Deep learning-based automatic pipeline for quantitative assessment of thigh muscle morphology and fatty infiltration.

PURPOSE: Fast and accurate thigh muscle segmentation from MRI is important for quantitative assessment of thigh muscle morphology and composition. A novel deep learning (DL) based thigh muscle and surrounding tissues segmentation model was developed for fully automatic and reproducible cross-sectional area (CSA) and fat fraction (FF) quantification and tested in patients at 10 years after anterior cruciate ligament reconstructions. METHODS: A DL model combining UNet and DenseNet was trained and tested using manually segmented thighs from 16 patients (32 legs). Segmentation accuracy was evaluated using Dice similarity coefficients (DSC) and average symmetric surface distance (ASSD). A UNet model was trained for comparison. These segmentations were used to obtain CSA and FF quantification. Reproducibility of CSA and FF quantification was tested with scan and rescan of six healthy subjects. RESULTS: The proposed UNet and DenseNet had high agreement with manual segmentation (DSC >0.97, ASSD < 0.24) and improved performance compared with UNet. For hamstrings of the operated knee, the automated pipeline had largest absolute difference of 6.01% for CSA and 0.47% for FF as compared to manual segmentation. In reproducibility analysis, the average difference (absolute) in CSA quantification between scan and rescan was better for the automatic method as compared with manual segmentation (2.27% vs. 3.34%), whereas the average difference (absolute) in FF quantification were similar. CONCLUSIONS: The proposed method exhibits excellent accuracy and reproducibility in CSA and FF quantification compared with manual segmentation and can be used in large-scale patient studies.

Expansion of Lymphocytes from Prostatic Adenocarcinoma and Adjacent Nonmalignant Tissue.

Background: The evaluation of tumour-infiltrating lymphocytes (TILs) in solid malignancies has yielded insights into immune regulation within the tumour microenvironment and has also led to the development and optimisation of adoptive T cell therapies. Objectives: This study examined the in vitro expansion of TILs from prostate adenocarcinoma, as a preliminary step to evaluate the potential of TILs for adoptive T cell therapy. Design, Setting, and Participants. Malignant and adjacent nonmalignant tissues were obtained from fifteen men undergoing radical prostatectomy. Interventions. There were no study interventions. Outcome Measurements and Statistical Analysis. Expanded cells were analysed by flow cytometry, and the data was assessed for associations between cell subpopulations and expansion rate. Results: Tumour-infiltrating lymphocytes could be expanded to numbers that would be needed to generate a therapeutic infusion product from nine of 15 malignant specimens (60%). The CD4+ T cells predominated over CD8+ T cells (median 56.8% CD4+, 30.0% CD8+), and furthermore, faster TIL expansion was associated with a higher proportion of CD4+ T cells (median 69.8% in faster-growing cultures; 36.8% in slower-growing cultures). A higher proportion of CD3-CD56+ cells versus CD3+ cells was associated with slower TIL expansion in cultures from malignant specimens (median 13.3% in slower-growing cultures versus 2.05% in faster-growing cultures), but not from nonmalignant specimens. Conclusions: The expansion of TILs for potential therapeutic use is feasible. Our findings also indicate that further examination of TILs from prostate adenocarcinomas may yield insights into mechanisms of regulation of T cells within the tumour microenvironment. Further research is required to evaluate their therapeutic potential.

Phase II clinical trial of adoptive cell therapy for patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and low-dose interleukin-2.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown significant clinical benefit, but is limited by toxicities due to a requirement for post-infusion interleukin-2 (IL-2), for which high dose is standard. To assess a modified TIL protocol using lower dose IL-2, we performed a single institution phase II protocol in unresectable, metastatic melanoma. The primary endpoint was response rate. Secondary endpoints were safety and assessment of immune correlates following TIL infusion. Twelve metastatic melanoma patients were treated with non-myeloablative lymphodepleting chemotherapy, TIL, and low-dose subcutaneous IL-2 (125,000 IU/kg/day, maximum 9-10 doses over 2 weeks). All but one patient had previously progressed after treatment with immune checkpoint inhibitors. No unexpected adverse events were observed, and patients received an average of 6.8 doses of IL-2. By RECIST v1.1, two patients experienced a partial response, one patient had an unconfirmed partial response, and six had stable disease. Biomarker assessment confirmed an increase in IL-15 levels following lymphodepleting chemotherapy as expected and a lack of peripheral regulatory T-cell expansion following protocol treatment. Interrogation of the TIL infusion product and monitoring of the peripheral blood following infusion suggested engraftment of TIL. In one responding patient, a population of T cells expressing a T-cell receptor Vß chain that was dominant in the infusion product was present at a high percentage in peripheral blood more than 2 years after TIL infusion. This study shows that this protocol of low-dose IL-2 following adoptive cell transfer of TIL is feasible and clinically active. (ClinicalTrials.gov identifier NCT01883323.).

Neoadjuvant Chemotherapy of Ovarian Cancer Results in Three Patterns of Tumor-Infiltrating Lymphocyte Response with Distinct Implications for Immunotherapy.

Purpose: Some forms of chemotherapy can enhance antitumor immunity through immunogenic cell death, resulting in increased T-cell activation and tumor infiltration. Such effects could potentially sensitize tumors to immunotherapies, including checkpoint blockade. We investigated whether platinum- and taxane-based chemotherapy for ovarian cancer induces immunologic changes consistent with this possibility.Experimental Design: Matched pre- and post-neoadjuvant chemotherapy tumor samples from 26 high-grade serous carcinoma (HGSC) patients were analyzed by immunohistochemistry (IHC) for a large panel of immune cells and associated factors. The prognostic significance of post-chemotherapy TIL patterns was assessed in an expanded cohort (n = 90).Results: Neoadjuvant chemotherapy was associated with increased densities of CD3+, CD8+, CD8+ TIA-1+, PD-1+ and CD20+ TIL. Other immune subsets and factors were unchanged, including CD79a+ CD138+ plasma cells, CD68+ macrophages, and MHC class I on tumor cells. Immunosuppressive cell types were also unchanged, including FoxP3+ PD-1+ cells (putative regulatory T cells), IDO-1+ cells, and PD-L1+ cells (both macrophages and tumor cells). Hierarchical clustering revealed three response patterns: (i) TILhigh tumors showed increases in multiple immune markers after chemotherapy; (ii) TILlow tumors underwent similar increases, achieving patterns indistinguishable from the first group; and (iii) TILnegative cases generally remained negative. Despite the dramatic increases seen in the first two patterns, post-chemotherapy TIL showed limited prognostic significance.Conclusions: Chemotherapy augments pre-existing TIL responses but fails to relieve major immune-suppressive mechanisms or confer significant prognostic benefit. Our findings provide rationale for multipronged approaches to immunotherapy tailored to the baseline features of the tumor microenvironment. Clin Cancer Res; 23(4); 925-34. ©2016 AACR.

Selective loss of bi-directional synaptic plasticity in the direct and indirect striatal output pathways accompanies generation of parkinsonism and l-DOPA induced dyskinesia in mouse models.

Parkinsonian symptoms arise due to over-activity of the indirect striatal output pathway, and under-activity of the direct striatal output pathway. l-DOPA-induced dyskinesia (LID) is caused when the opposite circuitry problems are established, with the indirect pathway becoming underactive, and the direct pathway becoming over-active. Here, we define synaptic plasticity abnormalities in these pathways associated with parkinsonism, symptomatic benefits of l-DOPA, and LID. We applied spike-timing dependent plasticity protocols to cortico-striatal synapses in slices from 6-OHDA-lesioned mouse models of parkinsonism and LID, generated in BAC transgenic mice with eGFP targeting the direct or indirect output pathways, with and without l-DOPA present. In naïve mice, bidirectional synaptic plasticity, i.e. LTP and LTD, was induced, resulting in an EPSP amplitude change of approximately 50% in each direction in both striatal output pathways, as shown previously. In parkinsonism and dyskinesia, both pathways exhibited unidirectional plasticity, irrespective of stimulation paradigm. In parkinsonian animals, the indirect pathway only exhibited LTP (LTP protocol: 143.5±14.6%; LTD protocol 177.7±22.3% of baseline), whereas the direct pathway only showed LTD (LTP protocol: 74.3±4.0% and LTD protocol: 63.3±8.7%). A symptomatic dose of l-DOPA restored bidirectional plasticity on both pathways to levels comparable to naïve animals (Indirect pathway: LTP protocol: 124.4±22.0% and LTD protocol: 52.1±18.5% of baseline. Direct pathway: LTP protocol: 140.7±7.3% and LTD protocol: 58.4±6.0% of baseline). In dyskinesia, in the presence of l-DOPA, the indirect pathway exhibited only LTD (LTP protocol: 68.9±21.3% and LTD protocol 52.0±14.2% of baseline), whereas in the direct pathway, only LTP could be induced (LTP protocol: 156.6±13.2% and LTD protocol 166.7±15.8% of baseline). We conclude that normal motor control requires bidirectional plasticity of both striatal outputs, which underlies the symptomatic benefits of l-DOPA. Switching from bidirectional to unidirectional plasticity drives global changes in striatal pathway excitability, and underpins parkinsonism and dyskinesia.

Generation of a model of L-DOPA-induced dyskinesia in two different mouse strains.

Prolonged use of the dopamine precursor L-DOPA for the treatment of Parkinson's disease commonly results in abnormal involuntary movements, which are termed L-DOPA-induced dyskinesia (LID). Over-activity at corticostriatal synapses onto neurons of the direct and indirect striatal output pathways has been implicated in the development of dyskinesia, but it has proved difficult to investigate the pathways separately due to their morphological similarities. The recent development of bacterial artificial chromosome mice that express green fluorescent protein in either the direct or indirect pathway allows visual identification of the output neurons in each pathway. Here we describe the use of two different strains of these transgenic mice (pure FVB and FVB crossed with C57BL6) in the development of mouse models of L-DOPA-induced dyskinesia. This model will allow the direct and indirect pathways to be studied individually to delineate the cellular and molecular mechanisms underlying dyskinesias. These studies demonstrate that mouse strain impacts on behavioural responses and L-DOPA sensitivity. Therefore, when generating mouse models of LID, strain must be taken into consideration when choosing the L-DOPA dosing regimen.

Altered function of glutamatergic cortico-striatal synapses causes output pathway abnormalities in a chronic model of parkinsonism.

OBJECTIVE: In Parkinson's disease, chronic striatal dopamine depletion results in over-activity and under-activity of the indirect and direct striatal output pathways respectively. In this study, we investigated changes in the function of glutamatergic cortico-striatal synapses that contribute to abnormalities in striatal efferents. METHODS: Whole-cell recordings were performed in striatal slices prepared from adult bacterial artificial chromosome mice, chronically lesioned with 6-hydroxydopamine (6-OHDA). Paired pulse facilitation, spontaneous synaptic activity, the ratio of AMPAR to NMDAR-mediated components of excitatory postsynaptic currents, AMPAR and NMDAR kinetics, current-voltage relationship and intrinsic membrane properties were assessed in indirect and direct pathway medium spiny neurons (MSNs), which were identified on the basis of expression of GFP, driven by the promoters of A2A or D1 receptor expression. The trajectory of striatal efferents, with respect to selective targeting of the globus pallidus and substantia nigra was also compared in sham-operated versus 6-OHDA-lesioned mice. RESULTS: Dopamine depletion did not affect the number of pathway specific output neurons or the trajectory of striatal outputs. In sham-operated animals, cortico-striatal synapses of both striatal efferent populations exhibited paired pulse facilitation and similar ratios of AMPAR to NMDAR-mediated components of excitatory postsynaptic currents. Following striatal dopamine depletion, indirect pathway neurons exhibited decreased levels of paired pulse facilitation, enhanced sensitivity to presynaptic stimulation and an increase in the relative contribution of NMDAR to the EPSC but no change in spontaneous synaptic activity. In sham-operated mice, neurons of the direct pathway exhibited lower firing frequency compared to the indirect pathway following current injection. However, in 6-OHDA-lesioned mice, in the direct pathway, firing threshold was reduced, spike frequency adaptation developed and the frequency of spontaneous activity was also reduced. In addition, changes in the properties of NMDAR kinetics suggest that these receptors were desensitised. DISCUSSION: Increased synchronicity between pre and postsynaptic neurons, as indicated by decreased paired pulse facilitation, and increased sensitivity to extracellular stimulation, combined with an increase in the contribution of NMDARs to the EPSC at cortico-striatal synapses, may contribute to the over-activity of indirect pathway neurons in the parkinsonian striatum. In contrast, a decrease in spontaneous activity, postsynaptic desensitisation to excitatory stimuli and spike frequency adaptation of cortico-striatal synapses may underlie under-activity of the direct pathway.

Psychometric testing of the Chinese quality of recovery score.

BACKGROUND: We evaluated a Chinese translation of the quality of recovery (QoR) score for measuring health status in patients after surgery and anesthesia. METHODS: The Chinese QoR score was developed by a panel of linguistic experts using a series of forward and backward translations. We then compared the psychometric performance of the Chinese QoR score with the original English version in bilingual Chinese patients undergoing a variety of surgeries. Using a crossover design, 210 patients were randomly assigned to complete both versions of the QoR score, 1 h apart, in the morning after surgery according to one of the two sequences: Chinese followed by the English QoR score or the reverse order. Test-retest and interrater reliabilities were calculated by comparing scores administered 6-8 h later and those completed by the patients and duty nurses, respectively. RESULTS: Patient responses to the Chinese QoR score agreed well with the English version (weighted kappa statistic, kappaw=0.92). Both QoR scores correlated with patient satisfaction scores (Chinese version, rho=0.82; English version, rho=0.79) confirming convergent validity. There was also significant negative correlation between the QoR scores and female gender, duration of hospital stay, or magnitude of surgery (discriminant construct validity). We found the Chinese QoR score has good internal consistency (Cronbach's rho=0.91), interobserver (kappaw=0.77), and test-retest reliability (kappaw=0.83). These values were similar to those of the original English version (Cronbach's rho=0.89, interobserver kappaw=0.84, test-retest reliability kappaw=0.88). CONCLUSION: The Chinese QoR score is conceptually, semantically, and operationally equivalent to the English version. Both scales provide valid, reliable, and responsive assessment of the QoR after surgery and anesthesia.