A phase Ib trial of pembrolizumab plus paclitaxel or flat-dose capecitabine in 1st/2nd line metastatic triple-negative breast cancer.

Chemoimmunotherapy with anti-programmed cell death 1/ligand 1 and cytotoxic chemotherapy is a promising therapeutic modality for women with triple-negative breast cancer, but questions remain regarding optimal chemotherapy backbone and biomarkers for patient selection. We report final outcomes from a phase Ib trial evaluating pembrolizumab (200 mg IV every 3 weeks) with either weekly paclitaxel (80 mg/m2 weekly) or flat-dose capecitabine (2000 mg orally twice daily for 7 days of every 14-day cycle) in the 1st/2nd line setting. The primary endpoint is safety (receipt of 2 cycles without grade III/IV toxicities requiring discontinuation or ≥21-day delays). The secondary endpoint is efficacy (week 12 objective response). Exploratory aims are to characterize immunologic effects of treatment over time, and to evaluate novel biomarkers. The trial demonstrates that both regimens meet the pre-specified safety endpoint (paclitaxel: 87%; capecitabine: 100%). Objective response rate is 29% for pembrolizumab/paclitaxel (n = 4/13, 95% CI: 10-61%) and 43% for pembrolizumab/capecitabine (n = 6/14, 95% CI: 18-71%). Partial responses are observed in two subjects with chemo-refractory metaplastic carcinoma (both in capecitabine arm). Both regimens are associated with significant peripheral leukocyte contraction over time. Response is associated with clinical PD-L1 score, non-receipt of prior chemotherapy, and the H&E stromal tumor-infiltrating lymphocyte score, but also by a novel 27 gene IO score and spatial biomarkers (lymphocyte spatial skewness). In conclusion, pembrolizumab with paclitaxel or capecitabine is safe and clinically active. Both regimens are lymphodepleting, highlighting the competing immunostimulatory versus lymphotoxic effects of cytotoxic chemotherapy. Further exploration of the IO score and spatial TIL biomarkers is warranted. The clinical trial registration is NCT02734290.

Paediatric adverse event rating scale: a measure of safety or efficacy? Novel analysis from the MADDY study.

OBJECTIVE: The Pediatric Adverse Event Rating Scale (PAERS) measured adverse events of children aged 6-12 years with ADHD and emotional dysregulation in the Micronutrients for ADHD in Youth (MADDY) study, an eight week multi-site randomized clinical trial of a broad-spectrum multinutrient treatment. Treatment sensitivity of the PAERS was assessed by calculating the treatment difference in change of the item scores from baseline to end of the RCT. METHODS: Principal component analysis retained 14 "adverse events" (out of 43 in the PAERS) that reflected ADHD symptoms and emotional dysregulation and was used to group the variables of interest. A combined score ranging from 0 to 5 was created based on symptom presence, functional impairment, and severity. Mean score change was calculated from baseline to week 8 by treatment (multinutrient vs placebo) with intention-to-treat and per-protocol samples. The study has been registered on clinicaltrials.gov as Micronutrients for ADHD in Youth (MADDY) Study, trial registration # NCT03252522 (https://clinicaltrials.gov/ct2/show/NCT03252522). RESULTS: The 126 children in the ITT sample had a mean age of 9.8 (SD = 1.7), with majority (73%) male, and 72% diagnosed with ADHD prior to the study screening. Baseline presence of PAERS symptoms was similar between treatment groups: the highest proportion was ADHD symptoms, followed by Irritable symptoms. The micronutrient group showed a greater decrease (improvement) in the mean anxiety combined score than the placebo group with a between-group difference in change of -0.36 (95% CI: -0.67, -0.04; p = .03) with ITT data and -0.48 (95% CI: -0.81, -0.15; p = .005) with per-protocol (n = 93) data. CONCLUSION: The multinutrient supplement did not result in more adverse events than placebo, suggesting it is a safe intervention. In addition to assessing actual adverse events, the PAERS may be a useful adjunct outcome measure for ADHD behaviors.

Changes in T-cell subsets and clonal repertoire during chemoimmunotherapy with pembrolizumab and paclitaxel or capecitabine for metastatic triple-negative breast cancer.

BACKGROUND: Chemoimmunotherapy is a standard treatment for triple-negative breast cancer (TNBC), however, the impacts of different chemotherapies on T-cell populations, which could correlate with clinical activity, are not known. Quantifying T-cell populations with flow cytometry and T-cell receptor (TCR) immunosequencing may improve our understanding of how chemoimmunotherapy affects T-cell subsets, and to what extent clonal shifts occur during treatment. TCR immunosequencing of intratumoral T cells may facilitate the identification and monitoring of putatively tumor-reactive T-cell clones within the blood. METHODS: Blood and tumor biopsies were collected from patients with metastatic TNBC enrolled in a phase Ib clinical trial of first or second-line pembrolizumab with paclitaxel or capecitabine. Using identical biospecimen processing protocols, blood samples from a cohort of patients treated for early-stage breast cancer were obtained for comparison. Treatment-related immunological changes in peripheral blood and intratumoral T cells were characterized using flow cytometry and TCR immunosequencing. Clonal proliferation rates of T cells were compared based on intratumoral enrichment. RESULTS: When combined with pembrolizumab, paclitaxel and capecitabine resulted in similar time-dependent lymphodepletions across measured peripheral T-cell subsets. Their effects were more modest than that observed following curative-intent dose-dense anthracycline and cyclophosphamide (ddAC) (average fold-change in CD3+ cells, capecitabine: -0.42, paclitaxel: -0.56, ddAC: -1.21). No differences in T-cell clonality or richness were observed following capecitabine or paclitaxel-based treatments. Regression modeling identified differences in the emergence of novel T-cell clones that were not detected at baseline (odds compared with ddAC, capecitabine: 0.292, paclitaxel: 0.652). Pembrolizumab with paclitaxel or capecitabine expanded T-cell clones within tumors; however, these clones did not always expand within the blood. Proliferation rates within the blood were similar between clones that were enriched and those that were not enriched within tumors. CONCLUSION: Chemoimmunotherapy for metastatic TNBC with pembrolizumab and capecitabine or paclitaxel resulted in similar peripheral T-cell subset lymphodepletion without altering T-cell clonal diversity. Regression modeling methods are applicable in immune monitoring studies, such as this to identify the odds of novel T-cell clones emerging during treatment, and proliferation rates of tumor-enriched T-cell clones.

A Phase Ib Study of Preoperative, Locoregional IRX-2 Cytokine Immunotherapy to Prime Immune Responses in Patients with Early-Stage Breast Cancer.

PURPOSE: To evaluate the safety and feasibility of preoperative locoregional cytokine therapy (IRX-2 regimen) in early-stage breast cancer, and to evaluate for intratumoral and peripheral immunomodulatory activity. PATIENTS AND METHODS: Sixteen patients with stage I-III early-stage breast cancer (any histology type) indicated for surgical lumpectomy or mastectomy were enrolled to receive preoperative locoregional immunotherapy with the IRX-2 cytokine biological (2 mL subcutaneous × 10 days to periareolar skin). The regimen also included single-dose cyclophosphamide (300 mg/m2) on day 1 to deplete T-regulatory cells and oral indomethacin to modulate suppressive myeloid subpopulations. The primary objective was to evaluate feasibility (i.e., receipt of therapy without surgical delays or grade 3/4 treatment-related adverse events). The secondary objective was to evaluate changes in stromal tumor-infiltrating lymphocyte score. The exploratory objective was to identify candidate pharmacodynamic changes for future study using a variety of assays, including flow cytometry, RNA and T-cell receptor DNA sequencing, and multispectral immunofluorescence. RESULTS: Preoperative locoregional cytokine administration was feasible in 100% (n = 16/16) of subjects and associated with increases in stromal tumor-infiltrating lymphocytes (P < 0.001). Programmed death ligand 1 (CD274) was upregulated at the RNA (P < 0.01) and protein level [by Ventana PD-L1 (SP142) and immunofluorescence]. Other immunomodulatory effects included upregulation of RNA signatures of T-cell activation and recruitment and cyclophosphamide-related peripheral T-regulatory cell depletion. CONCLUSIONS: IRX-2 is safe in early-stage breast cancer. Potentially favorable immunomodulatory changes were observed, supporting further study of IRX-2 in early-stage breast cancer and other malignancies.

Implementation and Validation of an Automated Flow Cytometry Analysis Pipeline for Human Immune Profiling.

Automated reagent preparation, sample processing, and data acquisition have increased the rate at which flow cytometry data can be generated. Furthermore, advances in technology and flow cytometry instrumentation continually increase the complexity and dimensionality of this data. Together, this leads to increased pressure on manual data analysis, which has inherent limitations including subjectivity of the analyst and the length of time needed for data processing. These issues can create bottlenecks in the data processing workflow and potentially compromise data quality. To address these issues, as well as the challenges associated with manual gating in a high-volume human immune profiling laboratory, we sought to implement an automated analysis pipeline. In this report, we discuss considerations for selecting an automated analysis method, the process of implementing an automated pipeline, and detail our successful incorporation of an automated gating strategy with flowDensity into our analysis workflow. This validated pipeline augments our laboratory's ability to provide rapid high-throughput immune profiling for patients participating in cancer immunotherapy clinical trials. © International Society for Advancement of Cytometry.

CpG preconditioning regulates miRNA expression that modulates genomic reprogramming associated with neuroprotection against ischemic injury.

Cytosine-phosphate-guanine (CpG) preconditioning reprograms the genomic response to stroke to protect the brain against ischemic injury. The mechanisms underlying genomic reprogramming are incompletely understood. MicroRNAs (miRNAs) regulate gene expression; however, their role in modulating gene responses produced by CpG preconditioning is unknown. We evaluated brain miRNA expression in response to CpG preconditioning before and after stroke using microarray. Importantly, we have data from previous gene microarrays under the same conditions, which allowed integration of miRNA and gene expression data to specifically identify regulated miRNA gene targets. CpG preconditioning did not significantly alter miRNA expression before stroke, indicating that miRNA regulation is not critical for the initiation of preconditioning-induced neuroprotection. However, after stroke, differentially regulated miRNAs between CpG- and saline-treated animals associated with the upregulation of several neuroprotective genes, implicating these miRNAs in genomic reprogramming that increases neuroprotection. Statistical analysis revealed that the miRNA targets were enriched in the gene population regulated in the setting of stroke, implying that miRNAs likely orchestrate this gene expression. These data suggest that miRNAs regulate endogenous responses to stroke and that manipulation of these miRNAs may have the potential to acutely activate novel neuroprotective processes that reduce damage.

Poly-ICLC preconditioning protects the blood-brain barrier against ischemic injury in vitro through type I interferon signaling.

Preconditioning with a low dose of harmful stimulus prior to injury induces tolerance to a subsequent ischemic challenge resulting in neuroprotection against stroke. Experimental models of preconditioning primarily focus on neurons as the cellular target of cerebral protection, while less attention has been paid to the cerebrovascular compartment, whose role in the pathogenesis of ischemic brain injury is crucial. We have shown that preconditioning with polyinosinic polycytidylic acid (poly-ICLC) protects against cerebral ischemic damage. To delineate the mechanism of poly-ICLC protection, we investigated whether poly-ICLC preconditioning preserves the function of the blood-brain barrier (BBB) in response to ischemic injury. Using an in vitro BBB model, we found that poly-ICLC treatment prior to exposure to oxygen-glucose deprivation maintained the paracellular and transcellular transport across the endothelium and attenuated the drop in transendothelial electric resistance. We found that poly-ICLC treatment induced interferon (IFN) ß mRNA expression in astrocytes and microglia and that type I IFN signaling in brain microvascular endothelial cells was required for protection. Importantly, this implicates a potential mechanism underlying neuroprotection in our in vivo experimental stroke model, where type I IFN signaling is required for poly-ICLC-induced neuroprotection against ischemic injury. In conclusion, we are the first to show that preconditioning with poly-ICLC attenuates ischemia-induced BBB dysfunction. This mechanism is likely an important feature of poly-ICLC-mediated neuroprotection and highlights the therapeutic potential of targeting BBB signaling pathways to protect the brain against stroke.

Toll-like receptor 7 preconditioning induces robust neuroprotection against stroke by a novel type I interferon-mediated mechanism.

BACKGROUND AND PURPOSE: Systemic administration of Toll-like receptor (TLR) 4 and TLR9 agonists before cerebral ischemia have been shown to reduce ischemic injury by reprogramming the response of the brain to stroke. Our goal was to explore the mechanism of TLR-induced neuroprotection by determining whether a TLR7 agonist also protects against stroke injury. METHODS: C57Bl/6, TNF(-/-), interferon (IFN) regulatory factor 7(-/-), or type I IFN receptor (IFNAR)(-/-) mice were subcutaneously administered the TLR7 agonist Gardiquimod (GDQ) 72 hours before middle cerebral artery occlusion. Infarct volume and functional outcome were determined after reperfusion. Plasma cytokine responses and induction of mRNA for IFN-related genes in the brain were measured. IFNAR(-/-) mice also were treated with the TLR4 agonist (lipopolysaccharide) or the TLR9 agonist before middle cerebral artery occlusion and infarct volumes measured. RESULTS: The results show that GDQ reduces infarct volume as well as functional deficits in mice. GDQ pretreatment provided robust neuroprotection in TNF(-/-) mice, indicating that TNF was not essential. GDQ induced a significant increase in plasma IFNα levels and both IRF7(-/-) and IFNAR(-/-) mice failed to be protected, implicating a role for IFN signaling in TLR7-mediated protection. CONCLUSIONS: Our studies provide the first evidence that TLR7 preconditioning can mediate neuroprotection against ischemic injury. Moreover, we show that the mechanism of protection is unique from other TLR preconditioning ligands in that it is independent of TNF and dependent on IFNAR.

Inhibition of amiodarone-induced lung fibrosis but not alveolitis by angiotensin system antagonists.

Earlier work in this laboratory showed that amiodarone induces apoptosis in alveolar epithelial cells by a mechanism inhibitable by angiotensin system antagonists. A variety of recent studies suggests a critical role for alveolar epithelial cell apoptosis in the pathogenesis of lung fibrosis. On this basis we hypothesized that amiodarone-induced alveolar epithelial cell apoptosis and lung fibrosis in vivo might be inhibitable by the angiotensin converting enzyme inhibitor captopril or the angiotensin receptor antagonist losartan. Amiodarone-induced lung fibrosis was induced in male Wistar rats by oral administration over six months. Replicate groups of rats received captopril or losartan in addition to amiodarone. Apoptosis was detected by increased total lung activity of caspase 3 and in situ end labeling (ISEL) of fragmented DNA. Collagen was localized and quantitated by the picrosirius red technique. Alveolar epithelial cell apoptosis was detected in amiodarone-treated animals as early as three weeks after the start of amiodarone administration; by six months exposure, the incidence of alveolar epithelial cell apoptosis was significantly reduced by coadministration of captopril or losartan. Alveolar wall collagen accumulation also was significantly attenuated by captopril (100%) or losartan (74%), but neither agent blunted the accumulation of alveolar macrophages evoked by amiodarone (5.3-fold at 6 months). Lung neutrophil content was unchanged by amiodarone treatment for three weeks or six months. These results indicate that amiodarone induces alveolar epithelial cell apoptosis in vivo that is inhibitable by angiotensin antagonists. They also support the hypothesis that blockade of angiotensin formation or function attenuates amiodarone-induced lung fibrosis irrespective of the severity of alveolitis.

Apoptosis-dependent acute lung injury and repair after intratracheal instillation of noradrenaline in rats.

Earlier work in this laboratory showed that noradrenaline (NA) induces apoptosis in primary cultures of alveolar epithelial cells (AECs). Apoptosis of alveolar epithelial cells may promote the collapse of lung barrier function. On this basis we hypothesized that exogenous NA, administered by intratracheal (I.T.) instillation, might induce AEC apoptosis in vivo followed by acute lung injury. Delivery of NA (10 microM) I.T. into male Wistar rats increased labelling of both fragmented DNA, measured by in situ end labelling (ISEL), and the active form of caspase 3 (anti-Casp3) 6 and 20 h after administration (P < 0.05), but instillation of the vehicle alone (PBS) had no effect. Both ISEL and anti-Casp3 labelling were attenuated by concurrent I.T. delivery of the broad-spectrum caspase inhibitor ZVADfmk. After 6 h, most ISEL- and Casp3-positive cells were located in the surfaces of alveolar walls, but after 20 h more were found in alveolar spaces (P < 0.05). Instillation of NA also increased the bronchoalveolar lavage (BAL) content of fluorescent albumin (BODIPY-alb), which had previously been injected intravenously; the increase was reversed by concurrent ZVADfmk administration. These data suggest that NA-induced apoptosis of AECs in vivo is sufficient to invoke transient collapse of AEC barrier function that is rapidly repaired.

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo.

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.