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1.
Leuk Res ; 102: 106520, 2021 03.
Article in English | MEDLINE | ID: mdl-33611131

ABSTRACT

This study evaluated long-term immunophenotypic changes in circulating levels of 24 immune cell subsets through 4 years of continuous treatment with first-line ibrutinib (420 mg once daily) in 31 patients with chronic lymphocytic leukemia (CLL) from the RESONATE-2 study, and compared them with untreated age-matched healthy donors (n = 20). Ibrutinib progressively decreased total B-cell counts and preferentially targeted malignant CLL B cells over normal B cells. Elevated counts of chronically activated, exhausted, and effector memory T cells were normalized within 6-16 months, while naive T cells remained mostly within healthy donor range (HDR). Immunosuppressive regulatory T cells and myeloid-derived suppressor cells were normalized within the first 1-2 years and then plateaued. Additionally, ibrutinib restored low counts of innate cell populations associated with antitumor immunity: plasmacytoid dendritic cells were restored to HDR after 2 years, and classical monocyte counts progressively and continuously increased toward HDR. Ibrutinib also consistently preserved circulating mature natural killer cell counts. The data indicate that ibrutinib continuously exerted positive effects on immune cell populations throughout 4 years of treatment, consistent with improved clinical outcomes observed in patients. The normalization of adaptive and innate immune cell populations suggests that long-term ibrutinib treatment mediates restoration of immunity.


Subject(s)
Adenine/analogs & derivatives , Antineoplastic Agents/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukocytes, Mononuclear/drug effects , Piperidines/therapeutic use , Adenine/therapeutic use , Aged , Aged, 80 and over , Female , Humans , Immunophenotyping , Male
2.
Leuk Res ; 97: 106432, 2020 10.
Article in English | MEDLINE | ID: mdl-32911375

ABSTRACT

Ibrutinib positively modulates many T-cell subsets in chronic lymphocytic leukemia (CLL). To understand ibrutinib's effects on the broader landscape of immune cell populations, we comprehensively characterized changes in circulating counts of 21 immune blood cell subsets throughout the first year of treatment in patients with relapsed/refractory (R/R) CLL (n = 55, RESONATE) and previously untreated CLL (n = 50, RESONATE-2) compared with untreated age-matched healthy donors (n = 20). Ibrutinib normalized abnormal immune cell counts to levels similar to those of age-matched healthy donors. Ibrutinib significantly decreased pathologically high circulating B cells, regulatory T cells, effector/memory CD4+ and CD8+ T cells (including exhausted and chronically activated T cells), natural killer (NK) T cells, and myeloid-derived suppressor cells; preserved naive T cells and NK cells; and increased circulating classical monocytes. T-cell function was assessed in response to T-cell receptor stimulation in patients with R/R CLL (n = 21) compared with age-matched healthy donors (n = 18). Ibrutinib significantly restored T-cell proliferative ability, degranulation, and cytokine secretion. Over the same period, ofatumumab or chlorambucil did not confer the same spectrum of normalization as ibrutinib in multiple immune subsets. These results establish that ibrutinib has a significant and likely positive impact on circulating malignant and nonmalignant immune cells and restores healthy T-cell function.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/immunology , Salvage Therapy , T-Lymphocytes, Regulatory/immunology , Adenine/analogs & derivatives , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/administration & dosage , Case-Control Studies , Chlorambucil/administration & dosage , Drug Resistance, Neoplasm , Female , Follow-Up Studies , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Natural Killer T-Cells/drug effects , Natural Killer T-Cells/immunology , Neoplasm Recurrence, Local/pathology , Piperidines , Prognosis , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , T-Lymphocytes, Regulatory/drug effects
3.
J Pharmacol Exp Ther ; 372(3): 331-338, 2020 03.
Article in English | MEDLINE | ID: mdl-31871305

ABSTRACT

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cell-based occupancy assays that quantify the percentage of drug-inactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 ± 0.27 and 1.09 ± 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT: This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Benzamides/pharmacology , Models, Biological , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Cell Line , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Kinetics , Piperidines , Protein Binding
4.
Int J Med Sci ; 16(4): 519-528, 2019.
Article in English | MEDLINE | ID: mdl-31171903

ABSTRACT

Oesophageal adenocarcinoma (OAC) is an exemplar model of obesity-associated cancer. Response to neoadjuvant chemoradiotherapy (NA CRT) is a clinical challenge. We examined if visceral adipose tissue and obesity status alter radiosensitivity in OAC. The radioresistant (OE33R) and radioresponsive (OE33P) OAC isogenic model was cultured with adipose tissue conditioned media from three patient cohorts: non-cancer patients, surgery only OAC patients and NA CRT OAC patients. Cell survival was characterised by clonogenic assay, metabolomic profiling by nuclear magnetic resonance spectroscopy and adipokine receptor gene expression by qPCR. A retrospective in vivo study compared tumour response to NA CRT in normal weight (n=53) versus overweight/obese patients (n=148). Adipose conditioned media (ACM) from all patient cohorts significantly increased radiosensitivity in radioresistant OE33R cells. ACM from the NA CRT OAC cohort increased radiosensitivity in OE33P cells. Metabolomic profiling demonstrated separation of the non-cancer and surgery only OAC cohorts and between the non-cancer and NA CRT OAC cohorts. Gene expression profiling of OE33P versus OE33R cells demonstrated differential expression of the adiponectin receptor-1 (AR1), adiponectin receptor-2 (AR2), leptin receptor (LepR) and neuropilin receptor-1 (NRP1) genes. In vivo overweight/obese OAC patients achieved an enhanced tumour response following NA CRT compared to normal weight patients. This study demonstrates that visceral adipose tissue modulates the cellular response to radiation in OAC.


Subject(s)
Adenocarcinoma/radiotherapy , Esophageal Neoplasms/radiotherapy , Intra-Abdominal Fat/drug effects , Obesity, Abdominal/radiotherapy , Radiation Tolerance/drug effects , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Body Mass Index , Cell Line, Tumor , Cell Survival/drug effects , Culture Media, Conditioned/pharmacology , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Intra-Abdominal Fat/pathology , Male , Metabolomics , Obesity, Abdominal/genetics , Obesity, Abdominal/pathology , Receptors, Adiponectin/genetics , Receptors, Leptin/radiation effects
5.
Ophthalmol Ther ; 7(1): 173-183, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29508370

ABSTRACT

INTRODUCTION: To determine visual and anatomical outcomes of diabetic macular oedema (DMO) patients in a tertiary centre following conversion to aflibercept having been refractory to previous treatment with bevacizumab/ranibizumab. METHODS: A retrospective case series of patients with a diagnosis of DMO undergoing aflibercept intravitreal therapy for at least 6 months who had previous treatment with three consecutive bevacizumab/ranibizumab injections pre-switch. Exclusion criteria included other procedures affecting visual outcome performed within the treatment period. Outcomes measured included visual acuity (VA), central macular thickness (CMT) and injection frequency. RESULTS: Eighteen eyes of 13 patients were included. Mean VA pre-switch was 61.5 ± 13.8 letters and CMT was 433.2 ± 101.4. Mean number of prior bevacizumab/ranibizumab treatments was 11.3 ± 7.2. Mean follow-up post-switch was 22.5 months (SD 7.9). Mean VA improved from baseline by 4.8 letters at 6 months (p = 0.005), by 6.1 letters at 12 months (p = 0.006), by 7.9 letters (p = 0.004) at 18 months and by 6.4 letters (p = 0.1) at 24 months. Mean CMT decreased from baseline by 108.6 µm at 6 months (p = 0.01), 117.7 µm at 12 months (p = 0.0003), 158.0 µm at 18 months (p = 0.005) and by 123.3 µm at 24 months (p = 0.02). CONCLUSION: Switching to aflibercept in treatment-resistant DMO produces significant improvements in visual and anatomical outcomes, with eventual maintenance of VA levels.

6.
Inflammation ; 39(5): 1729-36, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27423204

ABSTRACT

In the midst of a worsening obesity epidemic, the incidence of obesity-associated morbidities, including cancer, diabetes, cardiac and liver disease is increasing. Insights into mechanisms underlying pathological obesity-associated inflammation are lacking. Both the omentum, the principal component of visceral fat, and liver of obese individuals are sites of excessive inflammation, but to date the T cell profiles of both compartments have not been assessed or compared in a patient cohort with obesity-associated disease. We have previously identified that omentum is enriched with inflammatory cytokines, chemokines and T cells. Here, we compared the inflammatory profile of T cells in the omentum and liver of patients with the obesity-associated malignancy oesophageal adenocarcinoma (OAC). Furthermore, we assessed the secreted cytokine profile in OAC patient serum, omentum and liver to assess systemic and local inflammation. We observed parallel T cell cytokine profiles and phenotypes in the omentum and liver of OAC patients, in particular CD69(+) and inflammatory effector memory T cells. This study reflects similar processes of inflammation and T cell activation in the omentum and liver, and may suggest common targets to modulate pathological inflammation at these sites.


Subject(s)
Intra-Abdominal Fat/pathology , Liver/pathology , Neoplasms/pathology , Obesity/pathology , T-Lymphocyte Subsets/immunology , Adenocarcinoma , Cytokines/analysis , Esophageal Neoplasms , Humans , Immunologic Memory , Inflammation/immunology , Inflammation/pathology , Intra-Abdominal Fat/immunology , Liver/immunology , Obesity/complications , Omentum/immunology , Omentum/pathology
7.
Mol Med ; 222016 05 23.
Article in English | MEDLINE | ID: mdl-27254108

ABSTRACT

Esophageal adenocarcinoma (EAC) has a poor prognosis and is increasing in incidence in many western populations. Neoadjuvant chemoradiation therapy (CRT) followed by surgery is increasingly the standard of care for locally advanced EAC; however, resistance to treatment is a significant clinical problem. The identification of both novel biomarkers predicting response to treatment and novel therapeutic targets to enhance the efficacy of CRT are key to improving survival rates in EAC. In this study we performed global microRNA (miRNA) profiling of pre-treatment EAC biopsies and identified 67 miRNA significantly altered in patients who are resistant to CRT. One of these miRNA, miR-187, was significantly decreased in pre-treatment EAC tumors from patients having a poor response to neoadjuvant CRT, highlighting downregulation of miR-187 as a potential mechanism of treatment resistance in EAC. In vitro, miR-187 was demonstrated to play a functional role in modulating sensitivity to X-ray radiation and cisplatin in EAC and its dysregulation was demonstrated to be due to chromosomal alterations. In vitro, miR-187 altered expression of a diverse array of pathways, including the immune regulator complement component 3 (C3), serum levels of which we have previously demonstrated to predict patient response to CRT. In vivo, expression of C3 was significantly increased in tumors from patients having a poor response to CRT. This study highlights for the first time a role for miR-187 as a novel biomarker of response to CRT and a potential therapeutic target for enhancing the efficacy of CRT in EAC.


Subject(s)
Adenocarcinoma/genetics , Cisplatin/pharmacology , Complement C3/genetics , Drug Resistance, Neoplasm , Esophageal Neoplasms/genetics , Gene Expression Profiling/methods , MicroRNAs/genetics , Adenocarcinoma/drug therapy , Adenocarcinoma/radiotherapy , Adult , Aged , Cell Line, Tumor , Chemoradiotherapy , Down-Regulation , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/radiotherapy , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged
8.
Immunol Cell Biol ; 94(6): 531-7, 2016 07.
Article in English | MEDLINE | ID: mdl-27046081

ABSTRACT

Obesity is a global health problem presenting serious risk of disease fuelled by chronic inflammation, including type 2 diabetes mellitus, cardiovascular disease, liver disease and cancer. Visceral fat, in particular the omentum and liver of obese individuals are sites of excessive inflammation. We propose that chemokine-mediated trafficking of pro-inflammatory cells to the omentum and liver contributes to local and subsequent systemic inflammation. Oesophagogastric adenocarcinoma (OAC) is an exemplar model of obesity and inflammation driven cancer. We have demonstrated that T cells actively migrate to the secreted factors from the omentum and liver of OAC patients and that both CD4(+) and CD8(+) T cells bearing the chemokine receptor CCR5 are significantly more prevalent in these tissues compared to matched blood. The CCR5 ligand and inflammatory chemokine MIP-1α is also secreted at significantly higher concentrations in the omentum and liver of our OAC patient cohort compared to matched serum. Furthermore, we report that MIP-1α receptor antagonism can significantly reduce T cell migration to the secreted factors from OAC omentum and liver. These novel data suggest that chemokine receptor antagonism may have therapeutic potential to reduce inflammatory T cell infiltration to the omentum and liver and in doing so, may ameliorate pathological inflammation in obesity and obesity-associated cancer.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Movement/immunology , Liver/pathology , Neoplasms/immunology , Obesity/complications , Omentum/pathology , Receptors, CCR1/antagonists & inhibitors , Adipose Tissue/pathology , Aged , Case-Control Studies , Chemokines/metabolism , Culture Media, Conditioned/pharmacology , Female , Humans , Male , Neoplasms/blood , Neoplasms/etiology , Receptors, CCR1/metabolism
9.
BMC Cancer ; 14: 907, 2014 Dec 03.
Article in English | MEDLINE | ID: mdl-25471892

ABSTRACT

BACKGROUND: Visceral obesity has a strong association with both the incidence and mortality of esophageal adenocarcinoma (EAC). Alterations in mitochondrial function and energy metabolism is an emerging hallmark of cancer, however, the potential role that obesity plays in driving these alterations in EAC is currently unknown. METHODS: Adipose conditioned media (ACM) was prepared from visceral adipose tissue taken from computed tomography-determined viscerally-obese and non-obese EAC patients. Mitochondrial function in EAC cell lines was assessed using fluorescent probes, mitochondrial gene expression was assessed using qPCR-based gene arrays and intracellular ATP levels were determined using a luminescence-based kit. Glycolysis and oxidative phosphophorylation was measured using Seahorse XF technology and metabolomic analysis was performed using 1H NMR. Expression of metabolic markers was assessed in EAC tumor biopsies by qPCR. RESULTS: ACM from obese EAC patients significantly increased mitochondrial mass and mitochondrial membrane potential in EAC cells, which was significantly associated with visceral fat area, and was coupled with a significant decrease in reactive oxygen species. This mitochondrial dysfunction was accompanied by altered expression of 19 mitochondrial-associated genes and significantly reduced intracellular ATP levels. ACM from obese EAC patients induced a metabolic shift to glycolysis in EAC cells, which was coupled with significantly increased sensitivity to the glycolytic inhibitor 2-deoxyglucose. Metabolomic profiling demonstrated an altered glycolysis and amino acid-related signature in ACM from obese patients. In EAC tumors, expression of the glycolytic marker PKM2 was significantly positively associated with obesity. CONCLUSION: This study demonstrates for the first time that ACM from viscerally-obese EAC patients elicits an altered metabolic profile and can drive mitochondrial dysfunction and altered energy metabolism in EAC cells in vitro. In vivo, in EAC patient tumors, expression of the glycolytic enzyme PKM2 is positively associated with obesity.


Subject(s)
Adenocarcinoma/physiopathology , Energy Metabolism , Esophageal Neoplasms/physiopathology , Intra-Abdominal Fat/physiology , Mitochondria/physiology , Obesity, Abdominal/physiopathology , Adenocarcinoma/complications , Adenocarcinoma/genetics , Adenosine Triphosphate/metabolism , Aged , Antimetabolites/pharmacology , Body Mass Index , Carrier Proteins/genetics , Cell Line, Tumor , Culture Media, Conditioned , Deoxyglucose/pharmacology , Esophageal Neoplasms/complications , Esophageal Neoplasms/genetics , Female , Gene Expression , Glycolysis/drug effects , Humans , Intra-Abdominal Fat/diagnostic imaging , Male , Membrane Potential, Mitochondrial , Membrane Proteins/genetics , Metabolome , Middle Aged , Mitochondria/genetics , Obesity, Abdominal/complications , Radiography , Reactive Oxygen Species/metabolism , Thyroid Hormones/genetics , Thyroid Hormone-Binding Proteins
10.
Nutr Res ; 33(3): 171-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23507222

ABSTRACT

Excess visceral adiposity is associated with increased gastrointestinal cancer risk. Evidence suggests that the systemic inflammation and dysmetabolism observed in visceral obesity underpins this association. Along with magnetic resonance imaging, computed tomography is a gold standard for abdominal fat quantification and is routinely available for gastrointestinal cancer research. However, no gender-specific cutoff values are currently available for classifying visceral obesity in white populations. Using the metabolic syndrome (MetSyn) as an indicator of obesity-associated dysmetabolism, this study aimed to establish pathologically relevant, gender-specific cut-off values for use in obesity-associated cancer research. Total, visceral and subcutaneous fat areas were calculated between the L3 and L4 invertebral space from computed tomography scans in a cohort of 170 males and 66 females undergoing gastrointestinal resection. Receiver operating characteristics analysis was used to determine cut-off values for total, visceral and subcutaneous fat areas associated with MetSyn. Linear regression was used to correlate these values with waist circumference. Visceral fat area (VFA) strongly correlated with the presence of MetSyn (P < .0001). The cut-off value for VFA associated with the presence of MetSyn was 163.8 cm(2) in males (83.6% sensitivity, 62.5% specificity) and 80.1 cm(2) for females (96% sensitivity, 73.2% specificity). The waist circumference corresponding to these VFA values was 96.1 cm in males and 83.2 cm in females. This study is the first to generate gender-specific and pathologically relevant cut-off values for VFA in patients with gastrointestinal cancer. In the field of obesity-associated research, this new anthropometric measure is of paramount importance for determining the accurate pathological obesity status of cancer patients.


Subject(s)
Gastrointestinal Neoplasms/etiology , Intra-Abdominal Fat/diagnostic imaging , Metabolic Syndrome/diagnostic imaging , Obesity/complications , Tomography, X-Ray Computed , Adult , Aged , Aged, 80 and over , Body Mass Index , Cohort Studies , Female , Humans , Intra-Abdominal Fat/pathology , Male , Metabolic Syndrome/pathology , Middle Aged , Obesity/pathology , ROC Curve , Reference Values , Risk Factors , Sensitivity and Specificity , Sex Factors , Waist Circumference
11.
Biotechniques ; 45(3): 283-92, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18778252

ABSTRACT

Genome-scale gene expression technologies are increasingly being applied for biological research as a whole and toxicological screening in particular. In order to monitor data quality and process drift, we adopted the use of two rat-tissue mixtures (brain, liver, kidney, and testis) previously introduced as RNA reference samples. These samples were processed every time a microarray experiment was hybridized, thereby verifying the comparability of the resulting expression data for cross-study comparison. This study presents the analysis of 21 technical replicates of these two mixed-tissue samples using Affymetrix RAE230_2 GeneChip over a period of 12 months. The results show that detection sensitivity, measured by the number of present and absent sequences, is robust, and data correlation, indicated by scatter plots, varies little over time. Receiver operating characteristic (ROC) curves show the sensitivity and specificity of the current measurements are consistent with arrays previously classified as well performing. Overall, this paper shows that the inclusion of standard samples during microarray labeling and hybridization experiments is useful to benchmark the performance of microarray experiments over time and allows discovery of any process drift that, if it occurs, may confound the comparison of these datasets.


Subject(s)
Evaluation Studies as Topic , Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis/methods , RNA/chemistry , Animals , Area Under Curve , Benchmarking/methods , Brain Chemistry , Kidney/chemistry , Liver/chemistry , Male , Organ Specificity , RNA/isolation & purification , ROC Curve , Rats , Rats, Sprague-Dawley , Reference Standards , Sensitivity and Specificity , Testis/chemistry
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