Systematic Evaluation of the Immune Environment of Small Intestinal Neuroendocrine Tumours

BACKGROUND: The immune tumour microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumours (siNET) have not been fully defined. METHODS: Herein, we studied 40 patients with primary and synchronous metastatic siNETs , and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. Additionally, matched FFPE tissue was obtained for multi-parametric immunohistochemistry (IHC) to determine the relative abundance and distribution of T-cell infiltrate. Tumour mutational burden (TMB) was also assessed and correlated with immune infiltration. RESULTS: Effector tumour infiltrating lymphocytes had a higher expression of PD-1 in the tumour microenvironment compared to the periphery. Additionally, CD8+ tumour infiltrating lymphocytes had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 and higher levels of PD-1 expression compared to normal tissue. IHC revealed that the majority of cases have {less than or equal to}10% intratumoural T cells but a higher number of peritumoural T cells, demonstrating an "exclusion" phenotype. Finally, we confirmed that siNETs have a low TMB compared to other tumour types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio. CONCLUSIONS: Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit "excluded" T cells into the tumour microenvironment to treat patients with siNETs

PP2A/B55 and Fcp1 regulate Greatwall and Ensa desphorylation during mitotic exit

Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, neither Fcp1 nor PP2A appear to essential to dephosphorylate the bulk of mitotic Cdk1 substrates following Cdk1 inhibition. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit

Multisite Investigation of Outcomes With Implementation of CYP2C19 Genotype-Guided Antiplatelet Therapy After Percutaneous Coronary Intervention

OBJECTIVES: This multicenter pragmatic investigation assessed outcomes following clinical implementation of CYP2C19 genotype-guided antiplatelet therapy after percutaneous coronary intervention (PCI). BACKGROUND: CYP2C19 loss-of-function alleles impair clopidogrel effectiveness after PCI. METHODS: After clinical genotyping, each institution recommended alternative antiplatelet therapy (prasugrel, ticagrelor) in PCI patients with a loss-of-function allele. Major adverse cardiovascular events (defined as myocardial infarction, stroke, or death) within 12 months of PCI were compared between patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy. Risk was also compared between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy. Cox regression was performed, adjusting for group differences with inverse probability of treatment weights. RESULTS: Among 1,815 patients, 572 (31.5%) had a loss-of-function allele. The risk for major adverse cardiovascular events was significantly higher in patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy (23.4 vs. 8.7 per 100 patient-years; adjusted hazard ratio: 2.26; 95% confidence interval: 1.18 to 4.32; p = 0.013). Similar results were observed among 1,210 patients with acute coronary syndromes at the time of PCI (adjusted hazard ratio: 2.87; 95% confidence interval: 1.35 to 6.09; p = 0.013). There was no difference in major adverse cardiovascular events between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy (adjusted hazard ratio: 1.14; 95% confidence interval: 0.69 to 1.88; p = 0.60). CONCLUSIONS: These data from real-world observations demonstrate a higher risk for cardiovascular events in patients with a CYP2C19 loss-of-function allele if clopidogrel versus alternative therapy is prescribed. A future randomized study of genotype-guided antiplatelet therapy may be of value

Multisite Investigation of Outcomes With Implementation of CYP2C19 Genotype-Guided Antiplatelet Therapy After Percutaneous Coronary Intervention

OBJECTIVES: This multicenter pragmatic investigation assessed outcomes following clinical implementation of CYP2C19 genotype-guided antiplatelet therapy after percutaneous coronary intervention (PCI). BACKGROUND: CYP2C19 loss-of-function alleles impair clopidogrel effectiveness after PCI. METHODS: After clinical genotyping, each institution recommended alternative antiplatelet therapy (prasugrel, ticagrelor) in PCI patients with a loss-of-function allele. Major adverse cardiovascular events (defined as myocardial infarction, stroke, or death) within 12 months of PCI were compared between patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy. Risk was also compared between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy. Cox regression was performed, adjusting for group differences with inverse probability of treatment weights. RESULTS: Among 1,815 patients, 572 (31.5%) had a loss-of-function allele. The risk for major adverse cardiovascular events was significantly higher in patients with a loss-of-function allele prescribed clopidogrel versus alternative therapy (23.4 vs. 8.7 per 100 patient-years; adjusted hazard ratio: 2.26; 95% confidence interval: 1.18 to 4.32; p = 0.013). Similar results were observed among 1,210 patients with acute coronary syndromes at the time of PCI (adjusted hazard ratio: 2.87; 95% confidence interval: 1.35 to 6.09; p = 0.013). There was no difference in major adverse cardiovascular events between patients without a loss-of-function allele and loss-of-function allele carriers prescribed alternative therapy (adjusted hazard ratio: 1.14; 95% confidence interval: 0.69 to 1.88; p = 0.60). CONCLUSIONS: These data from real-world observations demonstrate a higher risk for cardiovascular events in patients with a CYP2C19 loss-of-function allele if clopidogrel versus alternative therapy is prescribed. A future randomized study of genotype-guided antiplatelet therapy may be of value

The regulatory network and the dynamics of the mitotic switch.

<p>(<b>A</b>) Model of the Cdk1 activation switch. Cdk1 activity is regulated by inhibitory Tyr15 phosphorylation modulated through the activities of Wee1 and Cdc25. The activity of these modifying enzymes are regulated both directly and indirectly (through Gwl, Ensa/ARPP19 and PP2A/B55) by Cdk1. (<b>B–J</b>) Simulation of mitotic entry and exit using a mathematical model of the regulatory network with the assumption included that Gwl is dephosphorylated by an OA-insensitive phosphatase (left panels), by an OA-sensitive phosphatase (middle panels), or specifically by PP2A/B55 (right panels). The simulation of mitotic entry is shown from the initial condition obtained using Cdk1 inhibition and either removal of Cdk1 inhibition (B–D) or PP2A inhibition by OA (E–G) promotes mitotic entry. The mitotic exit (H–J) is shown from the initial condition corresponding to metaphase state and Cdk1 inhibition promotes mitotic exit.</p

Revised model of the Cdk1 activation loop.

<p>The regulation of Gwl dephosphorylation by PP2A/B55 and Ensa/ARPP19 dephosphorylation by Fcp1 are incorporated into the previous network shown on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004004#pgen-1004004-g001" target="_blank">Fig. 1A</a>. Since Fcp1 is inhibited in Cdk1 dependent manner, Ensa is regulated by a coherent feed-forward loop as well: Cdk1 both activates the phosphorylation (via Gwl) and inhibits the dephosphorylation (via Fcp1) of Ensa.</p

Characterizing Gwl, Ensa/ARPP19 and SP dephosphorylation during mitotic exit.

<p>(<b>A</b>) HeLa cells were synchronized in mitosis by Eg5 inhibition using 5 µM STLC and pretreated for one hour with 1 µM OA, 10 µM TC or both. Samples were taken for extraction and immunoblot analysis at indicated timepoints following treatment with 10 µM RO3306. (<b>B</b>) Quantification of relative Cdk substrate (phospho-SP) dephosphorylation in OA and TC treated cells. Error bars indicate standard deviation calculated from three independent experiments. (<b>C</b>) Immunoblot analysis of STLC arrested cells following one hour treatment with 100 nM Calyculin A (CalA). Extracts were taken at indicated times after Cdk1 inhibition by 10 µM RO3306. (<b>D</b>) MBP Kinase assays with immuno-precipitated Flag-Gwl that was transiently expressed in HeLa cells. Flag-Gwl was purified from STLC arrested cells before and 30 minutes after RO3306 treatment. Cells were pretreated for one hour with the indicated phosphatase inhibitors. (<b>E</b>) Quantification of MBP kinase activity following Cdk1 inhibition (+RO) relative to mitotic cells before Cdk1 inhibition (-RO). Error bars indicate the standard deviation calculated from three independent experiments.</p

Testing Gwl and Ensa/ARPP19 phosphorylation in the G2/M switch.

<p>(<b>A</b>) Experimental protocol: DT40 cdk1as cells are blocked in G2 phase by 1NMPP1. Mitosis is triggered either by removing 1NMPP1 from the media, or by treating the cells with 1 µM OA. (<b>B</b>) Immuno-fluorescence analysis of cdk1as cells before (0) and 5 minutes after (5) release from G2 by 1NMPP1 removal using DAPI and the indicated antibodies. (<b>C</b>) Immuno-fluorescence analysis of mitotic entry in 1NMPP1 arrested cdk1as cells at indicated timepoints after treatment with 1 µM OA. (<b>D</b>) Immuno-blot analysis of 1NMPP1 treated cdk1as cell extracts taken at indicated timepoints following OA treatment, or from cells treated with both 1 µM OA and 10 µM TC.</p

Identifying the phosphatases required for Gwl and Ensa/ARPP19.

<p>(A) Dephosphorylation of Gwl and SP sites in B55 depleted cells. HeLa cells were transfected with combinations of B55α and δ siRNAs and synchronized in mitosis with STLC as above (see Materials & Methods). Cell extracts were sampled before and 30 minutes after RO3306 treatment and analyzed by immuno-blotting with indicated antibodies. (B) Dephosphorylation of Gwl, Ensa/Arpp19 and SP sites in Fcp1 depleted cells. HeLa cells were transfected with Fcp1 siRNA and synchronized in mitosis with STLC (see Materials & Methods). Cell extracts were sampled before and 30 minutes after RO3306 treatment and analyzed by immune-blotting with indicated antibodies. (C) Gwl phosphatase assay with immuno-precipitated Flag-B55α. Purified his-Gwl was phosphorylated by Cdk2/cycA and γ³²P ATP. Cdk2/cycA was removed from the reaction by further purification with Ni-Agarose beads and radiolabeled phospho-Gwl was incubated with immunoprecipitated B55α. The presence of the PP2A/B55α complex in the immune-precipitate was confirmed by immuno-blotting. Phosphatase activity was measured by scintillation counting of released <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004004#pgen.1004004-Visconti1" target="_blank">[³²]</a>P phosphate. (D) Similar phosphatase assay as in (C) with recombinant <i>in vitro</i> phosphorylated Ensa and immuno-precipitated GFP-Fcp1.</p