Lysine 473 Regulates the Progression of SLC7A11, the Cystine/Glutamate Exchanger, through the Secretory Pathway.

System xc-, the cystine/glutamate exchanger, is a membrane transporter that plays a critical role in the antioxidant response of cells. Recent work has shown that System xc- localizes to the plasma membrane during oxidative stress, allowing for increased activity to support the production of glutathione. In this study, we used site-directed mutagenesis to examine the role of C-terminal lysine residues (K422, K472, and K473) of xCT (SLC7A11) in regulating System xc-. We observed that K473R exhibits loss of transporter activity and membrane localization and is 7.5 kD lower in molecular weight, suggesting that K473 regulates System xc- trafficking and is modified under basal conditions. After ruling out ubiquitination and neddylation, we demonstrated that unlike WT xCT, K473R lacks N- and O-glycosylation and is sequestered in the endoplasmic reticulum. Next, we demonstrated that K473Q, a constitutively acetylated lysine mimic, also exhibits loss of transporter activity, decreased membrane expression, and a 4 kD decrease in molecular weight; however, it is N- and O-glycosylated and localized to the endoplasmic reticulum and Golgi. These results suggest that acetylation and deacetylation of K473 in the endoplasmic reticulum and Golgi, respectively, serve to regulate the progression of the transporter through the biosynthetic pathway.

Elastofibroma Dorsi: Case Series of a Rare Chest Wall Pseudotumor.

BACKGROUND: Elastofibroma dorsi (EFD) is a pseudotumor of the thoracic wall that can be difficult to diagnose due to its rarity. Prompt recognition can limit unnecessary workup and expedite treatment. This study retrospectively analyzed patients with a diagnosis of EFD, discussing clinical presentations and surgical outcomes. METHODS: This is an IRB-approved single-center retrospective study of all patients with a diagnosis of elastofibroma at our institution between 2000 and 2022. RESULTS: Ten patients were identified to have a pathologic diagnosis of EFD since 2000, with half presenting in the last 5 years. Our cohort had an average age of 56.8 years and was 50% female. The average age of male subjects was younger than females, 49.6-64.0 years, respectively (p = 0.10). Eighty percent (8/10) of patients had unilateral EFDs and symptoms lasted 27.1 months on average prior to diagnosis. Surgical resection was performed on 66.67% (8/12) of masses, with 87.5% (7/8) of patients who underwent surgery reporting complete resolution of their symptoms and none reporting recurrence. CONCLUSIONS: Although EFD is a rare pseudotumor, its incidence may be increasing. As such, surgeons should be aware of the typical clinical presentation; specifically, a slow growing, predominantly unilateral, painful, subscapular mass with an inhomogeneous pattern on imaging. Originally thought to predominantly affect elderly women, our study shows that younger men may be at risk as well. If patients present with EFD, complete surgical resection should be performed to achieve favorable outcomes and resolution of symptoms.

Integrating Research into the Undergraduate Curriculum: 1. Early Research Experiences and Training.

Undergraduate research experiences have emerged as some of the most beneficial high-impact practices in education, providing clear benefits to students that include improved critical thinking and scientific reasoning, increased academic performance, and enhanced retention both within STEM majors and in college overall. These benefits extend to faculty members as well. Several disciplines, including neuroscience, have implemented research as part of their curriculum, yet many research opportunities target late stage undergraduates, despite evidence that early engagement can maximize the beneficial nature of such work. A 2019 Society for Neuroscience professional development workshop provided multiple examples of integrating research into an undergraduate curriculum, including early engagement (Fernandes, 2020). This article is the first in a series of three that expands upon the information presented in those workshop discussions, focusing on ways to promote early research opportunities. The benefits and challenges associated with early research engagement suggest thoughtful consideration of the best mechanisms for implementation are warranted; some options might include apprenticeship models or course-based approaches. Regardless of mechanism, early research can serve to initiate more prolonged, progressive, scaffolded experiences that span the academic undergraduate career.

Integrating Research into the Undergraduate Curriculum: 2. Scaffolding Research Skills and Transitioning toward Independent Research.

Undergraduate research experiences are widely regarded as high-impact practices that foster meaningful mentoring relationships, enhance retention and graduation, and stimulate postbaccalaureate enrollment in STEM graduate and professional programs. Through immersion in a mentored original research project, student develop and apply their skills in critical thinking, problem solving, intellectual independence, communication, collaboration, project ownership, innovation, and leadership. These skills are readily transferable to a wide array of future careers in and beyond STEM that are well-served by evidence-based approaches. The 2019 Society for Neuroscience meeting included a well-attended workshop on integrating research into the curriculum at primarily undergraduate institutions (PUIs). This article is the second of three articles that summarize, analyze, and expand the workshop discussions. In this second article, we specifically describe approaches to transitional research courses that prepare students for independent research experiences such as undergraduate research theses. Educators can intentionally scaffold research experience and skills across the curriculum, to foster participation in scientific research and enhance diversity, equity, and inclusivity in research training. This article provides an overview of important goals and considerations for intermediate undergraduate research experiences, specific examples from several institutions of transitional courses that scaffold research preparation using different structures, and a summary of lessons learned from these experiences.

Integrating Research into the Undergraduate Curriculum: 3. Research Training in the Upper-level Neuroscience Curriculum.

The benefits of undergraduate training in research are significant. Integration of such training into the undergraduate experience, however, can be challenging at institutions without extensive research programs, and may inadvertently exclude some populations of students. Therefore, inclusion of research into the academic curriculum ensures all students can access this important training. The 2019 annual meeting of the Society for Neuroscience included a workshop on integrating research into the curriculum at primarily undergraduate institutions (PUIs). In this last article of a three-part series, we describe models for integrating research into advanced stages of the undergraduate curriculum, specifically for juniors and seniors. First, we describe multiple models of faculty-mentored group-based research. Second, we detail a peer-mentored research system, in which seniors mentor groups of first through third year students. Third, we describe multiple examples of integrating research into "capstone" courses for seniors. Fourth, we describe models in which a senior thesis is a graduation requirement for all students. Lastly, we describe several models of implementing an optional honors thesis for students. Although similarities exist across these programs, their differences allow for specific secondary objectives to be met, which are often unique to institutions and/or departments. Therefore, for each of these examples, we describe the context, specific design, and required student assessments. We conclude by discussing some of the key successes and challenges of developing programs that facilitate undergraduate research by upper-level students, and suggest a number of concepts that should be considered by individuals developing and assessing new programs.

Initial Report of Second-Line FOLFIRI in Combination with Ramucirumab in Advanced Gastroesophageal Adenocarcinomas: A Multi-Institutional Retrospective Analysis.

BACKGROUND: The randomized phase III RAINBOW trial established paclitaxel (pac) plus ramucirumab (ram) as a global standard for second-line (2L) therapy in advanced gastric and gastroesophageal junction adenocarcinoma, together gastroesophageal adenocarcinoma (GEA). Patients (pts) receiving first-line (1L) FOLFOX often develop neuropathy that renders continued neurotoxic agents in the 2L setting unappealing and other regimens more desirable. As such, FOLFIRI-ram has become an option for patients with 2L GEA. FOLFIRI-ramucirumab (ram) has demonstrated safety and activity in 2L colorectal cancer, but efficacy/safety data in GEA are lacking. SUBJECTS, MATERIALS, AND METHODS: Patients with GEA treated with 2L FOLFIRI-ram between August 2014 and April 2018 were identified. Clinicopathologic data including oxaliplatin neurotoxicity rates/grades (G), 2L treatment response, progression-free survival (PFS), overall survival (OS), safety, and molecular features were abstracted from three U.S. academic institutions. Kaplan-Meier survival analysis was used to generate PFS/OS; the likelihood ratio test was used to determine statistical significance. RESULTS: We identified 29 pts who received 2L FOLFIRI-ram. All pts received 1L platinum + fluoropyrimidine, and 23 of 29 (79%) had post-1L neuropathy; 12 (41%) had G1, and 11 (38%) had G2. Patients were evenly split between esophagus/gastroesophageal junction (12; 41%) and gastric cancer (17; 59%). Among evaluable pts (26/29), the overall response rate was 23% (all partial response) with a disease control rate of 79%. Median PFS was 6.0 months and median OS was 13.4 months among all evaluable pts. Six- and 12-month OS were 90% (n = 18/20) and 41% (n = 7/17). There were no new safety signals. CONCLUSION: We provide the first data suggesting FOLFIRI-ram is a safe, non-neurotoxic regimen comparing favorably with the combination of pac + ram used in the seminal RAINBOW trial. IMPLICATIONS FOR PRACTICE: Results of this study provide initial support for the safety and efficacy of second-line (2L) FOLFIRI-ramucirumab (ram) after progression on first-line platinum/fluoropyrimidine in patients with gastroesophageal adenocarcinoma (GEA). The overall response, progression-free survival, overall survival, and toxicity profile compare favorably with paclitaxel (pac) + ram and highlight the importance of the ongoing phase II RAMIRIS trial examining FOLFIRI-ram versus pac + ram in 2L GEA (NCT03081143). FOLFIRI-ram may warrant consideration for inclusion as an alternate regimen in consensus guidelines for GEA.

Personalized Antibodies for Gastroesophageal Adenocarcinoma (PANGEA): A Phase II Study Evaluating an Individualized Treatment Strategy for Metastatic Disease.

The one-year and median overall survival (mOS) rates of advanced gastroesophageal adenocarcinomas (GEA) are ∼50% and <12 months, respectively. Baseline spatial and temporal molecular heterogeneity of targetable alterations may be a cause of failure of targeted/immunooncologic therapies. This heterogeneity, coupled with infrequent incidence of some biomarkers, has resulted in stalled therapeutic progress. We hypothesized that a personalized treatment strategy, applied at first diagnosis then serially over up to three treatment lines using monoclonal antibodies combined with optimally sequenced chemotherapy, could contend with these hurdles. This was tested using a novel clinical expansion-platform type II design with a survival primary endpoint. Of 68 patients by intention-to-treat, the one-year survival rate was 66% and mOS was 15.7 months, meeting the primary efficacy endpoint (one-sided P = 0.0024). First-line response rate (74%), disease control rate (99%), and median progression-free survival (8.2 months) were superior to historical controls. The PANGEA strategy led to improved outcomes warranting a larger randomized study. SIGNIFICANCE: This study highlights excellent outcomes achieved by individually optimizing chemotherapy, biomarker profiling, and matching of targeted therapies at baseline and over time for GEA. Testing a predefined treatment strategy resulted in improved outcomes versus historical controls. Therapeutic resistance observed in correlative analyses suggests that dual targeted inhibition may be beneficial.This article is highlighted in the In This Issue feature, p. 211.

Hydrogen peroxide triggers an increase in cell surface expression of system xc- in cultured human glioma cells.

System xc- exchanges extracellular cystine for intracellular glutamate across the plasma membrane of many cell types. One of the physiological roles of System xc- is to provide cystine for synthesis of the antioxidant glutathione. Here we report that hydrogen peroxide (H2O2) triggers the translocation of System xc- to the plasma membrane within 10 min of the initial exposure. Specifically, we observed a three-fold increase in 35S-l-cystine uptake following a 10 min exposure to 0.3 mM H2O2. This effect was dose-dependent with an EC50 for H2O2 of 65 µM. We then used cell surface biotinylation analysis to test the hypothesis that the increase in activity is due to an increased number of transporters on the plasma membrane. We demonstrated that the amount of transporter protein, xCT, localized to the plasma membrane doubles within 10 min of H2O2 exposure as a result of an increase in its delivery rate and a reduction in its internalization rate. In addition, we demonstrated that H2O2 triggered a rapid decrease in total cellular glutathione which recovered within 2 h of the oxidative insult. The kinetics of glutathione recovery matched the time course for the recovery of xCT cell surface expression and System xc- activity following removal of the oxidative insult. Collectively, these results suggest that oxidants acutely modulate the activity of System xc- by increasing its cell surface expression, and that this process may serve as an important mechanism to increase de novo glutathione synthesis during periods of oxidative stress.

Evaluation of the Association of Perioperative UGT1A1 Genotype-Dosed gFOLFIRINOX With Margin-Negative Resection Rates and Pathologic Response Grades Among Patients With Locally Advanced Gastroesophageal Adenocarcinoma: A Phase 2 Clinical Trial.

Importance: Patients with locally advanced gastroesophageal adenocarcinoma (ie, stage ≥T3 and/or node positive) have high rates of recurrence despite surgery and adjunctive perioperative therapies, which also have high toxicity profiles. Evaluation of pharmacogenomically dosed perioperative gFOLFIRINOX (fluorouracil, leucovorin, oxaliplatin, and UGT1A1 genotype-directed irinotecan) to optimize efficacy while limiting toxic effects may have value. Objective: To evaluate the coprimary end points of margin-negative (R0) resection rates and pathologic response grades (PRGs) of gFOLFIRINOX therapy among patients with locally advanced gastroesophageal adenocarcinoma. Design, Setting, and Participants: This single-group phase 2 trial, conducted at 2 academic medical centers from February 2014 to March 2019, enrolled 36 evaluable patients with locally advanced adenocarcinoma of the esophagus, gastroesophageal junction, and gastric body. Data analysis was conducted in May 2019. Interventions: Patients received biweekly gFOLFIRINOX (fluorouracil, 2400 mg/m2 over 46 hours; oxaliplatin, 85 mg/m2; irinotecan, 180 mg/m2 for UGT1A1 genotype 6/6, 135 mg/m2 for UGT1A1 genotype 6/7, or 90 mg/m2 for UGT1A1 genotype 7/7; and prophylactic peg-filgastrim, 6 mg) for 4 cycles before and after surgery. Patients with tumors positive for ERBB2 also received trastuzumab (6-mg/kg loading dose, then 4 mg/kg). Main Outcomes and Measures: Margin-negative resection rate and PRG. Results: A total of 36 evaluable patients (27 [78%] men; median [range] age, 66 [27-85] years; 10 [28%] with gastric body cancer; 24 [67%] with intestinal-type tumors; 6 [17%] with ERBB2-positive tumors; 19 [53%] with UGT1A1 genotype 6/6; 16 [44%] with genotype 6/7; and 1 [3%] with genotype 7/7) were enrolled. Of these, 35 (97%) underwent surgery; 1 patient (3%) died after completing neoadjuvant chemotherapy while awaiting surgery. Overall, R0 resection was achieved in 33 of 36 patients (92%); 2 patients (6%) with linitis plastica achieved R1 resection. Pathologic response grades 1, 2, and 3 occurred in 13 patients (36%), 9 patients (25%), and 14 patients (39%), respectively, and PRG 1 was observed in 11 of 24 intestinal-type tumors (46%). Median disease-free survival was 30.1 months (95% CI, 15.0 months to not reached), and median overall survival was not reached (95% CI, 8.3 months to not reached). There were no differences in outcomes by UGT1A1 genotype group. A total of 38 patients, including 2 (5%) with antral tumors, were evaluable for toxic effects. Grade 3 or higher adverse events occurring in 5% or more of patients during the perioperative cycles included diarrhea (7 patients [18%]; 3 of 19 patients [16%] with genotype 6/6; 2 of 16 patients [13%] with genotype 6/7; 2 of 3 patients [67%] with genotype 7/7), anemia (2 patients [5%]), vomiting (2 patients [5%]), and nausea (2 patients [5%]). Conclusions and Relevance: In this study, perioperative pharmacogenomically dosed gFOLFIRINOX was feasible, providing downstaging with PRG 1 in more than one-third of patients and an R0 resection rate in 92% of patients. Trial Registration: ClinicalTrials.gov Identifier: NCT02366819.

Developing a project-oriented introduction to neuroscience lab at hope college.

The Introduction to Neuroscience course at Hope College includes a three-hour laboratory period each week. Seven of the fifteen weeks of the lab are used for a lab project that is focused on understanding the effects of gonadal hormones on brain and behavior. Students perform ovariectomies and implant sham, estradiol, or testosterone capsules in rats and then carry out five experiments: 1) Sexual Behavior, 2) Spatial Learning using the Morris Water Maze, 3) The Size of the Sexually Dimorphic Nucleus, 4) Phosphorylation of NMDA Receptors, and 5) Long Term Potentiation in Hippocampal Slices. The experiments are designed to provide the students with experiences at different levels of neuroscience, while improving their skills in statistics, using the primary literature, and scientific writing. The students generate interesting and statistically significant data which they summarize in a journal style lab reports. Using a Self Assessment of Learning Gains tool, we learned that students perceive the lab project improves their ability to A) pose questions from more than one disciplinary perspective that can be addressed by collecting and evaluating scientific evidence, B) learn about complex science problems that require insight from more than one discipline, C) extract main points from a scientific article and develop a coherent summary, and D) write reports using scientific data as evidence. Based on our results, we believe an extended lab project in an introductory neuroscience course can be used to engage students in neuroscience topics and help them develop the skills and habits of neuroscientists.

Obtaining Knowledge in Pathology Reports Through a Natural Language Processing Approach With Classification, Named-Entity Recognition, and Relation-Extraction Heuristics.

PURPOSE: Robust institutional tumor banks depend on continuous sample curation or else subsequent biopsy or resection specimens are overlooked after initial enrollment. Curation automation is hindered by semistructured free-text clinical pathology notes, which complicate data abstraction. Our motivation is to develop a natural language processing method that dynamically identifies existing pathology specimen elements necessary for locating specimens for future use in a manner that can be re-implemented by other institutions. PATIENTS AND METHODS: Pathology reports from patients with gastroesophageal cancer enrolled in The University of Chicago GI oncology tumor bank were used to train and validate a novel composite natural language processing-based pipeline with a supervised machine learning classification step to separate notes into internal (primary review) and external (consultation) reports; a named-entity recognition step to obtain label (accession number), location, date, and sublabels (block identifiers); and a results proofreading step. RESULTS: We analyzed 188 pathology reports, including 82 internal reports and 106 external consult reports, and successfully extracted named entities grouped as sample information (label, date, location). Our approach identified up to 24 additional unique samples in external consult notes that could have been overlooked. Our classification model obtained 100% accuracy on the basis of 10-fold cross-validation. Precision, recall, and F1 for class-specific named-entity recognition models show strong performance. CONCLUSION: Through a combination of natural language processing and machine learning, we devised a re-implementable and automated approach that can accurately extract specimen attributes from semistructured pathology notes to dynamically populate a tumor registry.

Circulating Tumor DNA Sequencing Analysis of Gastroesophageal Adenocarcinoma.

PURPOSE: Gastroesophageal adenocarcinoma (GEA) has a poor prognosis and few therapeutic options. Utilizing a 73-gene plasma-based next-generation sequencing (NGS) cell-free circulating tumor DNA (ctDNA-NGS) test, we sought to evaluate the role of ctDNA-NGS in guiding clinical decision-making in GEA. EXPERIMENTAL DESIGN: We evaluated a large cohort (n = 2,140 tests; 1,630 patients) of ctDNA-NGS results (including 369 clinically annotated patients). Patients were assessed for genomic alteration (GA) distribution and correlation with clinicopathologic characteristics and outcomes. RESULTS: Treatment history, tumor site, and disease burden dictated tumor-DNA shedding and consequent ctDNA-NGS maximum somatic variant allele frequency. Patients with locally advanced disease having detectable ctDNA postoperatively experienced inferior median disease-free survival (P = 0.03). The genomic landscape was similar but not identical to tissue-NGS, reflecting temporospatial molecular heterogeneity, with some targetable GAs identified at higher frequency via ctDNA-NGS compared with previous primary tumor-NGS cohorts. Patients with known microsatellite instability-high (MSI-High) tumors were robustly detected with ctDNA-NGS. Predictive biomarker assessment was optimized by incorporating tissue-NGS and ctDNA-NGS assessment in a complementary manner. HER2 inhibition demonstrated a profound survival benefit in HER2-amplified patients by ctDNA-NGS and/or tissue-NGS (median overall survival, 26.3 vs. 7.4 months; P = 0.002), as did EGFR inhibition in EGFR-amplified patients (median overall survival, 21.1 vs. 14.4 months; P = 0.01). CONCLUSIONS: ctDNA-NGS characterized GEA molecular heterogeneity and rendered important prognostic and predictive information, complementary to tissue-NGS.See related commentary by Frankell and Smyth, p. 6893.

Targeted Therapies for Targeted Populations: Anti-EGFR Treatment for EGFR-Amplified Gastroesophageal Adenocarcinoma.

Previous anti-EGFR trials in unselected patients with gastroesophageal adenocarcinoma (GEA) were resoundingly negative. We identified EGFR amplification in 5% (19/363) of patients at the University of Chicago, including 6% (8/140) who were prospectively screened with intention-to-treat using anti-EGFR therapy. Seven patients received ≥1 dose of treatment: three first-line FOLFOX plus ABT-806, one second-line FOLFIRI plus cetuximab, and three third/fourth-line cetuximab alone. Treatment achieved objective response in 58% (4/7) and disease control in 100% (7/7) with a median progression-free survival of 10 months. Pretreatment and posttreatment tumor next-generation sequencing (NGS), serial plasma circulating tumor DNA (ctDNA) NGS, and tumor IHC/FISH for EGFR revealed preexisting and/or acquired genomic events, including EGFR-negative clones, PTEN deletion, KRAS amplification/mutation, NRAS, MYC, and HER2 amplification, and GNAS mutations serving as mechanisms of resistance. Two evaluable patients demonstrated interval increase of CD3+ infiltrate, including one who demonstrated increased NKp46+, and PD-L1 IHC expression from baseline, suggesting an immune therapeutic mechanism of action. EGFR amplification predicted benefit from anti-EGFR therapy, albeit until various resistance mechanisms emerged.Significance: This paper highlights the role of EGFR inhibitors in EGFR-amplified GEA-despite negative results in prior unselected phase III trials. Using serial ctDNA and tissue NGS, we identified mechanisms of primary and acquired resistance in all patients, as well as potential contribution of antibody-dependent cell-mediated cytotoxicity to their clinical benefit. Cancer Discov; 8(6); 696-713. ©2018 AACR.See related commentary by Strickler, p. 679This article is highlighted in the In This Issue feature, p. 663.

Cultivation of an interdisciplinary, research-based neuroscience minor at hope college.

Hope College is an undergraduate liberal arts college with an enrollment of approximately 3,000 students. In the spring of 2005, we began to offer an interdisciplinary neuroscience minor program that is open to all students. The objective of this program is to introduce students to the field of neuroscience, and to do so in such a way as to broaden students' disciplinary perspectives, enhance communication and quantitative skills, and increase higher-level reasoning skills by encouraging collaboration among students who have different disciplinary backgrounds. This is a research-based program that culminates in a one-year capstone research course. Here we present the story of the program development at Hope College, including a description of our newly developed curriculum, our initial assessment data, and the lessons we have learned in developing this program.