Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable.

PURPOSE: Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. EXPERIMENTAL DESIGN: MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. RESULTS: MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. CONCLUSIONS: We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.

A reversible epigenetic memory of inflammatory injury controls lineage plasticity and tumor initiation in the mouse pancreas.

Inflammation is essential to the disruption of tissue homeostasis and can destabilize the identity of lineage-committed epithelial cells. Here, we employ lineage-traced mouse models, single-cell transcriptomic and chromatin analyses, and CUT&TAG to identify an epigenetic memory of inflammatory injury in the pancreatic acinar cell compartment. Despite resolution of pancreatitis, our data show that acinar cells fail to return to their molecular baseline, with retention of elevated chromatin accessibility and H3K4me1 at metaplasia genes, such that memory represents an incomplete cell fate decision. In vivo, we find this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second insult but increased tumorigenesis with an oncogenic Kras mutation. The lowered threshold for oncogenic transformation, in turn, can be restored by blockade of MAPK signaling. Together, we define the chromatin dynamics, molecular encoding, and recall of a prolonged epigenetic memory of inflammatory injury that impacts future responses but remains reversible.

Predicting tumour content of liquid biopsies from cell-free DNA.

BACKGROUND: Liquid biopsy is a minimally-invasive method of sampling bodily fluids, capable of revealing evidence of cancer. The distribution of cell-free DNA (cfDNA) fragment lengths has been shown to differ between healthy subjects and cancer patients, whereby the distributional shift correlates with the sample's tumour content. These fragmentomic data have not yet been utilised to directly quantify the proportion of tumour-derived cfDNA in a liquid biopsy. RESULTS: We used statistical learning to predict tumour content from Fourier and wavelet transforms of cfDNA length distributions in samples from 118 cancer patients. The model was validated on an independent dilution series of patient plasma. CONCLUSIONS: This proof of concept suggests that our fragmentomic methodology could be useful for predicting tumour content in liquid biopsies.

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

TET2 guards against unchecked BATF3-induced CAR T cell expansion.

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

What Every Gastroenterologist Should Know About Gastrointestinal NETs.

Gastrointestinal neuroendocrine tumors are increasingly common. Practitioners should examine these lesions carefully found on routine endoscopy. Obtaining accurate neuroendocrine tumors stage and grade is critical to patient assessment and management, and assistance from advanced endoscopists may be needed.

Use of endoluminal vacuum therapy after anastomotic leak in a gynecologic oncology patient with rectosigmoid resection: A case report.

•Anastomotic leak is an infrequent complication after colon resection and is associated with high morbidity and mortality.•Endoluminal vacuum therapy (EVAT) promotes wound closure by covering anastomotic leaks intraluminally and applying vacuum.•EVAT has been shown to be safe with mild adverse events.•EVAT should be considered in hemodynamically stable gynecologic oncology patients with a confined anastomotic leak.

Serum Pepsinogen as a Biomarker for Gastric Cancer in the United States: A Nested Case-Control Study Using the PLCO Cancer Screening Trial Data.

BACKGROUND: Gastric cancer lacks specific symptoms, resulting in diagnosis at later stages and high mortality. Serum pepsinogen is a biomarker for atrophic gastritis, a gastric cancer precursor, and may be useful to detect persons at increased risk of gastric cancer. METHODS: The Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial was conducted in the United States between 1993 and 2001. ELISA-based pepsinogen tests were conducted on prediagnostic serum samples of 105 PLCO participants who developed gastric cancer and 209 age, sex, and race-matched controls. Pepsinogen positive (PG+) was defined as pepsinogen I ≤ 70 µg/L and pepsinogen I/II ratio ≤3.0. Results of conditional logistic regression models, and sensitivity and specificity, of PG+ for gastric cancer are reported. RESULTS: Gastric cancer cases were more likely to be PG+ (31.4% vs. 5.5%, P < 0.001) at baseline than controls. Compared to PG-, PG+ was associated with an 8.5-fold increased risk for gastric cancer [95% confidence interval (CI) = 3.8-19.4]. This risk remained significant after adjusting for Helicobacter pylori, family history of gastric cancer, education, smoking, and BMI (aOR, 10.6; 95% CI, 4.3-26.2). In subgroup analysis, PG+ individuals were 11-fold more like to develop non-cardia gastric cancer (OR, 11.1; 95% CI, 4.3-28.8); conversely, they were not significantly more likely to develop cardia gastric cancer (OR, 2.0; 95% CI = 0.3-14.2). PG+ status yielded low sensitivity but high specificity for both noncardia (44.3%; 93.6%) and cardia gastric cancer (5.7%; 97.2%). CONCLUSIONS: Prediagnostic serum pepsinogen levels from a large, prospective cohort study were associated with risk of gastric cancer, particularly noncardia gastric cancer. IMPACT: PG status may identify individuals at higher risk of noncardia gastric cancer for targeted screening or interventions. See related commentary by Zhou and Huang, p. 1257.

Devices for esophageal function testing.

BACKGROUND AND AIMS: Esophageal function testing is an integral component of the evaluation of refractory GERD and esophageal motility disorders. This review summarizes the current technologies available for esophageal function testing, including the functional luminal imaging probe (FLIP), high-resolution esophageal manometry (HRM), and multichannel intraluminal impedance (MII) and pH monitoring. METHODS: We performed a MEDLINE, PubMed, and MAUDE database literature search to identify pertinent clinical studies through March 2021 using the following key words: esophageal manometry, HRM, esophageal impedance, FLIP, MII, and esophageal pH testing. Technical data were gathered from traditional and web-based publications, proprietary publications, and informal communications with pertinent vendors. The report was drafted, reviewed, and edited by the American Society for Gastrointestinal Endoscopy Technology Committee and approved by the Governing Board of the American Society for Gastrointestinal Endoscopy. RESULTS: FLIP is a high-resolution impedance planimetry system used for pressure and dimension measurement in the esophagus, pylorus, and anal sphincter. FLIP provides complementary information to HRM for esophageal motility disorders, especially achalasia. The Chicago classification, based on HRM data, is a widely adopted algorithmic scheme used to diagnose esophageal motility disorders. MII detects intraluminal bolus movement and, combined with pH measurement or manometry, provides information on acid and non-acid gastroesophageal reflux and bolus transit in patients with refractory GERD and for preoperative evaluation for anti-reflux procedures. CONCLUSIONS: Esophageal function testing techniques (FLIP, HRM, and MII-pH) have diagnostic and prognostic value in the evaluation of esophageal motility disorders and refractory GERD. Newer technologies and classification systems have enabled an increased understanding of these diseases.

Scaffold attachment factor B1 regulates androgen degradation pathways in prostate cancer.

The nuclear matrix protein Scaffold Attachment Factor B1 (SAFB1, SAFB) can act in prostate cancer (PCa) as an androgen receptor (AR) co-repressor that functions through epigenetic silencing of AR targets, such as prostate specific antigen (PSA, KLK3). Genomic profiling of SAFB1-silenced PCa cells indicated that SAFB1 may play a role in modulating intracrine androgen levels through the regulation of UDP-glucuronosyltransferase (UGT) genes, which inactivate steroid hormones. Gene silencing of SAFB1 resulted in increased levels of free dihydrotesterosterone (DHT), and increased resistance to the AR inhibitor enzalutamide. SAFB1 silencing suppressed expression of the UDP-glucuronosyltransferase family 2 member B15 gene (UGT2B15) and the closely related UGT2B17 gene, which encode proteins that irreversibly inactivate testosterone (T) and DHT. Analysis of human data indicated that genomic loss at the SAFB locus, or down-regulation of expression of the SAFB gene, is associated with aggressive PCa. These findings identify SAFB1 as an important regulator of androgen catabolism in PCa and suggest that loss or inactivation of this protein may promote AR activity by retention of active androgen in tumor cells.

Endoscopic therapies for gallbladder drainage.

BACKGROUND AND AIMS: Endoscopic management of acute cholecystitis has expanded in patients who are considered nonoperative candidates. Traditionally managed with percutaneous cholecystostomy (PC), improvement in techniques and devices has led to increased use of endoscopic methods for gallbladder drainage. This document reviews technical aspects of endoscopic transpapillary gallbladder drainage (ET-GBD) and EUS-guided GBD (EUS-GBD) as well as their respective technical/clinical success and adverse event rates. Available comparative data are also reviewed among nonsurgical gallbladder drainage techniques (PC, ET-GBD, and EUS-GBD). METHODS: The MEDLINE database was searched through March 2021 for relevant articles by using keywords including "acute cholecystitis," "interventional EUS," "percutaneous cholecystostomy," "transpapillary gallbladder drainage," "EUS-guided gallbladder drainage," "lumen-apposing metal stent," "gallbladder stenting," and "endoscopic gallbladder drainage." The manuscript was drafted by 2 authors and reviewed by members of the American Society for Gastrointestinal Endoscopy Technology Committee and subsequently by the American Society for Gastrointestinal Endoscopy Governing Board. RESULTS: Multiple studies have demonstrated acceptable outcomes comparing PC and both endoscopic gallbladder drainage techniques, ET-GBD and EUS-GBD. Published data suggest that endoscopic gallbladder drainage techniques may be associated with lower rates of adverse events and improved quality of life. However, there are important clinical considerations for choosing among these treatment options, requiring a multidisciplinary and collaborative approach to therapeutic decision-making in these patients. CONCLUSIONS: The implementation of EUS-GBD and ET-GBD in high-risk surgical patients with acute cholecystitis may result in favorable outcomes when compared with PC. Further improvements in techniques and training should lead to more widespread acceptance and dissemination of these treatment options.

Lumen-apposing metal stents (with videos).

BACKGROUND AND AIMS: Lumen-apposing metal stents (LAMSs) are a novel class of devices that have expanded the spectrum of endoscopic GI interventions. LAMSs with their dumbbell configuration, short saddle length, and large inner luminal diameter provide favorable stent characteristics to facilitate anastomosis formation between the gut lumen and adjacent structures. METHODS: The MEDLINE database was searched through April 2021 for articles related to LAMSs by using additional relevant keywords such as "walled-off pancreatic necrosis," "pseudocysts," "pancreatic fluid collection," "cholecystitis," "gastroenterostomy," in addition to "endoscopic treatment" and "endoscopic management," among others. RESULTS: This technology review describes the full spectrum of LAMS designs and delivery systems, techniques for deployment, procedural outcomes, safety, training issues, and financial considerations. CONCLUSIONS: Although LAMSs were initially introduced for drainage of pancreatic pseudocysts and walled-off necrosis, the versatility of these devices has led to a variety of off-label uses including gallbladder drainage, enteric bypass with the creation of gastroenterostomies, and treatment of luminal GI strictures.

Clinical and economic impacts of large volume delayed sampling and pathogen reduction technology platelet processing strategies in the United States.

BACKGROUND: Large volume delayed sampling (LVDS) and pathogen reduction technology (PRT) are strategies for platelet processing to minimize transfusion of contaminated platelet components (PCs). This study holistically compares the economic and clinical impact of LVDS and PRT in the United States. STUDY DESIGN AND METHODS: A decision model was constructed to simulate collection, processing, and use of PCs and to compare processing strategies: PRT with 5-day shelf life, LVDS with 7-day shelf life (LVDS7), and LVDS with 5-day shelf life extended to 7 days with secondary testing (LVDS5/2). Target population was adults requiring two or more transfusions. Collection, processing, storage, and distribution data were obtained from the National Blood Collection and Utilization Survey and published literature. Patient outcomes associated with transfusions were obtained from AABB guidelines, meta-analyses, and other published clinical studies. Costs were obtained from reimbursement schedules and other published sources. RESULTS: Given 10,000 donated units, 9512, 9511, and 9651 units of PRT, LVDS5/2, and LVDS7 PCs were available for transfusion, respectively. With these units, 1502, 2172, and 2329 transfusions can be performed with similar levels of adverse events. Assuming 30 transfusions a day, a hospital would require 69,325, 47,940, and 45,383 units of PRT, LVDS5/2, and LVDS7 platelets to perform these transfusions. The mean costs to perform transfusions were significantly higher with PRT units. CONCLUSIONS: Compared with PRT, LVDS strategies were associated with lower costs and higher PC availability while patients experienced similar levels of adverse events. Increased utilization of LVDS has the potential to improve efficiency, expand patient access to platelets, and reduce health care costs.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Tumor fraction-guided cell-free DNA profiling in metastatic solid tumor patients.

BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches. CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.