Ozone Responsive Gene Expression as a Model for Describing Repeat Exposure Response Trajectories and Interindividual Toxicodynamic Variability In Vitro.

Inhaled chemical/material exposures are a ubiquitous part of daily life around the world. There is a need to evaluate potential adverse effects of both single and repeat exposures for thousands of chemicals and an exponentially larger number of exposure scenarios (eg, repeated exposures). Meeting this challenge will require the development and use of in vitro new approach methodologies (NAMs); however, 2 major challenges face the deployment of NAMs in risk assessment are (1) characterizing what apical outcome(s) acute assays inform regarding the trajectory to long-term events, especially under repeated exposure conditions, and (2) capturing interindividual variability as it informs considerations of potentially susceptible and/or vulnerable populations. To address these questions, we used a primary human bronchial epithelial cell air-liquid interface model exposed to ozone (O3), a model oxidant and ubiquitous environmental chemical. Here we report that O3-induced proinflammatory gene induction is attenuated in repeated exposures thus demonstrating that single acute exposure outcomes do not reliably represent the trajectory of responses after repeated or chronic exposures. Further, we observed 10.1-, 10.3-, 14.2-, and 7-fold ranges of induction of interleukin (IL)-8, IL-6, heme oxygenase 1, and cyclooxygenase 2 transcripts, respectively, within in our population of 25 unique donors. Calculation of sample size estimates that indicated that 27, 24, 299, and 13 donors would be required to significantly power similar in vitro studies to identify a 2-fold change in IL-8, IL-6, HMOX1, and cyclooxygenase 2 transcript induction, respectively, to inform considerations of the uncertainty factors to reflect variability within the human population for in vitro studies.

Ipilimumab, nivolumab, and brentuximab vedotin combination therapies in patients with relapsed or refractory Hodgkin lymphoma: phase 1 results of an open-label, multicentre, phase 1/2 trial.

BACKGROUND: Recognising that the immune suppressive microenvironment promotes tumour growth in Hodgkin lymphoma, we hypothesised that activating immunity might augment the activity of targeted chemotherapy. We evaluated the safety and activity of combinations of brentuximab vedotin with nivolumab or ipilimumab, or both in patients with relapsed or refractory Hodgkin lymphoma. METHODS: In this multicentre, open-label, phase 1/2 trial, patients with relapsed or refractory Hodgkin lymphoma aged 18 years or older who had relapsed after at least one line of therapy, with an Eastern Cooperative Oncology Group performance status of 2 or lower, and adequate organ and marrow function, with no pulmonary dysfunction were eligible for inclusion. Phase 1 primary objectives were to determine the maximum tolerated dose and dose limiting toxicities of brentuximab vedotin combined with ipilimumab (ipilimumab group), nivolumab (nivolumab group), or both (triplet therapy group) using a 3 + 3 dose escalation design with expansion cohorts. During the dose escalation phase, patients were enrolled sequentially into one of six cohorts: in the ipilimumab group fixed brentuximab vedotin 1·8 mg/kg with ipilimumab 1 mg/kg (cohort A) or 3 mg/kg (cohort B); in the nivolumab group fixed nivolumab 3 mg/kg with brentuximab vedotin 1·2 mg/kg (cohort D) or 1·8 mg/kg (cohort E); and in the triplet therapy group fixed nivolumab 3 mg/kg and ipilimumab 1 mg/kg with brentuximab vedotin 1·2 mg/kg (cohort G) or 1·8 mg/kg (cohort H). Additional patients were enrolled in the expansion phase at the same doses of cohorts B, E, and H. All drugs were given intravenously; brentuximab vedotin and nivolumab were given every 3 weeks, ipilimumab was given every 6 weeks in the ipilimumab group and every 12 weeks in the triplet therapy group. All eligible and treated patients were included in the analysis. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01896999. The phase 2, randomised portion of the trial is still enrolling. FINDINGS: Between March 7, 2014, and Dec 28, 2017, 64 patients were enrolled; two patients in the ipilimumab group and one patient in the nivolumab group were excluded due to ineligibility after enrolment and 61 were evaluable. A total of six dose limiting toxicities were reported in four patients, and the doses used in cohorts B, E, and H were established as maximum tolerated doses and patients were subsequently enrolled onto expansion cohorts (C, F, and I) with these schedules. There were ten (43%) grade 3-4 treatment related adverse events in the ipilimumab group, three (16%) in the nivolumab group, and 11 (50%) in the triplet therapy group including: eight (13%) of 64 patients reporting rash, and colitis, gastritis, pancreatitis and arthritis, and diabetic ketoacidosis each occurring in one (2%) patient. There were two (3%) treatment related deaths, one in the nivolumab group and one in the triplet therapy group. The overall response rate was 76% (95% CI 53-92) in the ipilimumab group, 89% (65-99) in the nivolumab group, and 82% (60-95) in the triplet therapy group, and the complete response rate was 57% (95% CI 34-78%) in the ipilimumab group, 61% (36-83%) in the nivolumab group, and 73% (50-89%) in the triplet therapy group. With a median follow-up of 2·6 years (IQR 1·8-2·9) in the ipilimumab group, 2·4 years (2·2-2·6) in the nivolumab group, and 1·7 years (1·6-1·9) in the triplet therapy group, median progression-free survival is 1·2 years (95% CI 1·7-not reached) in the ipilimumab group, but was not reached in the other two treatment groups. Median overall survival has not been reached in any of the groups. INTERPRETATION: There are clear differences in activity and toxicity of the three combination regimens. The tolerability and preliminary activity for the two most active regimens, brentuximab vedotin with nivolumab and the triplet therapy, are being compared in a randomised phase 2 trial (NCT01896999). FUNDING: Eastern Cooperative Oncology Group-American College of Radiology Imaging Network and the National Cancer Institute of the National Institutes of Health.

Ibrutinib plus lenalidomide and rituximab has promising activity in relapsed/refractory non-germinal center B-cell-like DLBCL.

The outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) is poor, particularly in patients ineligible for stem cell transplantation or who fail induction therapy or salvage therapy. The phase 1b portion of this open-label, dose-escalation (3+3+3 design) study examined the maximum tolerated dose (MTD) and preliminary safety and activity of the regimen in transplant-ineligible adults with histologically confirmed relapsed/refractory DLBCL after at least 1 prior therapy. Patients received once-daily 560 mg ibrutinib, 375 mg/m2 intravenous rituximab day 1 of cycles 1 to 6, and 10, 15, 20, or 25 mg lenalidomide days 1 to 21 of each 28-day cycle. Forty-five patients were treated; median time since diagnosis was 14.1 months, and 51% of the patients had non-germinal center B-cell-like (non-GCB) DLBCL, 33% had transformed DLBCL, 60% were refractory, and 27% were primary refractory. Because of dose-limiting toxicities, a de-escalation cohort (10 mg lenalidomide) was initiated, and with subsequent re-escalation up to 25 mg lenalidomide, the MTD was not reached. In response-evaluable patients, the overall response rate (ORR) was 44% (complete response [CR], 28%); among them, the ORR was 65% (CR, 41%) in non-GCB and 69% and 56% in relapsed (n = 16) and secondary refractory (n = 27) disease, respectively. Overall and for non-GCB, median response duration was 15.9 months, with 2 patients receiving therapy beyond 3 years. Phase 2 was initiated with 20 mg lenalidomide in relapsed/refractory non-GCB, whereas the phase 1b 25-mg lenalidomide cohort was being completed; an additional 25-mg cohort in phase 2 is currently ongoing. This study was registered at www.clinicaltrials.gov as #NCT02077166.

Clinicopathologic correlates of MYD88 L265P mutation and programmed cell death (PD-1) pathway in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) patients have a poorer prognosis than systemic lymphoma. Gain-of-function MYD88 c.794T > C (p. L265P) mutation and programed cell death-1 (PD-1) pathway alterations are potential targetable pathways. Our study objective was to determine the clinicopathologic correlates of MYD88 mutation and PD-1 alterations in PCNSL and the impact of Epstein-Barr virus (EBV) infection. We studied 53 cases including 13 EBV-associated (EBVpos) PCNSL, 49% harbored MYD88 mutation, none seen in EBVpos PCNSL. MYD88 protein expression did not correlate with MYD88 mutation. T-cell and macrophage infiltration was common. All PD-L1-positive tumors were EBVpos. Two PD-L1 positive tumors showed 9p24.1/PD-L1 locus alterations by Fluorescence In Situ Hybridization. T cells and macrophages expressed PD-1 and/or PD-L1 in 98% and 83% cases, respectively. MYD88 mutation or protein expression and PD-1 or PD-L1 expression did not predict outcome. We hypothesize that EBVpos PCNSL has a distinct activation mechanism, independent of genetic alterations.

A new cell culture exposure system for studying the toxicity of volatile chemicals at the air-liquid interface.

A cell culture exposure system (CCES) was developed to expose cells established at an air-liquid interface (ALI) to volatile chemicals. We characterized the CCES by exposing indigo dye-impregnated filter inserts inside culture wells to 125 ppb ozone (O3) for 1 h at flow rates of 5 and 25 mL/min/well; the reaction of O3 with an indigo dye produces a fluorescent product. A 5-fold increase in fluorescence at 25 mL/min/well versus 5 mL/min/well was observed, suggesting higher flows were more effective. We then exposed primary human bronchial epithelial cells (HBECs) to 0.3 ppm acrolein for 2 h at 3, 5, and 25 mL/min/well and compared our results against well-established in vitro exposure chambers at the U.S. EPA's Human Studies Facility (HSF Chambers). We measured transcript changes of heme oxygenase-1 (HMOX1) and interleukin-8 (IL-8), as well as lactate dehydrogenase (LDH) release, at 0, 1, and 24 h post-exposure. Comparing responses from HSF Chambers to the CCES, differences were only observed at 1 h post-exposure for HMOX1. Here, the HSF Chamber produced a ∼6-fold increase while the CCES at 3 and 5 mL/min/well produced a ∼1.7-fold increase. Operating the CCES at 25 mL/min/well produced a ∼4.5-fold increase; slightly lower than the HSF Chamber. Our biological results, supported by our comparison against the HSF Chambers, agree with our fluorescence results, suggesting that higher flows through the CCES are more effective at delivering volatile chemicals to cells. This new CCES will be deployed to screen the toxicity of volatile chemicals in EPA's chemical inventories.

ERK1/2 and p38 regulate inter-individual variability in ozone-mediated IL-8 gene expression in primary human bronchial epithelial cells.

Inter-individual variability is observed in all biological responses; however this variability is difficult to model and its underlying mechanisms are often poorly understood. This issue currently impedes understanding the health effects of the air pollutant ozone. Ozone produces pulmonary inflammation that is highly variable between individuals; but reproducible within a single individual, indicating undefined susceptibility factors. Studying inter-individual variability is difficult with common experimental models, thus we used primary human bronchial epithelial cells (phBECs) collected from many different donors. These cells were cultured, exposed to ozone, and the gene expression of the pro-inflammatory cytokine IL-8 was measured. Similar to in vivo observations, we found that ozone-mediated IL-8 expression was variable between donors, but reproducible within a given donor. Recent evidence suggests that the MAP kinases ERK1/2 and p38 mediate ozone-induced IL-8 transcription, thus we hypothesized that differences in their activation may control IL-8 inter-individual variability. We observed a significant correlation between ERK1/2 phosphorylation and IL-8 expression, suggesting that ERK1/2 modulates the ozone-mediated IL-8 response; however, we found that simultaneous inhibition of both kinases was required to achieve the greatest IL-8 inhibition. We proposed a "dimmer switch" model to explain how the coordinate activity of these kinases regulate differential IL-8 induction.

Baseline Chromatin Modification Levels May Predict Interindividual Variability in Ozone-Induced Gene Expression.

Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual's environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (O3)-mediated induction in an air-liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression-histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan-acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and 5-hydroxymethylcytosine (5-hmC)-and the variability in the O3-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in O3-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an "epigenetic seed and soil" model in which chromatin modification states between individuals differ in the relative abundance of specific modifications (the "soil") that govern how receptive the gene is to toxicant-mediated cellular signals (the "seed") and thus regulate the magnitude of exposure-related gene induction.

Oncogenic BRAF(V600E) Induces Clastogenesis and UVB Hypersensitivity.

The oncogenic BRAF(V600E) mutation is common in melanomas as well as moles. The roles that this mutation plays in the early events in the development of melanoma are poorly understood. This study demonstrates that expression of BRAF(V600E) is not only clastogenic, but synergizes for clastogenesis caused by exposure to ultraviolet radiation in the 300 to 320 nM (UVB) range. Expression of BRAF(V600E) was associated with induction of Chk1 pS280 and a reduction in chromatin remodeling factors BRG1 and BAF180. These alterations in the Chk1 signaling pathway and SWI/SNF chromatin remodeling pathway may contribute to the clastogenesis and UVB sensitivity. These results emphasize the importance of preventing sunburns in children with developing moles.

Toxicities and outcomes among septuagenarians and octogenarians with diffuse large B-cell lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone therapy.

The diagnosis of non-Hodgkin lymphoma (NHL) is increasingly common among the elderly and it is well recognized that this patient population may benefit from therapy. No guidelines exist for chemotherapy dosing in the elderly population, and a clear assessment of treatment toxicity and benefits has not been previously reported. In this single-institution study, we report the toxicities and treatment outcomes of septuagenarians and octogenarians with large cell lymphoma treated with chemo-immunotherapy with or without radiation, as primary therapy with curative intent. We identified 37 patients over the age of 70 years diagnosed with large cell lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) and compared their experience with 65 patients aged less than 70 years. Our retrospective analysis suggests that elderly patients are more susceptible to treatment-related toxicity despite more frequent chemotherapy dose reductions and greater utilization of supportive care. While our aged patients experienced greater frequency of hospitalization during R-CHOP treatment, the vast majority were able to receive relative chemotherapy dose-intensity greater than 70% and experienced similar rates of complete remission.

Consolidative therapy with stem cell transplantation improves survival of patients with mantle cell lymphoma after any induction regimen.

Intensive induction regimen followed by high-dose chemotherapy and autologous stem cell transplantation (auto-SCT) is frequently used to improve outcomes in patients with mantle-cell lymphoma. The comparative impact of conventional vs intensive induction regimen before transplantation is unknown. Forty-eight patients with mantle-cell lymphoma receiving SCT at our institution between January 2000 and December 2010 were included in this study. At the time of initial presentation, 43 (89.5%) had stage IV disease and 18 (37.5%) received more than one chemotherapy regimen before transplantation. Forty patients underwent auto-SCT and 7 had allogeneic SCT (allo-SCT); 1 patient had an allo-SCT for relapsed disease after auto-SCT. At the time of this analysis (median follow-up of 6 years from diagnosis and 4 years from transplantation), 40 patients (88%) were alive with a 5-year disease-free survival of 74.8%. Age, disease stage, number of regimens pre-SCT, pre-SCT disease status, and type of SCT had no impact on long-term outcomes. Importantly, there were no differences among the types of induction regimen on outcomes in this cohort receiving SCT. Based on our data, we believe that future studies should focus on strategies to prevent disease relapse rather than comparing induction regimens before stem cell transplantation.

High-dose chemotherapy followed by autologous stem cell transplantation for relapsed or refractory Hodgkin lymphoma: prognostic features and outcomes.

Between January 1990 and April 2001, 115 patients received high-dose chemotherapy (HDT) followed by autologous stem cell transplantation (ASCT) for relapsed or refractory Hodgkin lymphoma (HL). With a median follow-up of 58 months (range, 1 - 175 months), 5-year progression-free survival (PFS) and overall survival (OS) were 46% and 58%, respectively. Twelve patients with primary refractory disease had a 5-year PFS of 41% and OS of 58%, not significantly different from those of the remaining cohort. Early and overall regimen related mortality were 7% and 16%, respectively. Male gender (P = 0.04) and a time to relapse (TTR) < 12 months (P = 0.03) were associated with decreased OS by univariate analysis. In multivariate analysis, TTR < 12 months remained statistically significant (P = 0.04). We have confirmed that HDT and ASCT result in long-term survival for a proportion of patients with relapsed or refractory HL. All patients, including those with primary refractory disease, benefited from HDT and ASCT.

Stochastic bifurcation in a driven laser system: experiment and theory.

We analyze the effects of stochastic perturbations in a physical example occurring as a higher-dimensional dynamical system. The physical model is that of a class- B laser, which is perturbed stochastically with finite noise. The effect of the noise perturbations on the dynamics is shown to change the qualitative nature of the dynamics experimentally from a stochastic periodic attractor to one of chaoslike behavior, or noise-induced chaos. To analyze the qualitative change, we apply the technique of the stochastic Frobenius-Perron operator [L. Billings et al., Phys. Rev. Lett. 88, 234101 (2002)] to a model of the experimental system. Our main result is the identification of a global mechanism to induce chaoslike behavior by adding stochastic perturbations in a realistic model system of an optics experiment. In quantifying the stochastic bifurcation, we have computed a transition matrix describing the probability of transport from one region of phase space to another, which approximates the stochastic Frobenius-Perron operator. This mechanism depends on both the standard deviation of the noise and the global topology of the system. Our result pinpoints regions of stochastic transport whereby topological deterministic dynamics subjected to sufficient noise results in noise-induced chaos in both theory and experiment.

Multi-scale continuum mechanics: from global bifurcations to noise induced high-dimensional chaos.

Many mechanical systems consist of continuum mechanical structures, having either linear or nonlinear elasticity or geometry, coupled to nonlinear oscillators. In this paper, we consider the class of linear continua coupled to mechanical pendula. In such mechanical systems, there often exist several natural time scales determined by the physics of the problem. Using a time scale splitting, we analyze a prototypical structural-mechanical system consisting of a planar nonlinear pendulum coupled to a flexible rod made of linear viscoelastic material. In this system both low-dimensional and high-dimensional chaos is observed. The low-dimensional chaos appears in the limit of small coupling between the continua and oscillator, where the natural frequency of the primary mode of the rod is much greater than the natural frequency of the pendulum. In this case, the motion resides on a slow manifold. As the coupling is increased, global motion moves off of the slow manifold and high-dimensional chaos is observed. We present a numerical bifurcation analysis of the resulting system illustrating the mechanism for the onset of high-dimensional chaos. Constrained invariant sets are computed to reveal a process from low-dimensional to high-dimensional transitions. Applications will be to both deterministic and stochastic bifurcations. Practical implications of the bifurcation from low-dimensional to high-dimensional chaos for detection of damage as well as global effects of noise will also be discussed.

FDG PET in the follow-up management of patients with newly diagnosed Hodgkin and non-Hodgkin lymphoma after first-line chemotherapy.

PURPOSE: The purpose of this study was to evaluate the accuracy of PET imaging for predicting recurrence of disease and determining fields of radiation therapy for patients with lymphoma after first-line chemotherapy. METHODS AND MATERIALS: The study population included 40 patients with lymphoma, newly diagnosed, staged and treated with either chemotherapy alone or combined modality therapy at this institution. PET findings were correlated with CT findings and radiation ports. Treatment and follow-up course were analyzed to determine patterns of failure. RESULTS: Twenty-eight of 40 patients (70%) were treated with chemotherapy alone, 12 of 40 (30%) were treated with combined modality therapy. Of the patients who received chemotherapy alone, 21 (75%) had a negative follow-up PET scan at the original site of disease, and 5 of these 21 (24%) recurred within the original site of disease. Of the patients who received combined modality therapy, 10 (83%) had a negative follow-up PET scan at the original site of disease and none recurred within the original site of disease. CONCLUSIONS: A negative PET scan after completion of therapy does not exclude the presence of residual microscopic disease and does not indicate complete remission. A higher recurrence rate in patients who were treated with chemotherapy alone compared with combined modality therapy suggests that some of these patients may benefit from aggressive radiation therapy planned at initial staging. The radiation treatment volumes may be better planned from the initial staging PET study because a negative follow-up PET scan after chemotherapy cannot exclude residual microscopic disease.

Peripheral T-cell lymphomas: clinical features and prognostic factors of 92 cases defined by the revised European American lymphoma classification.

The purpose of this study was to better define the clinical features and natural history of peripheral T-cell lymphomas (PTCL) entities included in the Revised European American lymphoma (REAL) classification. Cases of PTCL were retrieved from the records of the Department of Pathology and classified according to the REAL classification. In addition, cases of anaplastic large cell lymphoma (ALCL) were divided into classical, small cell, and primary cutaneous subtypes, and immunostaining for the anaplastic large-cell kinase (ALK) protein was performed on all cases of ALCL. Clinical features, response to therapy and survival were abstracted. Ninety-two cases of PTCL with adequate clinical information were retrieved. There were 40 cases of ALCL (30 classical, 7 small cell variant, 3 primary cutaneous), 28 PTCL, unspecified, 13 angioimmunoblastic T-cell lymphoma and 11 with other entities. The patients had a median age of 48 years with a range of 6-84 and had an estimated overall survival (OS) of 49% and progression-free survival (PFS) of 22% at 5 years. The International Prognostic Index (IPI) was a significant prognostic factor for both progression-free and OS. Histology was a significant predictor of PFS with anaplastic large cell having the best prognosis. ALK expression was not associated with an improved progression-free or overall-survival in patients with systemic T-cell ALCL. In conclusion, the REAL classification describes distinct PTCL entities. The IPI is the most important predictor of progression-free and OS in patients with PTCL. ALK expression may not provide prognostic information for systemic ALCL.

2-deoxy-2-[F-18]fluoro-D-glucose imaging with positron emission tomography for initial staging of Hodgkin's disease and lymphoma.

PURPOSE: To assess the accuracy of 2-Deoxy-2-[F-18] Fluoro-D-Glucose positron emission imaging (FDG-PET) for staging Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) compared to conventional staging (CS) and to evaluate the impact on patient management. METHODS: Forty-five consecutive patients with lymphoma underwent whole-body FDG-PET imaging for initial staging. Discordant lesions were verified with biopsy or clinical follow-up. The impact on staging and management was reviewed retrospectively. RESULTS: A total of 129 sites of disease were identified, and 88 of those were concordant. FDG-PET and conventional staging demonstrated 24 and 17 additional sites, respectively. FDG-PET correctly upstaged five patients and down-staged two patients (16% total), leading to a change in therapy in 6/45 (13%) patients. However, FDG-PET understaged three patients (7%), correctly staged by conventional staging modalities. Assuming that the addition of FDG-PET to conventional staging modalities is 100% accurate for staging lymphoma, the accuracy of FDG-PET alone was 91%, compared to 84% for conventional staging modalities. CONCLUSIONS: FDG-PET is a noninvasive and efficient imaging modality for staging patients with lymphoma and should be used in conjunction with conventional staging modalities, as they appear complementary.