Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3ß-hydroxysteroid dehydrogenase (3ßHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3ßHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY ¼ funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A.

APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Accuracy and safety of 101 consecutives neurosurgical procedures for newly diagnosed central nervous system lymphomas: a single-institution experience.

INTRODUCTION: Brain surgery is required to ascertain the diagnosis of central nervous system lymphoma. We assessed the diagnostic yield and safety of the surgical procedures, the predictors of postoperative morbidity, and of overall survival. METHODS: Observational single-institution retrospective cohort study (1992-2020) of 101 consecutive adult patients who underwent stereotactic biopsy, open biopsy, or resection for a newly diagnosed central nervous system lymphoma. RESULTS: The diagnostic yield was 100% despite preoperative steroid administration in 48/101 cases (47.5%). A preoperative Karnofsky Performance Status score less than 70 (p = 0.006) was an independent predictor of a new postoperative focal neurological deficit (7/101 cases, 6.9%). A previous history of hematological malignancy (p = 0.049), age 65 years or more (p = 0.031), and new postoperative neurological deficit (p < 0.001) were independent predictors of a Karnofsky Performance Status score decrease 20 points or more postoperatively (13/101 cases, 12.9%). A previous history of hematological malignancy (p = 0.034), and preoperative Karnofsky Performance Status score less than 70 (p = 0.024) were independent predictors of postoperative hemorrhage (13/101 cases, 12.9%). A preoperative Karnofsky Performance Status score less than 70 (p = 0.019), and a previous history of hematological malignancy (p = 0.014) were independent predictors of death during hospital stay (8/101 cases, 7.9%). In the 82 immunocompetent patients harboring a primary central nervous system lymphoma, age 65 years or more (p = 0.044), and time to hematological treatment more than 21 days (p = 0.008), were independent predictors of a shorter overall survival. A dedicated hematological treatment (p < 0.001) was an independent predictor of a longer overall survival. CONCLUSION: Brain biopsy is feasible with low morbidity for central nervous system lymphomas. Postoperatively, patients should be promptly referred for hematological treatment initiation.

Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.

OBJECTIVES: Venetoclax combined with hypomethylating agents is a new therapeutic strategy frequently used for treating AML patients who are not eligible for conventional chemotherapy. However, high response rates are heterogeneous due to different mechanisms mediating resistance to venetoclax such as up-regulation of MCL-1 expression. We thus tested the anti-leukemic activity of S63845, a specific MCL-1 inhibitor. METHODS: Apoptosis induces by S63845 with or without venetoclax was evaluated in primary AML samples and in AML cell lines co-cultured or not with bone marrow (BM) mesenchymal stromal cells. Sensitivity of leukemic cells to S63845 was correlated to the expression level of BCL-2, MCL-1, and BCL-XL determined by Western Blot and mass spectrometry-based proteomics. RESULTS: We observed that even if MCL-1 expression is weak compared to BCL-2, S63845 induces apoptosis of AML cells and strongly synergizes with venetoclax. Furthermore, AML cells resistant to venetoclax are highly sensitive to S63845. Interestingly, the synergistic effect of S63845 toward venetoclax-mediated apoptosis of AML cells is still observed in a context of interaction with the BM microenvironment that intrinsically mediates resistance to BCL2 inhibition. CONCLUSION: These results are therefore of great relevance for clinicians as they provide the rational for combining BCL-2 and MCL-1 inhibition in AML.

Impact of genotype in relapsed and refractory acute myeloid leukaemia patients treated with clofarabine and cytarabine: a retrospective study.

The treatment of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains a challenge. Among salvage chemotherapy regimens, the clofarabine and cytarabine (CLARA) combination has been widely evaluated and has a favourable safety/efficacy balance. Predictive factors of efficacy in patients with R/R AML are unclear, particularly the impact of AML-related gene mutations. We report our single-centre experience on 34 R/R AML patients treated with CLARA, with a focus on the genetic characterization of our cohort. CLARA yielded a 47% response rate among this poor-prognosis AML population, while two patients (5·8%) died due to treatment-related toxicity. The two-year progression-free survival and overall survival rates were 29·4% and 35·3%, respectively. Nine patients (26%) had long-term response with a median follow-up of 39·5 months among the responders, of whom six underwent haematopoietic stem cell transplantation. Adverse karyotype did not correlate with response or survival, and secondary AML were more frequent among responders to CLARA, suggesting that this combination may successfully salvage R/R AML patients regardless of adverse prognostic markers. We also observed that a low mutational burden and absence of splice mutations correlated with prolonged survival after CLARA, suggesting that extensive genotyping may have prognostic implications in R/R AML.

Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, ß, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1-/- , Ampk ß1-/- and Ampk γ1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.

Aplastic anemia in the elderly: a nationwide survey on behalf of the French Reference Center for Aplastic Anemia.

Aplastic anemia is a rare but potentially life-threatening disease that may affect older patients. Data regarding the treatment of aplastic anemia in this ageing population remains scarce. We conducted a retrospective nationwide multicenter study in France to examine current treatments for aplastic anemia patients over 60 years old. Our aims were to evaluate efficacy and tolerance, and to analyze predictive factors for response and survival. Over the course of a decade, 88 patients (median age 68.5 years) were identified in 19 centers, with a median follow up of 2.7 years; 21% had very severe and 36% severe aplastic anemia. We analyzed 184 treatment lines, mostly involving the standard combination of anti-thymocyte globulin and cyclosporine-A (33%), which was also the most frequent first-line treatment (50%). After first-line therapy, 32% of patients achieved a complete response, and 15% a partial response. Responses were significantly better in first line and in patients with good performance status, as well as in those that had followed an anti-thymocyte globulin and cyclosporine-A regimen (overall response rate of 70% after first-line treatment). All treatments were well tolerated by patients, including over the age of 70. Three-year survival was 74.7% (median 7.36 years). Age, Charlson comorbidity index and very severe aplastic anemia were independently associated with mortality. Age, per se, is not a limiting factor to aplastic anemia treatment with anti-thymocyte globulin and cyclosporine-A; this regimen should be used as a first-line treatment in elderly patients if they have a good performance status and low comorbidity index score.

AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells.

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

Tandem haematopoietic stem cell transplantation for High Risk relapsed/refractory Hodgkin Lymphoma: a LYSA study.

Tandem stem cell transplantation (SCT) is an option for high-risk relapsed/refractory Hodgkin Lymphoma (HL) patients. We evaluated the tolerance/efficacy of double autologous or autologous SCT (ASCT) followed by allogenic SCT (alloSCT) in 120 HL patients prospectively registered on a French nationwide database. Median age was 26 (14-56) years. Complete remission rate was 60%, including 33% after a single line, and another 27% after two or more salvage regimens. Partial response rate was 32%, and 8% suffered treatment failure. Overall, 115 (96%) patients underwent a first ASCT, and 73 (61%) had a tandem SCT, including alloSCT in 44 (60%) and ASCT in 29 (40%). The median follow-up was 43 months (4.8-73.7 months). The two-year progression-free survival rate for the whole population and for patients receiving tandem transplant was 56% (95% confidence interval [CI]: 46-65%) and 71% (95% CI: 49-84%), respectively. Among tandem transplants, we observed 20 deaths (17%), 10 of which were transplant-related (6 alloSCT and 4 ASCT). We suggest that tandem SCT is efficient in high-risk relapsed/refractory HL patients, although transplant-related mortality remains high. The benefit of tandem SCT should be balanced with the efficacy of Brentuximab vedotin-based post-transplant consolidative strategies in high-risk relapsed/refractory HL patients.

Urgent Chemotherapy in Sepsis-Like Shock Related to Hematologic Malignancies.

OBJECTIVES: Hematologic malignancies may result in multiple organ involvement including pulmonary and renal dysfunctions, and the less common acute circulatory failure. We herein addressed the outcome of patients with sepsis-like shock related to aggressive hematologic malignancies. DESIGN: A 10-year (2007-2016) monocenter retrospective study. SETTINGS: A medical ICU in a tertiary care center. PATIENTS: Patients with circulatory shock requiring vasopressors and who subsequently received chemotherapy. Shock was presumably related to the underlying malignancy after ruling out an ongoing or new-onset infectious process. The extent and time course of organ failures was assessed by a modified Sequential Organ Failure Assessment score devoid of the platelet component. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Seventeen patients were included, including 13 with non-Hodgkin lymphoma, two with hyperleukocytic acute myeloid leukemia, and two with "Human Herpes virus 8"-associated multicentric Castleman's disease. The following associated conditions prompted urgent administration of chemotherapy: tumor lysis syndrome (n = 10), hemophagocytic lymphohistiocytosis (n = 3), compressive bulky tumor (n = 3), pulmonary involvement (n = 3), and disseminated intravascular coagulation (n = 1). Following the initiation of chemotherapy, a number of patients died rapidly from untractable multiple organ failure. In contrast, chemotherapy led to a fast and dramatic improvement in organ failures in early survivors, as shown by the decrease in the modified Sequential Organ Failure Assessment score. However, the overall outcome was poor since only four and three patients could be discharged alive from the ICU and the hospital, and three and two patients remained alive at 6 months and 1 year. CONCLUSIONS: Multiple organ dysfunction syndrome related to hematologic malignancies is associated with a dismal outcome. A chemotherapy trial may provide a fast prognostic assessment of the reversibility of organ failure.

Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells.

Internal tandem duplication of the Fms-like tyrosine kinase-3 receptor (FLT3) internal tandem duplication (ITD) is found in 30% of acute myeloid leukemia (AML) and is associated with a poor outcome. In addition to tyrosine kinase inhibitors, therapeutic strategies that modulate the expression of FLT3-ITD are also promising. We show that AML samples bearing FLT3-ITD mutations are more sensitive to proteasome inhibitors than wild-type samples and this sensitivity is strongly correlated with a higher FLT3-ITD allelic burden. Using pharmacologic inhibitors of autophagy, specific downregulation of key autophagy proteins including Vps34, autophagy gene (Atg)5, Atg12, Atg13, biochemical, and microscopy studies, we demonstrated that proteasome inhibitors induced cytotoxic autophagy in AML cells. FLT3-ITD molecules were detectable within autophagosomes after bortezomib treatment indicating that autophagy induction was responsible for the early degradation of FLT3-ITD, which preceded the inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), PI3K/AKT, and STAT5 pathways, and subsequent activation of cell death. Moreover, proteasome inhibitors overcome resistance to quizartinib induced by mutations in the kinase domain of FLT3, suggesting that these compounds may prevent the emergence of mutant clones arising from tyrosine kinase inhibitor treatments. In xenograft mice models, bortezomib stimulated the conversion of LC3-I to LC3-II, indicating induction of autophagy in vivo, downregulated FLT3-ITD protein expression and improved overall survival. Therefore, selecting patients according to FLT3-ITD mutations could be a new way to detect a significant clinical activity of proteasome inhibitors in AML patients.

Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

Core-binding factor acute myeloid leukemia in first relapse: a retrospective study from the French AML Intergroup.

Although core-binding factor-acute myeloid leukemia (CBF-AML) (t[8;21] or inv[16]/t[16;16]) represents a favorable cytogenetic AML subgroup, 30% to 40% of these patients relapse after standard intensive chemotherapy. The encouraging results of gemtuzumab ozogamicin (GO) in newly diagnosed AML, and particularly in CBF-AML, incited us to retrospectively investigate the impact of GO-based salvage in these patients. We retrospectively analyzed the outcome of 145 patients with CBF-AML (59 t[8;21], 86 inv[16]/t[16;16]) in first relapse. As salvage, 48 patients received GO-based chemotherapy and 97 patients received conventional chemotherapy. Median age was 43 years (range, 16-76). Median first complete remission duration was 12.1 months (range, 2.1-93.6). Overall, second complete remission (CR2) rate was 88%. With a median follow-up from relapse of 3.5 years, the estimated 5-year disease-free survival (DFS) was 50% and 5-year overall survival (OS) was 51%. Older age and shorter first complete remission duration was associated with a shorter OS. Patients treated with GO had similar CR2 rate but significantly higher 5-year DFS (68% vs 42%; P = .05) and OS (65% vs 44%; P = .02). In multivariate analysis, GO salvage was still associated with a significant benefit in DFS and OS. In the 78 patients who received allogeneic hematopoietic stem cell transplantation in CR2, GO before transplant significantly improved posttransplant DFS and OS without excess of treatment-related mortality.

Efficacy and long-term toxicity of the rituximab-fludarabine-cyclophosphamide combination therapy in Waldenstrom's macroglobulinemia.

Waldenstrom's macroglobulinemia is generally treated with alkylating agents, purine analogs and monoclonal antibodies, alone or in combination. We report the outcomes of 82 patients (median age 61 years) treated with the RFC combination. Twenty-five patients were treatment-naive. RFC was administered every 4 weeks, for a median of five cycles. At treatment discontinuation, the overall response rate was 85.4%. The responses improved after treatment discontinuation in 25 patients, with a median time to best response achievement of 10.8 months, raising the major response rate (PR, VGPR and CR) from 64.6% to 76.8%. With a median follow-up of 47 months, the median progression-free survival time had not been reached (67% PFS at 48 months) and was influenced by age and treatment status before RFC. Likewise, the median time to next therapy had not been reached. Two cases of myelodysplastic syndrome/AML and 3 cases of transformation to aggressive lymphoma occurred. Thirteen patients died. The 3-year overall survival rate was 90%. Long-lasting cytopenias occurred in 19 patients. The RFC combination thus gave a high response rate and durable responses, even in heavily treatment-experienced patients. The high incidence of long-lasting cytopenia might be reduced by giving fewer courses and thereby minimizing myelotoxicity. Am. J. Hematol. 91:782-786, 2016. © 2016 Wiley Periodicals, Inc.