137 research outputs found
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Membrane Hsp70 â 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)âan essential element of the metastatic processâis 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 âcaptureâ of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)âcmHsp70.1âcan 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
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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
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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 sameâday antiâinflammatory response which may affect lowâgrade chronic inflammation and insulin resistance and benefit cardioâmetabolic health. The antiâinflammatory responses to intermittent gamesâbased exercise and to exercise in young people beyond 2âh postâexercise are unknown. Thus, the purpose of the present study is to examine the antiâinflammatory, glycemic and insulinemic response to gamesâbased activity in adolescents. Following ethical approval and familiarization, 39 adolescents (12.3±0.7 y) completed an exercise (E) and rested (R) trial in a counterbalanced, randomized crossover design. Following a standardized breakfast, participants completed 1âh gamesâbased activity. Capillary blood samples were taken at baseline, immediately and 1âh postâexercise and 30, 60 and 120âmin following a standardized lunch. A final blood sample was taken the next morning. Data were analyzed using repeated measures ANOVA. ILâ6 concentration was higher on day one of the exercise trial (E:3.4±0.4, R:2.7±0.4 pg.mLâ1; p=0.006), as was the antiâinflammatory ILâ6:TNFâα ratio (E:5.53±0.93, R:3.75±0.45; p=0.027). Levels of the antiâinflammatory cytokine ILâ10 increased on day two of the exercise trial (E:2.11±0.23, R:1.66±0.16 pg.mLâ1; 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.Lâ1x120 min; p<0.001). Gamesâbased activity stimulated an antiâinflammatory response up to 24 h postâexercise and improved insulin sensitivity in response to a standardized meal in healthy adolescents. These novel findings suggest that gamesâbased activity is an ecologically valid mode of exercise to elicit beneficial effects on cardioâmetabolic risk factors in young people
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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 âdetectionâ 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 âriskâ model combined the immunophenotypic features with PSA to predict whether a patient with PCa has high-risk disease (defined by the DâAmico 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
The Effect Of Breastfeeding On Child Development At 5 Years: A Cohort Study
Objective It is uncertain to what degree the relationship between breastfeeding and later cognitive development is a true biological effect, or is confounded by psychosocial factors. The study aim was to further investigate this relationship and the effect of duration of breast feeding on cognitive development. Methods A total of 3880 children were followed from birth. Breastfeeding duration was measured by questionnaire at 6 months of age and a Peabody Picture Vocabulary Test Revised (PPVT-R) was administered at 5 years. PPVT-R scores were adjusted for the effects of a large array of biological and psychosocial confounders. The relationship between breastfeeding and the mean PPVT-R scores were examined using analysis of variance and multiple linear regression. Results A strong positive relationship was demonstrated between breastfeeding and the PPVT-R scores with increasing scores with increased duration of breastfeeding. After adjusting for a wide range of biological and social factors, the adjusted mean for those breastfed for 6 months or more was 8.2 points higher for females and 5.8 points for males when compared to those never breastfed. Conclusion These findings suggest a significant benefit to child development is conferred by breastfeeding and is related independently to longer periods of breastfeeding
Analysis of Mutations in AARS2 in a Series of CSF1R-Negative Patients With Adult-Onset Leukoencephalopathy With Axonal Spheroids and Pigmented Glia
IMPORTANCE:
Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a frequent cause of adult-onset leukodystrophy known to be caused by autosomal dominant mutations in the CSF1R (colony-stimulating factor 1) gene. The discovery that CSF1R mutations cause ALSP led to more accurate prognosis and genetic counseling for these patients in addition to increased interest in microglia as a target in neurodegeneration. However, it has been known since the discovery of the CSF1R gene that there are patients with typical clinical and radiologic evidence of ALSP who do not carry pathogenic CSF1R mutations. These patients include those in whom the pathognomonic features of axonal spheroids and pigmented microglia have been found. Achieving a genetic diagnosis in these patients is important to our understanding of this disorder.
OBJECTIVE:
To genetically characterize a group of patients with typical features of ALSP who do not carry CSF1R mutations.
DESIGN, SETTING, AND PARTICIPANTS:
In this case series study, 5 patients from 4 families were identified with clinical, radiologic, or pathologic features of ALSP in whom CSF1R mutations had been excluded previously by sequencing. Data were collected between May 2014 and September 2015 and analyzed between September 2015 and February 2016.
MAIN OUTCOMES AND MEASURES:
Focused exome sequencing was used to identify candidate variants. Family studies, long-range polymerase chain reaction with cloning, and complementary DNA sequencing were used to confirm pathogenicity.
RESULTS:
Of these 5 patients, 4 were men (80%); mean age at onset of ALSP was 29 years (range, 15-44 years). Biallelic mutations in the alanyl-transfer (t)RNA synthetase 2 (AARS2) gene were found in all 5 patients. Frameshifting and splice site mutations were common, found in 4 of 5 patients, and sequencing of complementary DNA from affected patients confirmed that the variants were loss of function. All patients presented in adulthood with prominent cognitive, neuropsychiatric, and upper motor neuron signs. Magnetic resonance imaging in all patients demonstrated a symmetric leukoencephalopathy with punctate regions of restricted diffusion, typical of ALSP. In 1 patient, brain biopsy demonstrated axonal spheroids and pigmented microglia, which are the pathognomonic signs of ALSP.
CONCLUSIONS AND RELEVANCE:
This work indicates that mutations in the tRNA synthetase AARS2 gene cause a recessive form of ALSP. The CSF1R and AARS2 proteins have different cellular functions but overlap in a final common pathway of neurodegeneration. This work points to novel targets for research and will lead to improved diagnostic rates in patients with adult-onset leukoencephalopathy
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 Ă 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-ÎșB 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 (less than 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
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)âÎłârelated 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-Îłâdominant AML subtypes
Autologous stem cell transplantation with low-dose cyclophosphamide to improve mucosal healing in adults with refractory Crohn's disease: the ASTIClite RCT
Some text in this abstract has been reproduced from Lindsay J, Din S, Hawkey C, Hind D, Irving P, Lobo A, et al. OFR-9 An RCT of autologous stem-cell transplantation in treatment refractory Crohnâs disease (low-intensity therapy evaluation): ASTIClite. Gut 2021;70(Suppl. 4):A4.
Background
Treatment-refractory Crohnâs disease is characterised by chronic symptoms, poor quality of life and high costs to the NHS, and through days of work lost by patients. A previous trial of autologous haematopoietic stem cell transplant (HSCT) failed its end point of medication-free clinical remission for 3 months with no evidence of disease activity, and reported high toxicity. Subsequent studies suggest that HSCT achieves complete mucosal healing in 50% of patients, and that toxicity likely relates to the cyclophosphamide dose.
Objectives
The primary objective was to assess the efficacy of HSCTlite (HSCT with low-dose cyclophosphamide) compared with standard care for inducing regression of intestinal ulceration in patients with refractory Crohnâs disease at week 48. Secondary objectives included the assessment of disease activity, quality of life and regimen safety. Mechanistic objectives included immune reconstitution after HSCTlite.
Design
Two-arm, parallel-group randomised controlled trial with a 2â:â1 (interventionâ:âcontrol) allocation ratio.
Setting
Nine NHS trusts (eight trusts were recruitment sites; one trust was a treatment-only site).
Participants
Adults with treatment-refractory Crohnâs disease, for whom surgery was inappropriate or who had declined surgery.
Interventions
The intervention treatment was HSCTlite using cyclophosphamide, and the control was any current available treatment for Crohnâs disease, apart from stem cell transplantation.
Main outcomes
The primary outcome was treatment success at week 48 [mucosal healing (Simple Endoscopic Score for Crohnâs Disease ulcer subscore of 0) without surgery or death], assessed by central readers blinded to allocation and timing of assessment. Key secondary outcomes were clinical remission, Simple Endoscopic Score for Crohnâs Disease scores at week 48, change in Crohnâs Disease Activity Index scores and safety.
Results
The trial was halted owing to Suspected unexpected serious adverse events that took place after randomising 23 patients (HSCTlite arm, nâ=â13; usual-care arm, nâ=â10). Ten out of the 13 patients randomised to the HSCTlite arm received the intervention and nine (one death) reached the 48-week follow-up. In the usual-care arm 9 out of the 10 patients randomised reached the 48-week follow-up (one ineligible). The primary outcome was available for 7 out of 10 HSCTlite patients (including the patient who died) and six out of nine usual-care patients. Absence of endoscopic ulceration without surgery or death was reported in three out of seven (43%) HSCTlite patients, compared with zero out of six (0%) usual-care patients. Centrally read Simple Endoscopic Score for Crohnâs Disease scores [mean (standard deviation)] were 10.8 (6.3) and 10.0 (6.1) at baseline, compared with 2.8 (2.9) and 18.7 (9.1) at week 48, in the HSCT and usual-care arms, respectively. Clinical remission (Crohnâs Disease Activity Index scores of <â150) occurred in 57% and 17% of patients in the HSCTlite and usual-care arms, respectively, at week 48. Serious adverse events were more frequent in the HSCTlite arm [38 in 13 (100%) patients] than in the usual-care arm [16 in 4 (40%) patients]. Nine suspected unexpected serious adverse reactions were reported in six HSCTlite patients, including three cases of delayed renal failure due to proven thrombotic microangiopathy. Two HSCTlite patients died.
Conclusions
Within the limitations of reduced patient recruitment and numbers of patients assessed, HSCTlite meaningfully reduced endoscopic disease activity, with three patients experiencing resolution of ulceration. Suspected unexpected serious adverse reactions, particularly relating to thrombotic microangiopathy, make this regimen unsuitable for future clinical use.
Limitations
The early trial closure prevented complete recruitment, and the impact of the coronavirus pandemic prevented completion of some study investigations. Small participant numbers meant analysis could only be descriptive.
Future work
Owing to undetermined aetiology of thrombotic microangiopathy, further trials of HSCTlite in this population are not considered appropriate. Priorities should be to determine optimal treatment strategies for patients with refractory Crohnâs disease, including those with a stoma or multiple previous resections
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