Safety and efficacy of autologous haematopoietic stem-cell transplantation with low-dose cyclophosphamide mobilisation and reduced intensity conditioning versus standard of care in refractory Crohn's disease (ASTIClite): an open-label, multicentre, randomised controlled trial.

BACKGROUND: A previous controlled trial of autologous haematopoietic stem-cell transplantation (HSCT) in patients with refractory Crohn's disease did not meet its primary endpoint and reported high toxicity. We aimed to assess the safety and efficacy of HSCT with an immune-ablative regimen of reduced intensity versus standard of care in this patient population. METHODS: This open-label, multicentre, randomised controlled trial was conducted in nine National Health Service hospital trusts across the UK. Adults (aged 18-60 years) with active Crohn's disease on endoscopy (Simplified Endoscopic Score for Crohn's Disease [SES-CD] ulcer sub-score of ≥2) refractory to two or more classes of biological therapy, with no perianal or intra-abdominal sepsis or clinically significant comorbidity, were recruited. Participants were centrally randomly assigned (2:1) to either HSCT with a reduced dose of cyclophosphamide (intervention group) or standard care (control group). Randomisation was stratified by trial site by use of random permuted blocks of size 3 and 6. Patients in the intervention group underwent stem-cell mobilisation (cyclophosphamide 1 g/m2 with granulocyte colony-stimulating factor (G-CSF) 5 µg/kg) and stem-cell harvest (minimum 2·0 × 106 CD34+ cells per kg), before conditioning (fludarabine 125 mg/m2, cyclophosphamide 120 mg/kg, and rabbit anti-thymocyte globulin [thymoglobulin] 7·5 mg/kg in total) and subsequent stem-cell reinfusion supported by G-CSF. Patients in the control group continued any available conventional, biological, or nutritional therapy. The primary outcome was absence of endoscopic ulceration (SES-CD ulcer sub-score of 0) without surgery or death at week 48, analysed in the intention-to-treat population by central reading. This trial is registered with the ISRCTN registry, 17160440. FINDINGS: Between Oct 18, 2018, and Nov 8, 2019, 49 patients were screened for eligibility, of whom 23 (47%) were randomly assigned: 13 (57%) to the intervention group and ten (43%) to the control group. In the intervention group, ten (77%) participants underwent HSCT and nine (69%) reached 48-week follow-up; in the control group, nine (90%) reached 48-week follow-up. The trial was halted in response to nine reported suspected unexpected serious adverse reactions in six (46%) patients in the intervention group, including renal failure due to proven thrombotic microangiopathy in three participants and one death due to pulmonary veno-occlusive disease. At week 48, absence of endoscopic ulceration without surgery or death was reported in three (43%) of seven participants in the intervention group and in none of six participants in the control group with available data. Serious adverse events were more frequent in the intervention group (38 in 13 [100%] patients) than in the control group (16 in four [40%] patients). A second patient in the intervention group died after week 48 of respiratory and renal failure. INTERPRETATION: Although HSCT with an immune-ablative regimen of reduced intensity decreased endoscopic disease activity, significant adverse events deem this regimen unsuitable for future clinical use in patients with refractory Crohn's disease. FUNDING: Efficacy and Mechanism Evaluation Programme, a Medical Research Council and National Institute for Health Research partnership.

Immunohistochemical, Flow Cytometric, and ELISA-Based Analyses of Intracellular, Membrane-Expressed, and Extracellular Hsp70 as Cancer Biomarkers.

The major stress-inducible 70 kDa heat shock (stress) protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumor cells and thus might serve as a tumor-specific biomarker of aggressive disease and/or therapeutic resistance. We have previously shown that, in contrast to normal cells, tumor cells present Hsp70 on their plasma membrane. In order to elucidate the role of intracellular, membrane-bound and extracellular Hsp70 as a potential tumor biomarker in cancer, herein we describe protocols for the staining of cytosolic Hsp70 in tumor formalin-fixed paraffin-embedded (FFPE) sections from patients with glioblastoma multiforme using immunohistochemistry, for detecting the expression of plasma membrane-bound Hsp70 by a range of cancer-derived cells using multi-parametric flow cytometry using the cmHsp70.1 monoclonal antibody (mAb) and for the measurement of free and vesicular-associated Hsp70 in the circulation of patients with cancer using a unique enzyme-linked immunosorbent assay (ELISA).

Effect of adiposity and physical fitness on cardiometabolic risk factors in adolescents: A 2-year longitudinal study.

Although risk factors for cardiometabolic diseases begin to present in young people, the association between physical fitness and adiposity with traditional and novel risk factors for cardiometabolic diseases across adolescence remains relatively unknown. Following ethical approval, fifty-two adolescents (age 11.6 ± 0.6 years; 32 girls) were recruited for a 2-years longitudinal study. Adiposity was assessed based on sum of skinfolds, waist circumference and body mass index, and physical fitness as distance run on the multi-stage fitness test (MSFT). Risk factors for cardiometabolic diseases (pro- and anti-inflammatory cytokines, plasma insulin, Homeostatic Model Assessment of Insulin Resistance - HOMA-IR, blood pressure) were measured following an overnight fast. Relationships between independent and response variables were analysed using multi-level modelling (final combined models were created using the stepwise backward elimination method). Plasma insulin concentration and HOMA-IR were positively associated with adiposity and inversely associated with distance run on the MSFT (all p < 0.05). The final combined models for plasma insulin concentration and HOMA-IR contained main effects for age, skinfolds and distance run on the MSFT. Levels of the anti-inflammatory cytokine IL-10 were inversely related to the sum of skinfolds (p = 0.046), whereas there was a trend for levels of the pro-inflammatory cytokine TNF-α to be positively related to the sum of skinfolds (p = 0.056). Adiposity and physical fitness are important, independent, determinants of metabolic health in adolescents. Furthermore, adiposity influences levels of pro- and anti-inflammatory cytokines in adolescence, with greater adiposity associated with a poorer inflammatory profile. The present study demonstrates an independent effect of physical fitness on metabolic health longitudinally across adolescence. It is therefore recommended that future work develops therapeutic interventions that reduce adiposity and enhance physical fitness in adolescents, to promote lifelong health.

Carnosine protects stimulus-secretion coupling through prevention of protein carbonyl adduction events in cells under metabolic stress.

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress. We also report that carnosine, a histidine containing dipeptide, prevented 65-90% of 4-hydroxynonenal and 3-nitrotyrosine adduction events, and that this in turn preserved mitochondrial function and protected stimulus-secretion coupling in cells exposed to metabolic stress. Carnosine therefore offers significant therapeutic potential against metabolic diseases.

Optimizing Cell Synchronization Using Nocodazole or Double Thymidine Block.

Cell synchronization is crucial when studying events that take place at specific points of the cell cycle. Several chemical agents can be used to achieve the cell culture synchronization but not all type of cells respond equally to a given concentration of these drugs. Here we describe a simple optimization method to select concentrations and timings for nocodazole or thymidine treatments using fluorescence staining. In addition, we provide detailed protocols to arrest an asynchronous culture of either suspension or adherent cells in G1/S or in G2/M.

Prostate Cancer: Early Detection and Assessing Clinical Risk Using Deep Machine Learning of High Dimensional Peripheral Blood Flow Cytometric Phenotyping Data.

Detecting the presence of prostate cancer (PCa) and distinguishing low- or intermediate-risk disease from high-risk disease early, and without the need for potentially unnecessary invasive biopsies remains a significant clinical challenge. The aim of this study is to determine whether the T and B cell phenotypic features which we have previously identified as being able to distinguish between benign prostate disease and PCa in asymptomatic men having Prostate-Specific Antigen (PSA) levels < 20 ng/ml can also be used to detect the presence and clinical risk of PCa in a larger cohort of patients whose PSA levels ranged between 3 and 2617 ng/ml. The peripheral blood of 130 asymptomatic men having elevated Prostate-Specific Antigen (PSA) levels was immune profiled using multiparametric whole blood flow cytometry. Of these men, 42 were subsequently diagnosed as having benign prostate disease and 88 as having PCa on biopsy-based evidence. We built a bidirectional Long Short-Term Memory Deep Neural Network (biLSTM) model for detecting the presence of PCa in men which combined the previously-identified phenotypic features (CD8+CD45RA-CD27-CD28- (CD8+ Effector Memory cells), CD4+CD45RA-CD27-CD28- (CD4+ Effector Memory cells), CD4+CD45RA+CD27-CD28- (CD4+ Terminally Differentiated Effector Memory Cells re-expressing CD45RA), CD3-CD19+ (B cells), CD3+CD56+CD8+CD4+ (NKT cells) with Age. The performance of the PCa presence 'detection' model was: Acc: 86.79 ( ± 0.10), Sensitivity: 82.78% (± 0.15); Specificity: 95.83% (± 0.11) on the test set (test set that was not used during training and validation); AUC: 89.31% (± 0.07), ORP-FPR: 7.50% (± 0.20), ORP-TPR: 84.44% (± 0.14). A second biLSTM 'risk' model combined the immunophenotypic features with PSA to predict whether a patient with PCa has high-risk disease (defined by the D'Amico Risk Classification) achieved the following: Acc: 94.90% (± 6.29), Sensitivity: 92% (± 21.39); Specificity: 96.11 (± 0.00); AUC: 94.06% (± 10.69), ORP-FPR: 3.89% (± 0.00), ORP-TPR: 92% (± 21.39). The ORP-FPR for predicting the presence of PCa when combining FC+PSA was lower than that of PSA alone. This study demonstrates that AI approaches based on peripheral blood phenotyping profiles can distinguish between benign prostate disease and PCa and predict clinical risk in asymptomatic men having elevated PSA levels.

TP53 abnormalities correlate with immune infiltration and associate with response to flotetuzumab immunotherapy in AML.

Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 37% to 46% of acute myeloid leukemia (AML) with adverse-risk cytogenetics and correlate with primary induction failure, high risk of relapse, and dismal prognosis. Herein, we aimed to characterize the immune landscape of TP53-mutated AML and determine whether TP53 abnormalities identify a patient subgroup that may benefit from immunotherapy with flotetuzumab, an investigational CD123 × CD3 bispecific dual-affinity retargeting antibody (DART) molecule. The NanoString PanCancer IO360 assay was used to profile 64 diagnostic bone marrow (BM) samples from patients with TP53-mutated (n = 42) and TP53-wild-type (TP53-WT) AML (n = 22) and 45 BM samples from patients who received flotetuzumab for relapsed/refractory (R/R) AML (15 cases with TP53 mutations and/or 17p deletion). The comparison between TP53-mutated and TP53-WT primary BM samples showed higher expression of IFNG, FOXP3, immune checkpoints, markers of immune senescence, and phosphatidylinositol 3-kinase-Akt and NF-κB signaling intermediates in the former cohort and allowed the discovery of a 34-gene immune classifier prognostic for survival in independent validation series. Finally, 7 out of 15 patients (47%) with R/R AML and TP53 abnormalities showed complete responses to flotetuzumab (<5% BM blasts) on the CP-MGD006-01 clinical trial (NCT #02152956) and had significantly higher tumor inflammation signature, FOXP3, CD8, inflammatory chemokine, and PD1 gene expression scores at baseline compared with nonresponders. Patients with TP53 abnormalities who achieved a complete response experienced prolonged survival (median, 10.3 months; range, 3.3-21.3 months). These results encourage further study of flotetuzumab immunotherapy in patients with TP53-mutated AML.

Identifying prostate cancer and its clinical risk in asymptomatic men using machine learning of high dimensional peripheral blood flow cytometric natural killer cell subset phenotyping data.

We demonstrate that prostate cancer can be identified by flow cytometric profiling of blood immune cell subsets. Herein, we profiled natural killer (NK) cell subsets in the blood of 72 asymptomatic men with Prostate-Specific Antigen (PSA) levels < 20 ng ml-1, of whom 31 had benign disease (no cancer) and 41 had prostate cancer. Statistical and computational methods identified a panel of eight phenotypic features ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text]) that, when incorporated into an Ensemble machine learning prediction model, distinguished between the presence of benign prostate disease and prostate cancer. The machine learning model was then adapted to predict the D'Amico Risk Classification using data from 54 patients with prostate cancer and was shown to accurately differentiate between the presence of low-/intermediate-risk disease and high-risk disease without the need for additional clinical data. This simple blood test has the potential to transform prostate cancer diagnostics.

With an estimated 1.8 million new cases in 2018 alone, prostate cancer is the fourth most common cancer in the world. Catching the disease early increases the chances of survival, but this cancer remains difficult to detect. The best diagnostic test currently available measures the blood level of a protein called the prostate-specific antigen (PSA for short). Heightened amounts of PSA may mean that the patient has cancer, but 15% of individuals with prostate cancer have normal levels of the protein, and many healthy people can have high amounts of PSA. This blood test is therefore not widely accepted as a reliable diagnostic tool. Other methods exist to detect prostate cancer, yet their results are limited. A small piece of the prostate can be taken for analysis, but results from this invasive procedure are often incorrect. Scans can help to spot a tumor, but they are not accurate enough to be conclusive on their own. New tests are therefore urgently needed. Prostate cancer is often associated with changes in the immune system that can be detected through a blood test. In particular, the appearance of a type of white blood (immune) cells called natural killer cells may be altered. Yet, it was unclear whether measurements based on these cells could help to detect prostate cancer and assess the severity of the disease. Here, Hood, Cosma et al. collected and examined the natural killer cells of 72 participants with slightly elevated PSA levels and no other symptoms. Amongst these, 31 individuals had prostate cancer and 41 were healthy. These biological data were then used to produce computer models that could detect the presence of the disease, as well as assess its severity. The algorithms were developed using machine learning, where previous patient information is used to make prediction on new data. This work resulted in a new detection tool which was 12.5% more accurate than the PSA test in detecting prostate cancer; and in a detection tool that was 99% accurate in predicting the risk of the disease (in terms of clinical significance) in individuals with prostate cancer. Although these new approaches first need to be validated in the clinic before being deployed, they could ultimately improve the detection and diagnosis of prostate cancer, saving lives and reducing the need for further tests.

Immune landscapes predict chemotherapy resistance and immunotherapy response in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Multi-Stage Fitness Test Performance, V ˙ O2 Peak and Adiposity: Effect on Risk Factors for Cardio-Metabolic Disease in Adolescents.

The role of physical activity in determining the metabolic health of adolescents is poorly understood, particularly concerning the effect on low-grade chronic inflammation (chronic elevation of pro-inflammatory cytokines IL-1ß, IL-6, TNF-α and acute phase protein CRP, which is implicated in the etiology of atherosclerosis) and anti-inflammatory mediators such as IL-10. Furthermore, there is limited information on the mediating effects of performance on the multi-stage fitness test (MSFT), V ˙ O2 peak and adiposity on risk factors for cardio-metabolic disease in adolescents. Purpose: To examine the effect of performance on the MSFT, V ˙ O2 peak and adiposity on risk factors for cardio-metabolic diseases in adolescents. Methods: Following ethical approval, 121 adolescents (11.3 ± 0.8 year) completed the study. Risk factors for cardio-metabolic disease (circulating inflammatory cytokines, blood glucose and plasma insulin concentrations) was assessed using a fasted capillary blood sample. Participants were separated into quartiles based upon distance ran during the MSFT, the blood lactate response to submaximal exercise, V ˙ O2 peak (determined during an uphill graded treadmill test), and adiposity (determined as the sum of four skinfolds). The blood lactate response to submaximal exercise and V02 peak were measured in a sub-group of participants. Data were analyzed using two-way between-subjects ANCOVA and multiple linear regression. Results: Participants with the lowest performance on the MSFT had higher blood concentrations of IL-6 (3.25 ± 0.25 pg mL-1) and IL-1ß (4.78 ± 0.54 pg mL-1) and lower concentrations of IL-10 (1.80 ± 0.27 pg mL-1) when compared with all other quartiles (all p < 0.05). Yet, when categorized into V ˙ O2 peak quartiles, no differences existed in any of the inflammatory mediators (all p > 0.05). Performance on the MSFT was the only predictor of IL-6 (ß = -0.291, p = 0.031), IL-1ß (ß = -0.405, p = 0.005), IL-10 (ß = 0.325, p = 0.021) and fasted blood glucose (ß = -0.545, p < 0.001) concentrations. Adiposity was the only predictor of plasma insulin concentration (ß = 0.515, p < 0.001) and blood pressure (diastolic: ß = 0.259, p = 0.042; mean arterial pressure: ß = 0.322, p = 0.011). Conclusion: Enhanced performance on the MSFT, but not V ˙ O2 peak, was associated with a favorable inflammatory profile in adolescents; whilst adiposity adversely affected plasma insulin, diastolic and mean arterial blood pressure. These findings demonstrate that enhancing performance on the MSFT and maintaining a healthy body composition are a potential therapeutic intervention for the attenuation of risk factors for cardio-metabolic diseases in adolescents.

Autologous stem cell transplantation in refractory Crohn's disease - low intensity therapy evaluation (ASTIClite): study protocols for a multicentre, randomised controlled trial and observational follow up study.

BACKGROUND: Intestinal inflammation in Crohn's disease (CD) is caused by mucosal immune system reactivity to luminal antigen and results in debilitating symptoms, reduced quality of life, impaired work productivity and significant health care costs. Not all patients respond to conventional and biologic therapies, with chronic inflammation ensuing. Although surgical resection may be required, disease frequently returns and surgery may not be an option, or may be declined. Case reports suggest potential benefit after haematopoietic stem cell transplant (HSCT) for patients with refractory CD. The ASTIC trial asked whether HSCT could cure CD. Few patients achieved the primary endpoint of clinical remission for 3 months, off all medication with no evidence of active disease, and there were a high number of adverse events (AEs) and serious adverse events (SAEs), including one patient death. However, beneficial effects were observed in some aspects of disease activity. The ASTIClite trial will investigate these potential benefits and safety using a lower intensity regimen than ASTIC. METHODS: Ninety-nine participants will be recruited from secondary care IBD centres in the UK into a multicentre, randomised controlled trial (RCT, ASTIClite) and an observational follow-up, and randomised to autologous HSCT versus standard care (ratio 2:1). The primary endpoint is treatment success at week 48, defined as mucosal healing without surgery or death. Secondary endpoints relating to efficacy, safety and mechanistic analyses will be evaluated at week 8, 14, 24, 32, 40 and 48. Long-term safety of the low intensity HSCT regimen forms the primary endpoint for the EBMT follow-up study and will be assessed annually for 4-7 years. DISCUSSION: ASTIClite will compare HSCTlite with standard care with respect to safety, efficacy and quality of life, and capture outcomes allowing findings to be generalised to current clinical practice in the UK. It will also provide significant mechanistic insights into the immunological consequences of HSCTlite and its impact on treatment outcomes. The observational follow-up will provide information, which is currently unavailable for this population. TRIAL REGISTRATION: The ASTIClite RCT was registered on 31st October 2017 ( ISRCTN17160440 ) and the EBMT follow-up study on 19th January 2018 ( ISRCTN31981313 ).

Cytokine, glycemic, and insulinemic responses to an acute bout of games-based activity in adolescents.

An acute bout of endurance exercise in adults stimulates a same-day anti-inflammatory response which may affect low-grade chronic inflammation and insulin resistance and benefit cardio-metabolic health. The anti-inflammatory responses to intermittent games-based exercise and to exercise in young people beyond 2 hours post-exercise are unknown. Thus, the purpose of the present study is to examine the anti-inflammatory, glycemic and insulinemic response to games-based activity in adolescents. Following ethical approval and familiarization, 39 adolescents (12.3 ± 0.7 years) completed an exercise (E) and rested (R) trial in a counterbalanced, randomized crossover design. Following a standardized breakfast, participants completed 1-hour games-based activity. Capillary blood samples were taken at baseline, immediately and 1 hour post-exercise, and 30, 60 and 120 minutes following a standardized lunch. A final blood sample was taken the next morning. Data were analyzed using repeated measures ANOVA. IL-6 concentration was higher on day one of the exercise trial (E:3.4 ± 0.4, R:2.7 ± 0.4 pg/mL; P = 0.006), as was the anti-inflammatory IL-6:TNF-α ratio (E:5.53 ± 0.93, R:3.75 ± 0.45; P = 0.027). Levels of the anti-inflammatory cytokine IL-10 increased on day two of the exercise trial (E:2.11 ± 0.23, R:1.66 ± 0.16 pg/mL; P = 0.032). Insulin sensitivity was enhanced on the exercise trial with a reduction in iAUC following the standardized lunch (E:2310 ± 834, R:3122 ± 1443 mU/L × 120 minutes; P < 0.001). Games-based activity stimulated an anti-inflammatory response up to 24 hours post-exercise and improved insulin sensitivity in response to a standardized meal in healthy adolescents. These novel findings suggest that games-based activity is an ecologically valid mode of exercise to elicit beneficial effects on cardio-metabolic risk factors in young people.

Membrane Hsp70-A Novel Target for the Isolation of Circulating Tumor Cells After Epithelial-to-Mesenchymal Transition.

The presence of circulating tumor cells (CTCs) in the peripheral blood is a pre-requisite for progression, invasion, and metastatic spread of cancer. Consequently, the enumeration and molecular characterization of CTCs from the peripheral blood of patients with solid tumors before, during and after treatment serves as a valuable tool for categorizing disease, evaluating prognosis and for predicting and monitoring therapeutic responsiveness. Many of the techniques for isolating CTCs are based on the expression of epithelial cell surface adhesion molecule (EpCAM, CD326) on tumor cells. However, the transition of adherent epithelial cells to migratory mesenchymal cells (epithelial-to-mesenchymal transition, EMT)-an essential element of the metastatic process-is frequently associated with a loss of expression of epithelial cell markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the "capture" of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)-cmHsp70.1-can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression.

Immune-Phenotyping and Transcriptomic Profiling of Peripheral Blood Mononuclear Cells From Patients With Breast Cancer: Identification of a 3 Gene Signature Which Predicts Relapse of Triple Negative Breast Cancer.

Background: Interactions between the immune system and tumors are highly reciprocal in nature, leading to speculation that tumor recurrence or therapeutic resistance could be influenced or predicted by immune events that manifest locally, but can be detected systemically. Methods: Multi-parameter flow cytometry was used to examine the percentage and phenotype of natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), monocyte subsets and regulatory T (Treg) cells in the peripheral blood of of 85 patients with breast cancer (50 of whom were assessed before and after one cycle of anthracycline-based chemotherapy), and 23 controls. Transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) in 23 patients were generated using a NanoString gene profiling platform. Results: An increased percentage of immunosuppressive cells such as granulocytic MDSCs, intermediate CD14++CD16+ monocytes and CD127negCD25highFoxP3+ Treg cells was observed in patients with breast cancer, especially patients with stage 3 and 4 disease, regardless of ER status. Following neoadjuvant chemotherapy, B cell numbers decreased significantly, whereas monocyte numbers increased. Although chemotherapy had no effect on the percentage of Treg, MDSC and NK cells, the expression of inhibitory receptors CD85j, LIAR and NKG2A and activating receptors NKp30 and NKp44 on NK cells increased, concomitant with a decreased expression of NKp46 and DNAM-1 activating receptors. Transcriptomic profiling revealed a distinct group of 3 patients in the triple negative breast cancer (TNBC) cohort who expressed high levels of mRNA encoding genes predominantly involved in inflammation. The analysis of a large transcriptomic dataset derived from the tumors of patients with TNBC revealed that the expression of CD163, CXCR4, THBS1 predicted relapse-free survival. Conclusions: The peripheral blood immunome of patients with breast cancer is influenced by the presence and stage of cancer, but not by molecular subtypes. Furthermore, immune profiling coupled with transcriptomic analyses of peripheral blood cells may identify patients with TNBC that are at risk of relapse after chemotherapy.

Immune Reconstitution After Autologous Hematopoietic Stem Cell Transplantation in Crohn's Disease: Current Status and Future Directions. A Review on Behalf of the EBMT Autoimmune Diseases Working Party and the Autologous Stem Cell Transplantation In Refractory CD-Low Intensity Therapy Evaluation Study Investigators.

Patients with treatment refractory Crohn's disease (CD) suffer debilitating symptoms, poor quality of life, and reduced work productivity. Surgery to resect inflamed and fibrotic intestine may mandate creation of a stoma and is often declined by patients. Such patients continue to be exposed to medical therapy that is ineffective, often expensive and still associated with a burden of adverse effects. Over the last two decades, autologous hematopoietic stem cell transplantation (auto-HSCT) has emerged as a promising treatment option for patients with severe autoimmune diseases (ADs). Mechanistic studies have provided proof of concept that auto-HSCT can restore immunological tolerance in chronic autoimmunity via the eradication of pathological immune responses and a profound reconfiguration of the immune system. Herein, we review current experience of auto-HSCT for the treatment of CD as well as approaches that have been used to monitor immune reconstitution following auto-HSCT in patients with ADs, including CD. We also detail immune reconstitution studies that have been integrated into the randomized controlled Autologous Stem cell Transplantation In refractory CD-Low Intensity Therapy Evaluation trial, which is designed to test the hypothesis that auto-HSCT using reduced intensity mobilization and conditioning regimens will be a safe and effective means of inducing sustained control in refractory CD compared to standard of care. Immunological profiling will generate insight into the pathogenesis of the disease, restoration of responsiveness to anti-TNF therapy in patients with recurrence of endoscopic disease and immunological events that precede the onset of disease in patients that relapse after auto-HSCT.

Phenotype and Function of Activated Natural Killer Cells From Patients With Prostate Cancer: Patient-Dependent Responses to Priming and IL-2 Activation.

Background: Although immunotherapy has emerged as the "next generation" of cancer treatments, it has not yet been shown to be successful in the treatment of patients with prostate cancer, for whom therapeutic options remain limited to radiotherapy and androgen (hormone) deprivation therapy. Previous studies have shown that priming natural killer (NK) cells isolated from healthy individuals via co-incubation with CTV-1 cells derived from an acute lymphoblastic leukemia (ALL) enhances their cytotoxicity against human DU145 (metastatic) prostate cancer cells, but it remains unknown to what extent NK cells from patients with prostate cancer can be triggered to kill. Herein, we explore the phenotype of peripheral blood NK cells in patients with prostate cancer and compare the capacity of CTV-1 cell-mediated priming and IL-2 stimulation to trigger NK cell-mediated killing of the human PC3 (metastatic) prostate cancer cell line. Methods: The phenotype of resting, primed (co-incubation with CTV-1 cells for 17 h) and IL-2 activated (100 IU/ml IL-2 for 17 h) NK cells isolated from frozen-thawed peripheral blood mononuclear cell (PBMC) preparations from patients with benign disease (n = 6) and prostate cancer (n = 18) and their cytotoxicity against PC3 and K562 cells was determined by flow cytometry. Relationship(s) between NK cell phenotypic features and cytotoxic potential were interrogated using Spearman Rank correlation matrices. Results and Conclusions: NK cell priming and IL-2 activation of patient-derived NK cells resulted in similar levels of cytotoxicity, but distinct NK cell phenotypes. Importantly, the capacity of priming and IL-2 stimulation to trigger cytotoxicity was patient-dependent and mutually exclusive, in that NK cells from ~50% of patients preferentially responded to priming whereas NK cells from the remaining patients preferentially responded to cytokine stimulation. In addition to providing more insight into the biology of primed and cytokine-stimulated NK cells, this study supports the use of autologous NK cell-based immunotherapies for the treatment of prostate cancer. However, our findings also indicate that patients will need to be stratified according to their potential responsiveness to individual therapeutic approaches.

Immunohistochemical and Flow Cytometric Analysis of Intracellular and Membrane-Bound Hsp70, as a Putative Biomarker of Glioblastoma Multiforme, Using the cmHsp70.1 Monoclonal Antibody.

The major stress-inducible 70 kDa heat shock (stress) protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumor cells and thus might serve as a tumor-specific biomarker of aggressive disease. We have previously shown that, in contrast to normal cells, tumor cells present Hsp70 on their plasma membrane. In order to elucidate the role of intracellular and membrane-bound Hsp70 as a potential tumor biomarker in glioblastoma multiforme, herein, we describe protocols for the staining of cytosolic Hsp70 in tumor formalin fixed paraffin-embedded (FFPE) sections using immunohistochemistry, and for plasma membrane-bound Hsp70 by multi-parametric flow cytometry using the cmHsp70.1 monoclonal antibody (mAb).

Identifying the Presence of Prostate Cancer in Individuals with PSA Levels <20 ng ml-1 Using Computational Data Extraction Analysis of High Dimensional Peripheral Blood Flow Cytometric Phenotyping Data.

Determining whether an asymptomatic individual with Prostate-Specific Antigen (PSA) levels below 20 ng ml-1 has prostate cancer in the absence of definitive, biopsy-based evidence continues to present a significant challenge to clinicians who must decide whether such individuals with low PSA values have prostate cancer. Herein, we present an advanced computational data extraction approach which can identify the presence of prostate cancer in men with PSA levels <20 ng ml-1 on the basis of peripheral blood immune cell profiles that have been generated using multi-parameter flow cytometry. Statistical analysis of immune phenotyping datasets relating to the presence and prevalence of key leukocyte populations in the peripheral blood, as generated from individuals undergoing routine tests for prostate cancer (including tissue biopsy) using multi-parametric flow cytometric analysis, was unable to identify significant relationships between leukocyte population profiles and the presence of benign disease (no prostate cancer) or prostate cancer. By contrast, a Genetic Algorithm computational approach identified a subset of five flow cytometry features (CD8+CD45RA-CD27-CD28- (CD8+ Effector Memory cells); CD4+CD45RA-CD27-CD28- (CD4+ Terminally Differentiated Effector Memory Cells re-expressing CD45RA); CD3-CD19+ (B cells); CD3+CD56+CD8+CD4+ (NKT cells)) from a set of twenty features, which could potentially discriminate between benign disease and prostate cancer. These features were used to construct a prostate cancer prediction model using the k-Nearest-Neighbor classification algorithm. The proposed model, which takes as input the set of flow cytometry features, outperformed the predictive model which takes PSA values as input. Specifically, the flow cytometry-based model achieved Accuracy = 83.33%, AUC = 83.40%, and optimal ROC points of FPR = 16.13%, TPR = 82.93%, whereas the PSA-based model achieved Accuracy = 77.78%, AUC = 76.95%, and optimal ROC points of FPR = 29.03%, TPR = 82.93%. Combining PSA and flow cytometry predictors achieved Accuracy = 79.17%, AUC = 78.17% and optimal ROC points of FPR = 29.03%, TPR = 85.37%. The results demonstrate the value of computational intelligence-based approaches for interrogating immunophenotyping datasets and that combining peripheral blood phenotypic profiling with PSA levels improves diagnostic accuracy compared to using PSA test alone. These studies also demonstrate that the presence of cancer is reflected in changes in the peripheral blood immune phenotype profile which can be identified using computational analysis and interpretation of complex flow cytometry datasets.

Immune Cell Phenotyping Using Flow Cytometry.

Fluorescent immunophenotyping uses fluorescently-conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single-cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single-cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser-based technology which analyzes the physical and fluorescent properties of cells in suspension in real-time as they flow through the instrument. This approach has a number of advantages over other techniques that can be used for characterizing cell populations in single-cell suspensions, in that it can nonsubjectively interrogate up to millions of cells and acquire data on the presence of different cell subpopulations and phenotypical changes within these populations in seconds. This unit describes basic procedures for the direct and indirect immunofluorescent staining of surface and intracellular proteins that are expressed by lymphoid cells which have been isolated from tissues or blood. Protocols for the resolution of dead cells and for the fixation of cells are also included. This unit also provides essential information relating to the selection and titration of antibodies, fluorochrome choice, spectral overlap and compensation, the use of controls, and the standardization of data acquisition and analysis. It also highlights new technologies and platforms that can be used to interrogate the presence of cell subpopulations and their phenotype to an even greater depth.

Tumor imaging and targeting potential of an Hsp70-derived 14-mer peptide.

BACKGROUND: We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro. METHODOLOGY/PRINCIPAL FINDINGS: The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.