAP-expressing T cells were more suppressive than conventional Treg , but TIM-3-expressing T cells were not suppressive. Our results show that pembrolizumab does not modulate functions of Treg and mediates its immunostimulatory effects via the release of effector T cells from suppression. These findings may assist in the development of agents designed to intervene in IC pathways to overcome Treg resistance to ICI.
• Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy.

  Harrison, OJ; Torrens, C; Salhiyyah, K; Modi, A; Moorjani, N; Townsend, Paul A; Ohri, SK; Cagampang, F; Institute of Developmental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK (2019)

  OBJECTIVES: Bicuspid aortic valve disease is common and is associated with ascending aortic aneurysms. Vascular smooth muscle cell (VSMC) apoptosis is characteristic of the ascending aorta of bicuspid patients, and NOTCH1 gene mutations have also been linked to the disease. NOTCH signalling is a fundamental cell signalling pathway, which dictates cell fate decisions including apoptosis. Our objective was to elucidate the role of NOTCH signalling in VSMC apoptosis and differentiation in bicuspid aortopathy. METHODS: Ascending aortic biopsies were obtained from 19 bicuspid and 12 tricuspid aortic valve patients and were sub-classified into 4 groups according to the maximum ascending aortic diameter (aneurysmal ??45?mm). Apoptotic VSMCs were counted by light microscopy using a TUNEL assay. Gene expression of key regulators of NOTCH signalling (NOTCH1 and HES1), apoptosis (BAX and BCL-2) and VSMC differentiation (MYH11, CNN1 and MYH10) were quantified using quantitative real-time PCR. Primary VSMCs were cultured from 2 tricuspid aortic valve and 2 bicuspid aortic valve patients, NOTCH signalling was inhibited with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, and the gene expression was again quantified. RESULTS: The apoptotic cell count was significantly higher in bicuspid aortic valve patients (3.2 cells/50 000??m2 vs 1.1 cells/50 000??m2; P?=?0.033). There was a trend towards lower apoptotic cell count in the aneurysmal versus non-aneurysmal tricuspid and bicuspid groups and an increased ratio of proapoptotic gene expression, which was not statistically significant. This was associated with a 2.8-fold increase in contractile gene expression (P?=?0.026) and a 2.0-fold increase in NOTCH signalling gene expression in bicuspid versus tricuspid aortic valve patients (P?=?0.022). NOTCH inhibition in cultured VSMCs induced a similar pattern of increased proapoptotic and procontractile gene expressions. CONCLUSIONS: This preliminary study suggests that NOTCH activation in the non-aneurysmal bicuspid aortas may underlie aortopathy by influencing VSMC apoptosis and differentiation. NOTCH signalling manipulation may provide a therapeutic target for preventing aneurysms in bicuspid patients. Further studies with larger sample sizes are needed to substantiate the present findings.

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