Citations

Summary Objective Seizures develop in 80% of patients with anti– N ‐methyl‐ d ‐aspartate receptor (NMDAR) encephalitis, and these represent a major cause of morbidity and mortality. Anti‐NMDAR antibodies have been linked to memory loss in encephalitis; however, their role in seizures has not been established. We determined whether anti‐NMDAR antibodies from autoimmune encephalitis patients are pathogenic for seizures. Methods We performed continuous intracerebroventricular infusion of cerebrospinal fluid (CSF) or purified immunoglobulin (IgG) from the CSF of patients with anti‐NMDAR encephalitis or polyclonal rabbit anti‐NMDAR IgG, in male C57BL/6 mice. Seizure status during a 2‐week treatment was assessed with video‐electroencephalography. We assessed memory, anxiety‐related behavior, and motor function at the end of treatment and assessed the extent of neuronal damage and gliosis in the CA1 region of hippocampus. We also performed whole‐cell patch recordings from the CA1 pyramidal neurons in hippocampal slices of mice with seizures. Results Prolonged exposure to rabbit anti‐NMDAR IgG, patient CSF, or human IgG purified from the CSF of patients with encephalitis induced seizures in 33 of 36 mice. The median number of seizures recorded in 2 weeks was 13, 39, and 35 per mouse in these groups, respectively. We observed only 18 brief nonconvulsive seizures in 11 of 29 control mice (median seizure count of 0) infused with vehicle (n = 4), normal CSF obtained from patients with noninflammatory central nervous system (CNS) conditions (n = 12), polyclonal rabbit IgG (n = 7), albumin (n = 3), and normal human IgG (n = 3). We did not observe memory deficits, anxiety‐related behavior, or motor impairment measured at 2 weeks in animals treated with CSF from affected patients or rabbit IgG. Furthermore, there was no evidence of hippocampal cell loss or astrocyte proliferation in the same mice. Significance Our findings indicate that autoantibodies can induce seizures in anti‐NMDAR encephalitis and offer a model for testing novel therapies for refractory autoimmune seizures.

Abstract The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here, we compare a reversible model of epithelial–mesenchymal transition (EMT) induced by TGFβ to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGFβ-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal–epithelial transition (MET). Genetic knockout of autophagy-related 7 (ATG7) or pharmacologic inhibition of SYK activity with fostamatinib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, this study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells. Significance: These findings present inhibition of spleen tyrosine kinase as a therapeutic option to limit breast cancer metastasis by promoting systemic tumor dormancy. See related commentary by Farrington and Narla, p. 1756

Abstract Background: Although the need for palliative care is gaining recognition in Southeast Asia, knowledge about how decisions are made for children near the end of life remains sparse. Objective: To explore pediatric intensivists' attitudes and practices surrounding end-of-life care in Vietnam. Methods: This is a mixed-methods study conducted at a tertiary pediatric and neonatal intensive care unit in Hanoi. Physicians and nurses completed a quantitative survey about their views on end-of-life care. A subset of these providers participated in semistructured interviews on related topics. Analysis of surveys and interviews were conducted. Results were triangulated. Results: Sixty-eight providers (33 physicians and 35 nurses) completed the quantitative survey, and 18 participated in interviews. Qualitative data revealed three overarching themes with numerous subthemes and supporting quotations. The first theme was factors influencing providers' decision-making process to escalate or withdraw treatment. Quantitative data showed that 40% of providers valued the family's ability to pay to continue life-sustaining treatment. Second, communication dynamics in decision making were highlighted; 72% of providers would be willing to override a family's wishes to withdraw life-sustaining treatment. Third, provider perceptions of death varied, with 68% regarding their patients' deaths as a personal failure. Conclusions: We elicited and documented how pediatric intensivists in Vietnam currently think about and provide end-of-life care. These findings indicate a need to strengthen palliative care training, increase family involvement in decision making, implement standardized and official do-not-resuscitate documentation, and expand pediatric hospice services at the individual, hospital, and national levels in Vietnam.

This work presents a heterogeneous immunoassay using the integrated functionalities of a channel and droplets in a digital microfluidic (DMF) platform. Droplet functionality in DMF allows for the programmable manipulation of discrete sample and reagent droplets in the range of nanoliters. Pressure-driven channels become advantageous over droplets when sample must be washed, as the supernatant can be thoroughly removed in a convenient and rapid manner while the sample is immobilized. Herein, we demonstrate a magnetic bead-based, enzyme-linked immunosorbent assay (ELISA) using ~60 nL of human interleukin-6 (IL-6) sample. The wash buffer was introduced in the form of a wall-less virtual electrowetting channel by a syringe pump at the flow rate of 10 μL/min with ~100% bead retention rate. Critical parameters such as sample wash flow rate and bead retention rate were optimized for reliable assay results. A colorimetric readout was analyzed in the International Commission on Illumination (CIE) color space without the need for costly equipment. The concepts presented in this work are potentially applicable in rapid neonatal disease screening using a finger prick blood sample in a DMF platform.

PURPOSE Predictive biomarkers to identify patients with human epidermal growth factor receptor 2 (HER2)–positive breast cancer who may benefit from targeted therapy alone are required. We hypothesized that early measurements of tumor maximum standardized uptake values corrected for lean body mass (SULmax) on [ 18 F]fluorodeoxyglucose positron emission tomography/computed tomography would predict pathologic complete response (pCR) to neoadjuvant pertuzumab and trastuzumab (PT). PATIENTS AND METHODS Patients with stage II/III, estrogen receptor–negative, HER2-positive breast cancer received four cycles of neoadjuvant PT. [ 18 F]Fluorodeoxyglucose positron emission tomography/computed tomography was performed at baseline and 15 days after PT initiation (C1D15). Eighty evaluable patients were required to test the null hypothesis that the area under the curve of percentage of change in SULmax by C1D15 predicting pCR is less than or equal to 0.65, with a one-sided type I error rate of 10%. RESULTS Eighty-eight women were enrolled (83 evaluable), and 85% (75 of 88) completed all four cycles of PT. pCR after PT alone was 34%. Receiver operating characteristic analysis yielded an area under the curve of 0.76 (90% CI, 0.67 to 0.85), which rejected the null hypothesis. Between patients who obtained pCR versus not, a significant difference in median percent reduction in SULmax by C1D15 was observed (63.8% v 33.5%; P < .001), an SULmax reduction greater than or equal to 40% was more prevalent (86% v 46%; P < .001; negative predictive value, 88%; positive predictive value, 49%), and a significant difference in median C1D15 SULmax (1.6 v 3.9; P < .001) and higher proportion of C1D15 SULmax less than or equal to 3 (93% v 38%; P < .001; negative predictive value, 94%; positive predictive value, 55%) were observed. CONCLUSION Early changes in SULmax predict response to four cycles of PT in estrogen receptor–negative, HER2-positive breast cancer. Once optimized, this quantitative imaging strategy may facilitate a more tailored approach to therapy in this setting.

Abstract The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Beisi Xu, Shizhen Zhu, Xiaoling Zhang, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. MYC activation through enhancer hijacking or focal enhancer amplification drives a subset of high-risk pediatric neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA19.

Abstract Despite unprecedented successes with immune checkpoint blockade in advanced cancers, these agents provide durable clinical benefit in only a subset of patients. In addition, the clinical management of patients receiving immune checkpoint blockade remains quite challenging due to the unpredictable and kinetically heterogeneous responses which can manifest as late responses, pseudoprogression, or hyperprogression in subsets of patients. The dynamics and heterogeneity of programmed cell death protein ligand 1 (PD-L1, aka B7-H1) expression confound its use as a predictive biomarker in cancer immunotherapy, but blood-based biomarkers have the potential to predict responders and detect mechanisms of resistance to immunotherapy. We previously reported that Bim (BH3-only protein) is a downstream signaling molecule of the PD-1 pathway, and that Bim levels in circulating tumor-reactive T-cells reflected patient responses to anti-PD-1 therapy in melanoma. We also discovered the existence of soluble PD-L1 (sPD-L1) in the plasma of cancer patients and showed a correlation of sPD-L1 with cancer stages in renal cell carcinoma and with prognosis in metastatic melanoma. Here we evaluated the frequency of tumor-reactive T cells with Bim expression along with effector memory profile and sPD-L1 as biomarkers of response in a new cohort of patients with metastatic melanoma and lung cancer undergoing anti-PD-1 therapy. We recruited 100 cancer patients treated with anti-PD-1 therapy who had peripheral blood collected at baseline and at each subsequent radiographic tumor evaluation. Frequencies of Bim+ and effector memory CD8+ T cells were measured by flow cytometry in gated tumor-reactive CD11a high PD1+ CD8+ T cells. We also measured levels of sPD-L1 in plasma at baseline and serially during treatment with ELISA. Baseline T cell markers and sPD-L1 levels and their percent changes in patients who had a radiographic response were compared to those who had progressive disease (PD). We found that patients with objective response after 4 cycles of anti-PD-1 therapy had higher frequency of Bim+ CD8+ T cells at baseline compared to patients with PD (mean 43% vs. 30%, P=0.0484). The frequencies of Bim+ T cells decreased significantly after the first 3 months of treatment in responders compared with progressors (mean -16% vs. + 40% P=0.0111). The frequency of effector memory CD8+ T cells also dramatically increased in responders in 3-6 months after anti-PD-1 therapy (mean 89% vs. 9.56%, p=0.002). High baseline sPD-L1 was associated with progression on anti-PD-1 therapy (mean 2.8 ng/mL vs. 0.7 ng/mL, p=0.07) or development of immune-related toxicities and the levels increased by 12 weeks in patients progressing on therapy. Conclusion: Measurements of T cell biomarkers (Bim and effector memory) and sPD-L1 levels provide a new noninvasive way to predict and monitor patient responses to anti-PD-1 monotherapy in melanoma and lung cancers. Note: This abstract was not presented at the conference. Citation Format: Roxana S. Dronca, Aaron Mansfield, Xin Liu, Susan Harrington, Elizabeth Enninga, Svetomir Markovic, Lisa Kottschade, Rob Mcwilliams, Matthew Block, Wendy Nevala, Michael Thompson, Haidong Dong. Blood-based T cell biomarkers and soluble PD-L1 predict responses and immune-related toxicity to PD-1 blockade in melanoma and lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A38.

Abstract Background: Tumor surface PD-L1 (B7-H1) signals through T-cell surface receptor PD-1 to suppress antitumor immunity. PD-1 inhibitors like pembrolizumab block this pathway to prevent tumor escape from immunosurveillance. These PD-1 checkpoint inhibitors achieve objective response in 20-30% of patients with melanoma (1-3). As the majority of these patients are resistant to this treatment, there is a critical need to combat PD-1 checkpoint inhibitor resistance in melanoma. What is the mechanism of PD-1 checkpoint inhibitor resistance in melanoma? Our group has reported that soluble PD-L1 (sPD-L1, sB7-H1) is elevated in the serum of patients with PD-1 inhibitor-resistant melanoma and non-small cell lung cancer (NSCLC) (4). We have also shown that soluble PD-L1 is produced by cleavage from other tumor types and leads to apoptosis in CD8+ T-cells (5). The aim of this study is to confirm the source of sPD-L1 in human melanoma and its relation to outcomes. Methods: B7H1-expressing transgenic melanoma cells (Mel-B7H1) were treated with an array of protease inhibitors over 24 hours. Cells were then pelleted and supernatants were analyzed by ELISA for sPD-L1 (sB7-H1) production. Tumor serum samples were also analyzed by ELISA for sPD-L1 production.Tumor pathology slides from patients with melanoma (serum donors from the above study) were stained by immunohistochemistry with antibodies against proteases ADAM10 or ADAM17. Slides were visualized by microscopy for the presence of these antigens. Activated human CD8+ T-cells were cultured with recombinant human B7-H1 (PD-L1) protein, sPD-L1-rich, or sPD-L1-depleted supernatants from a tumor cell line in the presence of placebo or PD-1 checkpoint inhibitor antibodies. Cell survival was measured at 48 hours by flow cytometry.Results: ADAM10 inhibitor GI254023X, ADAM17 inhibitor TAPI-0, and broad metalloprotease inhibitor TAPI-2 inhibited Mel-B7H1 production of sB7H1 (p&lt;0.001, 0.095, and 0.001 respectively). Specific inhibitors of other metalloproteases did not significantly alter sB7H1 production. PD-L1 transcription levels were not significantly altered. Tumor pathology slides from patients with high serum sPD-L1 exhibited high ADAM10 and/or ADAM17 staining. Conversely, tumors from patients with low-sPD-L1levels tended to exhibit low ADAM10 and/or ADAM17 staining. Recombinant PD-L1 and PD-L1-rich supernatants induced cell death of CD8+ T-cells, whereas PBS control solution and PD-L1-depleted supernatants did not. Supraphysiologic doses of pembrolizumab decreased cell death in PD-L1-rich supernatant-treated CD8+ T-cells. Conclusions: Matrix metalloprotease inhibitors ADAM10 and ADAM17 mediate sPD-L1 production from melanoma cells. In samples from patients with melanoma, immunohistochemical staining of these proteases in tumor tissue correlates with serum sPD-L1 levels. As we have shown previously, sPD-L1 induces apoptosis of activated CD8+ T-cells, which may explain PD-1 inhibitor resistance in most patients with melanoma.We posit that sPD-L1 outcompetes PD-1 inhibitors to suppress tumor immunosurveillance. Our results suggest that the combination of ADAM10/17 inhibitor and PD-1 checkpoint inhibitor may overcome this mechanism of PD-1 resistance in melanoma and other cancers. Future studies will determine (A) how a combined ADAM10/17 inhibitor and PD-1 inhibitor may function in clinical use, (B) how tumor-derived sPD-L1 signals downstream to cause apoptosis in T-cells, and (C) whether surface ADAM10/17 predict surface PD-L1 staining in melanoma. References: 1. Ribas et al., Lancet Oncol 2015;16(8):908–18. 2. Hamid et al., Ann Oncol 2016;27:6. 3. Weber et al., Lancet Oncol 2015:16(4):375–84. 4. Dronca et al., J Clin Oncol 2017:35(15):11534. 5. Orme et al., AACR Adv Malig Lymphoma 2018. Citation Format: Jacob J. Orme, Khalid Jazieh, Susan Harrington, Matthew Ball, Tariq U. Azam, Xin (Cindy) Liu, Tiancheng Xie, Aaron Mansfield, Roxana S. Dronca, Haidong Dong. Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor immunity – A resistance mechanism to PD-1 checkpoint blockade in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A210.

ABSTRACTObjectives:The aim of the present study was to investigate the natural history of chronic pancreatitis (CP); patients in the North American Pancreatitis Study2 (NAPS2, adults) and INternational Study group of Pediatric Pancreatitis: In search for a cuRE (INSPPIRE, pediatric) were compared.Methods:Demographics, risk factors, disease duration, management and outcomes of 224 children and 1063 adults were compared using appropriate statistical tests for categorical and continuous variables.Results:Alcohol was a risk in 53% of adults and 1% of children (P < 0.0001); tobacco in 50% of adults and 7% of children (P < 0.0001). Obstructive factors were more common in children (29% vs 19% in adults, P = 0.001). Genetic risk factors were found more often in children. Exocrine pancreatic insufficiency was similar (children 26% vs adult 33%, P = 0.107). Diabetes was more common in adults than children (36% vs 4% respectively, P < 0.0001). Median emergency room visits, hospitalizations, and missed days of work/school were similar across the cohorts. As a secondary analysis, NAPS2 subjects with childhood onset (NAPS2‐CO) were compared with INSPPIRE subjects. These 2 cohorts were more similar than the total INSPPIRE and NAPS2 cohorts, including for genetic risk factors. The only risk factor significantly more common in the NAPS2‐CO cohort compared with the INSPPIRE cohort was alcohol (9% NAPS2‐CO vs 1% INSPPIRE cohorts, P = 0.011).Conclusions:Despite disparity in age of onset, children and adults with CP exhibit similarity in demographics, CP treatment, and pain. Differences between groups in radiographic findings and diabetes prevalence may be related to differences in risk factors associated with disease and length of time of CP.