An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.

Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aß) for growth and survival in the brain parenchyma. Melanoma-secreted Aß activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aß decreases brain metastatic burden. SIGNIFICANCE: Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aß as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. This article is highlighted in the In This Issue feature, p. 1171.

Efficacy and Safety of COVID-19 Convalescent Plasma in Hospitalized Patients: A Randomized Clinical Trial.

Importance: There is clinical equipoise for COVID-19 convalescent plasma (CCP) use in patients hospitalized with COVID-19. Objective: To determine the safety and efficacy of CCP compared with placebo in hospitalized patients with COVID-19 receiving noninvasive supplemental oxygen. Design, Setting, and Participants: CONTAIN COVID-19, a randomized, double-blind, placebo-controlled trial of CCP in hospitalized adults with COVID-19, was conducted at 21 US hospitals from April 17, 2020, to March 15, 2021. The trial enrolled 941 participants who were hospitalized for 3 or less days or presented 7 or less days after symptom onset and required noninvasive oxygen supplementation. Interventions: A unit of approximately 250 mL of CCP or equivalent volume of placebo (normal saline). Main Outcomes and Measures: The primary outcome was participant scores on the 11-point World Health Organization (WHO) Ordinal Scale for Clinical Improvement on day 14 after randomization; the secondary outcome was WHO scores determined on day 28. Subgroups were analyzed with respect to age, baseline WHO score, concomitant medications, symptom duration, CCP SARS-CoV-2 titer, baseline SARS-CoV-2 serostatus, and enrollment quarter. Outcomes were analyzed using a bayesian proportional cumulative odds model. Efficacy of CCP was defined as a cumulative adjusted odds ratio (cOR) less than 1 and a clinically meaningful effect as cOR less than 0.8. Results: Of 941 participants randomized (473 to placebo and 468 to CCP), 556 were men (59.1%); median age was 63 years (IQR, 52-73); 373 (39.6%) were Hispanic and 132 (14.0%) were non-Hispanic Black. The cOR for the primary outcome adjusted for site, baseline risk, WHO score, age, sex, and symptom duration was 0.94 (95% credible interval [CrI], 0.75-1.18) with posterior probability (P[cOR<1] = 72%); the cOR for the secondary adjusted outcome was 0.92 (95% CrI, 0.74-1.16; P[cOR<1] = 76%). Exploratory subgroup analyses suggested heterogeneity of treatment effect: at day 28, cORs were 0.72 (95% CrI, 0.46-1.13; P[cOR<1] = 93%) for participants enrolled in April-June 2020 and 0.65 (95% CrI, 0.41 to 1.02; P[cOR<1] = 97%) for those not receiving remdesivir and not receiving corticosteroids at randomization. Median CCP SARS-CoV-2 neutralizing titer used in April to June 2020 was 1:175 (IQR, 76-379). Any adverse events (excluding transfusion reactions) were reported for 39 (8.2%) placebo recipients and 44 (9.4%) CCP recipients (P = .57). Transfusion reactions occurred in 2 (0.4) placebo recipients and 8 (1.7) CCP recipients (P = .06). Conclusions and Relevance: In this trial, CCP did not meet the prespecified primary and secondary outcomes for CCP efficacy. However, high-titer CCP may have benefited participants early in the pandemic when remdesivir and corticosteroids were not in use. Trial Registration: ClinicalTrials.gov Identifier: NCT04364737.

Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Glycans, such as carbohydrate antigen 19-9, are biomarkers of PDAC and are emerging as important modulators of cancer phenotypes. Herein, we used a systems-based approach integrating glycomic analysis of the well-established KC mouse, which models early events in transformation, and analysis of samples from human pancreatic cancer patients to identify glycans with potential roles in cancer formation. We observed both common and distinct patterns of glycosylation in pancreatic cancer across species. Common alterations included increased levels of α-2,3-sialic acid and α-2,6-sialic acid, bisecting GlcNAc and poly-N-acetyllactosamine. However, core fucose, which was increased in human PDAC, was not seen in the mouse, indicating that not all human glycomic changes are observed in the KC mouse model. In silico analysis of bulk and single-cell sequencing data identified ST6 beta-galactoside alpha-2,6-sialyltransferase 1, which underlies α-2,6-sialic acid, as overexpressed in human PDAC, concordant with histological data showing higher levels of this enzyme at the earliest stages. To test whether ST6 beta-galactoside alpha-2,6-sialyltransferase 1 promotes pancreatic cancer, we created a novel mouse in which a pancreas-specific genetic deletion of this enzyme overlays the KC mouse model. The analysis of our new model showed delayed cancer formation and a significant reduction in fibrosis. Our results highlight the importance of a strategic systems approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer.

Pancreas transplantation from hepatitis C viremic donors to uninfected recipients.

Despite utilization of hepatitis C viremic organs for hepatitis C naïve recipients (HCV D+/R-) in other solid organ transplants, HCV viremic pancreata remain an unexplored source of donor organs. This study reports the first series of HCV D+/R- pancreas transplants. HCV D+/R- had shorter waitlist times compared to HCV D-/R-, waiting a mean of 16 days from listing for HCV-positive organs. HCV D+/R- had a lower match allocation sequence than HCV D-/R-, and this correlated with receipt of organs with a lower Pancreas Donor Risk Index (PDRI) score. All HCV D+/R- had excellent graft function with a mean follow-up of 438 days and had undetectable HCV RNA levels by a mean of 23 days after initiation of HCV-directed therapy. The rates of infectious complications, reoperation, readmission, rejection, and length of stay were not impacted by donor HCV status. A national review of potential ideal pancreas donors found that 37% of ideal HCV-negative pancreas allografts were transplanted, compared to only 5% of ideal HCV-positive pancreas allografts. The results of the current study demonstrate the safety of accepting HCV-positive pancreata for HCV-naïve recipients and advocates for increased utilization of ideal HCV-positive pancreas allografts.

PD-L1 engagement on T cells promotes self-tolerance and suppression of neighboring macrophages and effector T cells in cancer.

Programmed cell death protein 1 (PD-1) ligation delimits immunogenic responses in T cells. However, the consequences of programmed cell death 1 ligand 1 (PD-L1) ligation in T cells are uncertain. We found that T cell expression of PD-L1 in cancer was regulated by tumor antigen and sterile inflammatory cues. PD-L1+ T cells exerted tumor-promoting tolerance via three distinct mechanisms: (1) binding of PD-L1 induced STAT3-dependent 'back-signaling' in CD4+ T cells, which prevented activation, reduced TH1-polarization and directed TH17-differentiation. PD-L1 signaling also induced an anergic T-bet-IFN-γ- phenotype in CD8+ T cells and was equally suppressive compared to PD-1 signaling; (2) PD-L1+ T cells restrained effector T cells via the canonical PD-L1-PD-1 axis and were sufficient to accelerate tumorigenesis, even in the absence of endogenous PD-L1; (3) PD-L1+ T cells engaged PD-1+ macrophages, inducing an alternative M2-like program, which had crippling effects on adaptive antitumor immunity. Collectively, we demonstrate that PD-L1+ T cells have diverse tolerogenic effects on tumor immunity.

Small bowel neuroendocrine tumors: A critical analysis of diagnostic work-up and operative approach.

BACKGROUND: Small bowel neuroendocrine tumors (SB-NETs) are often small, multifocal, difficult to localize preoperatively, and can be overlooked during operative exploration. The optimal work-up and operative approach is unknown. METHODS: Patients who underwent resection of SB-NETs at a single-institution from 2000 to 2014 were included. Primary aim was to describe the diagnostic work-up and compare minimally invasive (MIS) to open resection. RESULTS: Ninety-three patients underwent resection for SB-NETs. About 71% were symptomatic and on average underwent three diagnostic tests: 45% had octreoscans (85% diagnostic yield); 11% had SB-enteroscopy (10% yield); 19% had capsule endoscopy (83% yield, but identified the correct tumor number in only 21%). About 27 pts underwent MIS versus 66 open. MIS pts were younger (56 vs. 61 yrs; P = 0.035), and less likely to have obstruction (4% vs. 24%; P = 0.019) and metastases (19% vs. 44%; P = 0.038). Compared to open, MIS had smaller (1.7 vs. 2.4 cm; P = 0.03) and fewer tumors resected (2 vs. 5; P = 0.049), but similar LN yield (13 vs. 12; P = 0.7). In non-metastatic, curative-intent resections, MIS still resected fewer tumors compared to open (1.5 vs. 4; P = 0.034). CONCLUSION: Capsule endoscopy may be better than small bowel enteroscopy at identifying occult SB-NETs, but may underestimate tumor burden. While MIS may be appropriate in select patients, recognizing the limitations of preoperative evaluation is critical for these tumors, as heightened operative vigilance is often required. J. Surg. Oncol. 2016;114:671-676. © 2016 Wiley Periodicals, Inc.

Is there an Optimal Surgical Approach to Neuroendocrine Tumors of the Ampulla? A Single Institution Experience Over 15 Years.

Neuroendocrine tumors (NETs) of the ampulla of Vater are rare neoplasms accounting for a small fraction of gastroenteropancreatic NETs. The optimal surgical approach remains controversial. This study aimed to define the optimal approach in patients with ampullary NETs. Patients who underwent resection of ampullary NETs from 2000 to 2014 were analyzed. Fourteen patients with well-differentiated, nonfunctional NETs were identified. The mean age was 60 ± 15 years, and nine (64%) were male. The mean tumor size was 1.6 ± 0.9 cm and the majority (61%) had an advanced tumor-node-metastasis stage. Eight (57%) patients underwent pancreaticoduodenectomy (PD) and six (43%) underwent transduodenal ampullectomy (TA). Although the mean tumor size was similar (1.7 vs 1.5 cm), the majority (75 vs 20%) of PD patients demonstrated a trend toward more aggressive tumors, characterized by advance T stage in 25 vs 0 per cent, lymph node positivity in 88 vs 17 per cent, and elevated proliferative index (Ki-67) in 25 vs 0 per cent. Complete resection (R0) was achieved in all PD patients versus 75 per cent in TA patients. There was no difference in major complication rate (50 vs 33%). In the TA group, one (17%) patient recurred and two (33%) patients died, whereas only one (13%) patient died in the PD group and no patients recurred. In conclusion, ampullary tumors can be aggressive tumors characterized by high regional lymph node involvement. A transduodenal approach may provide an inadequate oncological resection and thus has the potential to understage ampullary NETs. In the era of increasing therapeutic options for gastroenteropancreatic NETs, accurate staging is crucial and appropriate oncologic resection via PD for ampullary NETs should be considered.

A 15-year experience with gastric neuroendocrine tumors: Does type make a difference?

BACKGROUND: Gastric neuroendocrine tumors (GNETs) are rare and classified into three types by disease etiology and typical behavior. METHODS: The aim was to describe outcomes after GNET resection at a single institution from 2000 to 2014, stratified by tumor type. Given the small patient number, P-values were not assigned. RESULTS: Of 22 patients, 12 patients (55%) had Type 1, none (0%) had Type 2, and 10 (45%) had Type 3 tumors. Compared to Type 3, Type 1 patients were younger (mean age: 52 vs. 59 years) with similar rates of endoscopic resection (25% vs. 20%). Type 1 GNETs often had multiple tumors (60% vs. 10%) and were not poorly differentiated (0% vs. 11%). Only 33% of Type 1 had nodal metastases compared to 71% of Type 3. Type 1 GNETs presented with metastatic disease less often (17% vs. 40%). Three year recurrence-free survival was 33% for Type 1 compared to 86% for Type 3. Disease-specific survival at 3-years was 100% and 75% for Types 1 and 3, respectively. CONCLUSION: Type 1 GNETs are often indolent and multifocal without nodal involvement, but have high recurrence risk. Type 3 is more aggressive with increased nodal involvement; nodal evaluation should be routinely performed. Determination of GNET type is paramount to treating patients with this rare disease. J. Surg. Oncol. 2016;114:576-580. © 2016 Wiley Periodicals, Inc.

Pancreatic neuroendocrine tumors: Preoperative factors that predict lymph node metastases to guide operative strategy.

BACKGROUND: Enucleation and anatomic resection (central, distal, or pancreaticoduodenectomy) are surgical options for pancreatic neuroendocrine tumors. Depending on nodal-status, enucleation alone may not be oncologically appropriate. Preoperative factors predictive of nodal-involvement are not well defined. METHODS: Patients who underwent curative-intent enucleation or resection of non-metastatic, well/moderately differentiated tumors at a single institution (2000-2014) were included. The aim was to determine factors associated with nodal-metastases and recurrence-free survival. RESULTS: Of 195 patients undergoing resection, 164 met inclusion-criteria. Lymphadenectomy was performed in 131 (80%), and 32 (24%) had nodal-metastases. Receiver-operative-characteristics analysis revealed tumor size ≥2 cm was associated with nodal-involvement (AUC: 0.689; Sensitivity: 90%; Specificity: 53%). On multivariable analysis, male gender (OR: 3.16; 95%CI: 1.18-8.46; P = 0.02), head/uncinate location (HR: 5.37; 95%CI: 2.07-13.96; P = 0.001), and size ≥2 cm (HR: 6.52; 95%CI: 1.75-24.30; P = 0.005) were associated with nodal-positivity. Nodal-metastases (HR: 3.04; 95%CI: 1.04-8.91; P = 0.043) and advanced T-stage (HR: 5.39; 95%CI: 1.46-19.95; P = 0.012) were independently associated with decreased recurrence-free survival. Enucleation (n = 17; 10%) had more positive margins and similar complication rates, pancreatic fistula rates, and lengths of stay as anatomic resections. CONCLUSION: For pancreatic neuroendocrine tumors, male gender, head/uncinate location, and size ≥2 cm are associated with nodal-metastases. Nodal involvement is associated with decreased recurrence-free survival. Anatomic resection may be preferred in patients with these characteristics, as enucleation alone may under-stage patients and does not appear to be associated with an improved complication profile. J. Surg. Oncol. 2016;114:440-445. © 2016 Wiley Periodicals, Inc.

Symptomatic presentation as a predictor of recurrence in gastroenteropancreatic neuroendocrine tumors: A single institution experience over 15 years.

BACKGROUND AND OBJECTIVES: The prognostic implication of symptomatic presentation of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) remains unclear. METHODS: Patients who underwent resection of nonfunctional GEP-NETs (2000-2014) were analyzed. Primary outcomes were overall survival (OS) and distant recurrence-free survival (RFS). RESULTS: Symptomatic presentation was seen in 208 (61%) of 339 patients and was associated with younger age (55 vs. 59 yrs, P = 0.001), higher tumor grade (38% vs. 21%, P = 0.027), presence of lymphovascular invasion (58% vs. 33%, P < 0.001), presence of perineural invasion (54% vs. 29%, P = 0.002), and advanced disease (T3/T4/N1/M1 [63% vs. 44%, P = 0.002]), but not tumor size (2.6 vs. 2.5 cm, P = 0.74). Symptomatic presentation was associated with decreased 3-yr distant-RFS (80% vs. 89%, P = 0.012), but not OS. When accounting for adverse features, symptomatic presentation remained independently associated with reduced distant-RFS (HR 3.51, P = 0.007). On subgroup-analysis of advanced disease patients, symptomatology was associated with reduced 3-yr distant-RFS (67% vs. 79%, P = 0.012) and persisted as an independent risk-factor for decreased distant-RFS (HR 3.01, P = 0.027). CONCLUSIONS: Symptomatic presentation of GEP-NETs is associated with aggressive features and worse distant-RFS. Trials assessing adjuvant therapy for advanced GEP-NETs are needed, and symptomatic presentation may be considered as one inclusion criterion. Following resection, symptomatic presentation should be taken into account when planning surveillance. J. Surg. Oncol. 2016;114:163-169. © 2016 Wiley Periodicals, Inc.

Systemic Levamisole-Induced Vasculitis in a Cocaine User without Cutaneous Findings: A Consideration in Diagnosis.

Levamisole is a known immunomodulating agent frequently used as a cutting agent in cocaine consumed in the United States today. Numerous cases of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis connected with the use of levamisole-adulterated cocaine have previously been reported in the literature, classically characterized by a retiform purpuric rash. We report a case of a crack-cocaine user without cutaneous abnormalities who developed ANCA-associated glomerulonephritis that progressed to renal failure. This case demonstrates the difficulties in solidifying the diagnosis of levamisole-induced vasculitis in the absence of cutaneous findings and the need to pursue more testing to establish causality in ANCA-associated vasculitis that has potential for severe end-organ damage in patients who continue to use cocaine.

A dominant allele of Arabidopsis pectin-binding wall-associated kinase induces a stress response suppressed by MPK6 but not MPK3 mutations.

The plant cell wall is composed of a matrix of cellulose fibers, flexible pectin polymers, and an array of assorted carbohydrates and proteins. The receptor-like Wall-Associated Kinases (WAKs) of Arabidopsis bind pectin in the wall, and are necessary both for cell expansion during development and for a response to pathogens and wounding. Mitogen Activated Protein Kinases (MPKs) form a major signaling link between cell surface receptors and both transcriptional and enzyme regulation in eukaryotes, and Arabidopsis MPK6 and MPK3 indeed have important roles in development and the response to stress and pathogens. A dominant allele of WAK2 requires kinase activity and activates a stress response that includes an increased ROS accumulation and the up-regulation of numerous genes involved in pathogen resistance, wounding, and cell wall biogenesis. This dominant allele requires a functional pectin binding and kinase domain, indicating that it is engaged in a WAK signaling pathway. A null mutant of the major plasma membrane ROS-producing enzyme complex, rbohd/f does not suppress the WAK2cTAP-induced phenotype. A mpk6, but not a mpk3, null allele is able to suppress the effects of this dominant WAK2 mutation, thus distinguishing MPK3 and MPK6, whose activity previously was thought to be redundant. Pectin activation of gene expression is abated in a wak2-null, but is tempered by the WAK-dominant allele that induces elevated basal stress-related transcript levels. The results suggest a mechanism in which changes to the cell wall can lead to a large change in cellular responses and help to explain how pathogens and wounding can have general effects on growth.