Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2.

Targeted protein degradation is an emergent and rapidly evolving therapeutic strategy. In particular, biologics-based targeted degradation modalities (bioPROTACs) are relatively under explored compared to small molecules. Here, we investigate how target affinity, cellular localization, and valency of bioPROTACs impact efficacy of targeted degradation of the oncogenic phosphatase src-homology 2 containing protein tyrosine phosphatase-2 (SHP2). We identify bivalent recruitment of SHP2 by bioPROTACs as a broadly applicable strategy to improve potency. Moreover, we demonstrate that SHP2-targeted bioPROTACs can effectively counteract gain-of-function SHP2 mutants present in cancer, which are otherwise challenging to selectively target with small molecule constructs. Overall, this study demonstrates the utility of bioPROTACs for challenging targets, and further explicates design principles for therapeutic bioPROTACs.

cIAP1/2 Antagonism Induces Antigen-Specific T Cell-Dependent Immunity.

Checkpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naive T cells can receive costimulation not only via CD28 but also through TNF superfamily receptors that signal via NF-κB. Antagonists of the ubiquitin ligases cellular inhibitor of apoptosis protein (cIAP)1/2, also called second mitochondria-derived activator of caspases (SMAC) mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-κB signaling that mimics costimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Dendritic cell activation is enhanced by cIAP1/2 antagonism in vitro, and intratumoral dendritic cells show higher expression of MHC class II in tumors from cIAP1/2 antagonism-treated mice. In this study, we use in vivo mouse models of syngeneic pancreatic cancer that generate endogenous T cell responses ranging from moderate to poor. Across multiple models, cIAP1/2 antagonism has pleiotropic beneficial effects on antitumor immunity, including direct effects on tumor-specific T cells leading to overall increased activation, increased control of tumor growth in vivo, synergy with multiple immunotherapy modalities, and immunologic memory. In contrast to checkpoint blockade, cIAP1/2 antagonism does not increase intratumoral T cell frequencies. Furthermore, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent antitumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses.

Transforming Growth Factor-ß Blockade in Pancreatic Cancer Enhances Sensitivity to Combination Chemotherapy.

BACKGROUND & AIMS: Transforming growth factor-b (TGFb) plays pleiotropic roles in pancreatic cancer, including promoting metastasis, attenuating CD8 T-cell activation, and enhancing myofibroblast differentiation and deposition of extracellular matrix. However, single-agent TGFb inhibition has shown limited efficacy against pancreatic cancer in mice or humans. METHODS: We evaluated the TGFß-blocking antibody NIS793 in combination with gemcitabine/nanoparticle (albumin-bound)-paclitaxel or FOLFIRINOX (folinic acid [FOL], 5-fluorouracil [F], irinotecan [IRI] and oxaliplatin [OX]) in orthotopic pancreatic cancer models. Single-cell RNA sequencing and immunofluorescence were used to evaluate changes in tumor cell state and the tumor microenvironment. RESULTS: Blockade of TGFß with chemotherapy reduced tumor burden in poorly immunogenic pancreatic cancer, without affecting the metastatic rate of cancer cells. Efficacy of combination therapy was not dependent on CD8 T cells, because response to TGFß blockade was preserved in CD8-depleted or recombination activating gene 2 (RAG2-/-) mice. TGFß blockade decreased total α-smooth muscle actin-positive fibroblasts but had minimal effect on fibroblast heterogeneity. Bulk RNA sequencing on tumor cells sorted ex vivo revealed that tumor cells treated with TGFß blockade adopted a classical lineage consistent with enhanced chemosensitivity, and immunofluorescence for cleaved caspase 3 confirmed that TGFß blockade increased chemotherapy-induced cell death in vivo. CONCLUSIONS: TGFß regulates pancreatic cancer cell plasticity between classical and basal cell states. TGFß blockade in orthotropic models of pancreatic cancer enhances sensitivity to chemotherapy by promoting a classical malignant cell state. This study provides scientific rationale for evaluation of NIS793 with FOLFIRINOX or gemcitabine/nanoparticle (albumin-bound) paclitaxel chemotherapy backbone in the clinical setting and supports the concept of manipulating cancer cell plasticity to increase the efficacy of combination therapy regimens.

PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance.

Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA), making developmental regulators therapeutically attractive. Here we demonstrate diverse functions for pancreatic and duodenal homeobox 1 (PDX1), a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of pancreatic intraepithelial neoplasia (PanIN)-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor-suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing epithelial-to-mesenchymal transition (EMT), and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression.

Long-term durability of multibranched endovascular repair of thoracoabdominal and pararenal aortic aneurysms.

OBJECTIVE: The objective of this study was to assess the durability of multibranched endovascular repair of thoracoabdominal aortic aneurysms (TAAAs) and pararenal aortic aneurysms by examining the rates of late-occurring (beyond 30 days) complications. METHODS: There were 146 patients who underwent endovascular TAAA repair using a stent graft, with a total of 538 caudally oriented self-expanding branches. Four patients died in the perioperative period and were excluded, leaving 142 patients (mean age, 73 ± 8 years; 35 [24.7%] women). Follow-up included clinical examination and computed tomography angiography at 1 month, 6 months, and 12 months and yearly thereafter. RESULTS: Mean aneurysm diameter was 67 ± 9 mm. Sixty-seven TAAAs (47.2%) were Crawford type I, II, III, or V; 75 (52.8%) were type IV or pararenal. Three patients (2.1%) died >30 days after operation from perioperative complications. During a mean follow-up of 36 months (±28 months), there were four additional aneurysm-related deaths: one (0.7%) as a result of aneurysm rupture in the presence of untreatable type I endoleak, one (0.7%) after conversion to open repair for stent graft infection, one (0.7%) after occlusion of superior mesenteric artery and celiac branches, and one (0.7%) due to bilateral renal branch occlusion. There was one additional open conversion for stent graft infection (0.7%). Nineteen patients (13.3%) underwent 20 reinterventions for late-occurring complications, including 11 (7.7%) for renal branch occlusion or stenosis, 1 (0.7%) for mesenteric branch stenosis, 4 (2.8%) for graft limb occlusion, 1 (0.7%) for type IB endoleak (distal stent graft migration), and 1 (0.7%) for type III endoleak (fabric erosion); 2 (1.4%) open conversions were performed for stent graft infection. There were no late type IA endoleaks. By Kaplan-Meier analysis, freedom from aneurysm-related death was 91.1% and freedom from aneurysm-related death or reintervention was 76.8% at 5 years. The 5-year overall survival rate of 49.1% reflects the high rate of cardiopulmonary comorbidity. Although renal branch occlusion (23 occlusions of 256 renal branches [8.9%]) was the most common late complication, only five patients required permanent dialysis. CONCLUSIONS: Total endovascular repair of TAAAs and pararenal aortic aneurysms using axially oriented cuffs is safe, effective, and durable in the long term.

Multibranched endovascular aortic aneurysm repair in patients with and without chronic aortic dissections.

OBJECTIVE: The objective of this study was to compare multibranched endovascular aneurysm repair (MBEVAR) of postdissection thoracoabdominal aortic aneurysms (TAAAs) and pararenal aortic aneurysms (PRAAs) with MBEVAR of degenerative TAAAs and PRAAs and to assess the role played by the preoperative correction of potential complicating factors, such as true lumen compression and false lumen origin of vital branches, using adjunctive maneuvers. METHODS: From July 2005 to July 2017, there were 162 patients who underwent elective MBEVAR of TAAAs and PRAAs. Data on demographics, procedural details, and outcomes were collected prospectively. RESULTS: The mean age was 73 ± 8 years, and 119 of 162 (74%) were men; 19 of 162 (12%) had prior aortic dissections. Patients with dissections were younger (65 ± 11 years vs 74 ± 7 years; P = .002) and were less likely to have smoked (13/19 [68%] vs 135/143 [94%]; P = .002) or to have peripheral artery disease (0/19 [0%] vs 35/143 [24%]; P = .01) compared with those without dissections. Patients with prior dissections were more likely to have Crawford type II (10/19 [53%] vs 22/143 [15%]; P = .001) and type III (6/19 [32%] vs 16/143 [11%]; P = .03) TAAAs and were more likely to require at least one pre-MBEVAR adjunctive procedure (14/19 [74%] vs 55/143 [38%]; P = .006) compared with those without dissection. There was no difference in perioperative death, stroke, or paraplegia rates between the two groups. Median follow-up was 2.4 years (interquartile range, 0.8-4.7) and did not differ significantly between the two groups. There were no significant differences in branch vessel occlusion, endoleak rate, or aneurysm-related death between the two groups. CONCLUSIONS: Patients with chronic type B aortic dissection are more likely to have extensive aneurysms and more likely to require adjunctive procedures to provide the appropriate anatomic substrate for MBEVAR, but this does not appear to affect the conduct of MBEVAR or its outcomes.

Strict Control of Blood Glucose With an Intravenous Insulin Infusion Decreases the Risk of Post-operative Lower Extremity Weakness After Complex Endovascular Aortic Aneurysm Repair.

OBJECTIVE/BACKGROUND: It has previously been shown that post-operative lower extremity weakness (LEW) is associated with elevated blood and cerebrospinal fluid (CSF) glucose levels after branched endovascular aneurysms repair (BEVAR) of extensive aortic aneurysms. The purpose of this study was to determine whether a post-operative insulin infusion protocol (IIP) to achieve tight blood glucose control decreases the rate of LEW. METHODS: From October 2013, blood and CSF samples were collected pre-operatively, immediately post-operatively, and on post-operative day one in asymptomatic patients undergoing BEVAR. In July 2016, an IIP was initiated to maintain post-operative blood glucose levels <120 mg/dL for 48 h. Data on demographics, operative repair, complications, and outcomes were collected prospectively. RESULTS: Between October 2013 and April 2018, 43 patients underwent BEVAR. Twenty-two (group A) underwent BEVAR before initiation of the IIP. Of these, seven (32%) developed LEW within 48 h of repair. This was temporary in five (23%) and permanent in two (9%) patients. Post-operative blood glucose levels were significantly higher in patients with LEW compared with those without LEW (140 ± 27 mg/dL vs. 117 ± 16 mg/dL; p = .02). Post-operative CSF glucose levels were significantly higher in patients with LEW compared with those without LEW (102 ± 15 mg/dL vs. 77 ± 15 mg/dL; p = .001). The subsequent 21 patients (group B) underwent BEVAR after initiation of the IIP. No patient in group B developed LEW while on the IIP, but one (5%) developed paraplegia on post-operative day four. The rate of early LEW (<48 h post-operatively) was significantly lower after initiation of the IIP (32% in group A vs. 0% in group B; p = .009). There was no difference in demographics, comorbidities, or operative time between the groups. CONCLUSION: An IIP to control blood glucose after BEVAR is associated with a decreased rate of post-operative LEW. Tight control of blood glucose should be considered after any extensive aortic reconstruction to minimise the risk of post-operative LEW.

Cardiac mitochondrial metabolism may contribute to differences in thermal tolerance of red- and white-blooded Antarctic notothenioid fishes.

Studies in temperate fishes provide evidence that cardiac mitochondrial function and the capacity to fuel cardiac work contribute to thermal tolerance. Here, we tested the hypothesis that decreased cardiac aerobic metabolic capacity contributes to the lower thermal tolerance of the haemoglobinless Antarctic icefish, Chaenocephalus aceratus, compared with that of the red-blooded Antarctic species, Notothenia coriiceps. Maximal activities of citrate synthase (CS) and lactate dehydrogenase (LDH), respiration rates of isolated mitochondria, adenylate levels and changes in mitochondrial protein expression were quantified from hearts of animals held at ambient temperature or exposed to their critical thermal maximum (CTmax). Compared with C. aceratus, activity of CS, ATP concentration and energy charge were higher in hearts of N. coriiceps at ambient temperature and CTmax While state 3 mitochondrial respiration rates were not impaired by exposure to CTmax in either species, state 4 rates, indicative of proton leakage, increased following exposure to CTmax in C. aceratus but not N. coriiceps The interactive effect of temperature and species resulted in an increase in antioxidants and aerobic metabolic enzymes in N. coriiceps but not in C. aceratus Together, our results support the hypothesis that the lower aerobic metabolic capacity of C. aceratus hearts contributes to its low thermal tolerance.

The loss of hemoglobin and myoglobin does not minimize oxidative stress in Antarctic icefishes.

The unusual pattern of expression of hemoglobin (Hb) and myoglobin (Mb) among Antarctic notothenioid fishes provides an exceptional model system for assessing the impact of these proteins on oxidative stress. We tested the hypothesis that the lack of oxygen-binding proteins may reduce oxidative stress. Levels and activity of pro-oxidants and small-molecule and enzymatic antioxidants, and levels of oxidized lipids and proteins in the liver, oxidative skeletal muscle and heart ventricle were quantified in five species of notothenioid fishes differing in the expression of Hb and Mb. Levels of ubiquitinated proteins and rates of protein degradation by the 20S proteasome were also quantified. Although levels of oxidized proteins and lipids, ubiquitinated proteins, and antioxidants were higher in red-blooded fishes than in Hb-less icefishes in some tissues, this pattern did not persist across all tissues. Expression of Mb was not associated with oxidative damage in the heart ventricle, whereas the activity of citrate synthase and the contents of heme were positively correlated with oxidative damage in most tissues. Despite some tissue differences in levels of protein carbonyls among species, rates of degradation by the 20S proteasome were not markedly different, suggesting either alternative pathways for eliminating oxidized proteins or that redox tone varies among species. Together, our data indicate that the loss of Hb and Mb does not correspond with a clear pattern of either reduced oxidative defense or oxidative damage.

Rhesus monkeys (Macaca mulatta) remember agency information from past events and integrate this knowledge with spatial and temporal features in working memory.

The purpose of the present study was to examine whether rhesus monkeys remember information about their own agency-along with spatial, temporal and contextual properties-from a previously experienced event. In Experiment 1, rhesus monkeys (n = 4) used symbols to reliably indicate whether they had performed or observed an event on a computer screen. In Experiment 2, naïve and experienced monkeys (n = 8) reported agency information when stringent controls for perceptual and proprioceptive cues were included. In Experiment 3, five of the monkeys completed a task in which they reported agency information along with spatial and temporal features of events. Two monkeys performed this agency discrimination when they could not anticipate which memory test they would receive. There was also evidence that these features were integrated in memory. Implications of this research are discussed in relation to working memory, episodic memory and self-awareness in nonhuman animals.

The Thermodynamic Mechanism of Peptide-MHC Class II Complex Formation Is a Determinant of Susceptibility to HLA-DM.

Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306-319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non-compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by "sensing" flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.

Molecular drivers of mitochondrial membrane proliferation in response to cold acclimation in threespine stickleback.

Little is known about how the synthesis of mitochondrial phospholipids is integrated into mitochondrial biogenesis in fish or mammals. Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the addition of fatty acyl CoA to the sn-1 position of glycerol-3-phosphate, in what is considered the rate-limiting step in phospholipid biosynthesis. Previous studies have shown that mitochondrial volume density increases in oxidative skeletal muscle but not liver of Gasterosteus aculeatus (threespine stickleback) in response to cold acclimation. We hypothesized that maximal activity of GPAT would increase in oxidative skeletal muscle but not liver during cold acclimation, coinciding with mitochondrial biogenesis. GPAT activity was measured in liver and oxidative skeletal (pectoral adductor) muscle of threespine stickleback acclimated to 8°C or 20°C. In addition, mRNA levels of enzymes involved in phospholipid synthesis, including cytidine diphosphodiacylglycerol synthase-1 (CDS1), CDS2, GPAT1, GPAT2 and 1-acylglycerol 3-phosphate acyltransferase-2 (AGPAT2), were quantified in liver and pectoral muscle of stickleback harvested during cold acclimation. GPAT activity and transcript levels of AGPAT2 increased in response to cold acclimation in pectoral muscle but not liver. Transcript levels of GPAT1 increased in liver but not pectoral muscle. Overall our results suggest that the activity of GPAT, and possibly AGPAT as well, increase during cold acclimation and may contribute to mitochondrial phospholipid biosynthesis required for mitochondrial biogenesis.

A Community-Driven Implementation of the Body and Soul Program in Churches in the Twin Cities, Minnesota, 2011-2014.

BACKGROUND: African Americans have high disease and death rates due to cancer and cardiovascular disease. Health promotion efforts to improve diet have the potential to reduce these rates. COMMUNITY CONTEXT: Given their importance in the community and the extent of their reach, churches are effective avenues for health promotion efforts targeting African Americans. The objectives of this project were to promote healthy eating among African American church members, engage African American churches in the implementation of Body and Soul (an evidenced-based program that encourages healthy eating), and implement the program in the community with minimal resources. METHODS: From 2011 through 2014 we conducted a community engagement project to implement the 12-week Body and Soul program, which includes demonstrations of healthy recipes and peer counseling, in 20 churches. Participants (n = 310) completed baseline and follow-up surveys on their eating habits and experience with peer counseling. Church coordinators (n = 11) completed a survey evaluating the program. OUTCOME: Participants' weekly servings of fruit (baseline, 4.3; follow-up, 5.4; P < .001) and vegetables (baseline, 4.5; follow-up, 5.3; P < .001) increased. Church coordinators reported enthusiasm about Body and Soul at their church, and 10 of 11 church coordinators indicated that their pastor encouraged members to attend Body and Soul events. Program success was promoted by engaging the pastor in program activities and by scheduling events soon after church services. Implementation challenges were variation in peer counseling among churches and low turnout at follow-up events. INTERPRETATION: The project was successfully implemented in the 20 churches, and increases in healthy eating were observed. This project demonstrated that Body and Soul can be implemented in communities with little funds or other resources.

A Novel Gain-of-Signal Assay to Detect Targeted Protein Degradation.

Targeted protein degradation offers a promising avenue for expanding therapeutic development to previously inaccessible proteins of interest by regulating the target abundance rather than activity. However, current methods to screen for effective degraders serve as major bottlenecks for the development of degrader therapies. Here, we develop a novel assay platform for identification and characterization of macromolecules capable of inducing targeted degradation of oncogenic phosphatase SHP2. Unlike traditional reporter assays that utilize loss-of-signal readouts to detect degradation, our assay platform expresses a robust fluorescence signal in response to the depletion of a target protein and incorporates additional measures intended to prevent undesirable false positives. Using this gain-of-signal assay, we successfully identified novel macromolecule SHP2 degraders from a screen of 192 candidates and proposed design principles for further development of macromolecule degraders. This work demonstrates a proof of concept for gain-of-signal assays as a tool for screening targeted degrader candidates.

Tuft cells transdifferentiate to neural-like progenitor cells in the progression of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is partly initiated through the transdifferentiation of acinar cells to metaplastic ducts that act as precursors of neoplasia and cancer. Tuft cells are solitary chemosensory cells not found in the normal pancreas but arise in metaplasia and neoplasia, diminishing as neoplastic lesions progress to carcinoma. Metaplastic tuft cells (mTCs) function to suppress tumor progression through communication with the tumor microenvironment, but their fate during progression is unknown. To determine the fate of mTCs during PDA progression, we have created a lineage tracing model that uses a tamoxifen-inducible tuft-cell specific Pou2f3CreERT/+ driver to induce transgene expression, including the lineage tracer tdTomato or the oncogene Myc. mTC lineage trace models of pancreatic neoplasia and carcinoma were used to follow mTC fate. We found that mTCs, in the carcinoma model, transdifferentiate into neural-like progenitor cells (NRPs), a cell type associated with poor survival in PDA patients. Using conditional knock-out and overexpression systems, we found that Myc activity in mTCs is necessary and sufficient to induce this Tuft-to-Neuroendocrine-Transition (TNT).

Strategies to promote language inclusion at 17 CTSA hubs.

The prioritization of English language in clinical research is a barrier to translational science. We explored promising practices to advance the inclusion of people who speak languages other than English in research conducted within and supported by NIH Clinical Translational Science Award (CTSA) hubs. Key informant interviews were conducted with representatives (n = 24) from CTSA hubs (n = 17). Purposive sampling was used to identify CTSA hubs focused on language inclusion. Hubs electing to participate were interviewed via Zoom. Thematic analysis was performed to analyze interview transcripts. We report on strategies employed by hubs to advance linguistic inclusion and influence institutional change that were identified. Strategies ranged from translations, development of culturally relevant materials and consultations to policies and procedural changes and workforce initiatives. An existing framework was adapted to conceptualize hub strategies. Language justice is paramount to bringing more effective treatments to all people more quickly. Inclusion will require institutional transformation and CTSA hubs are well positioned to catalyze change.

Biomarkers of pembrolizumab efficacy in advanced anal squamous cell carcinoma: analysis of a phase II clinical trial and a cohort of long-term responders.

BACKGROUND: Recent trials suggest that programmed cell death 1 (PD-1)-directed immunotherapy may be beneficial for some patients with anal squamous cell carcinoma and biomarkers predictive of response are greatly needed. METHODS: This multicenter phase II clinical trial (NCT02919969) enrolled patients with metastatic or locally advanced incurable anal squamous cell carcinoma (n=32). Patients received pembrolizumab 200 mg every 3 weeks. The primary endpoint of the trial was objective response rate (ORR). Exploratory objectives included analysis of potential predictive biomarkers including assessment of tumor-associated immune cell populations with multichannel immunofluorescence and analysis of circulating tumor tissue modified viral-human papillomavirus DNA (TTMV-HPV DNA) using serially collected blood samples. To characterize the clinical features of long-term responders, we combined data from our prospective trial with a retrospective cohort of patients with anal cancer treated with anti-PD-1 immunotherapy (n=18). RESULTS: In the phase II study, the ORR to pembrolizumab monotherapy was 9.4% and the median progression-free survival was 2.2 months. Despite the high level of HPV positivity observed with circulating TTMV-HPV DNA testing, the majority of patients had low levels of tumor-associated CD8+PD-1+ T cells on pretreatment biopsy. Patients who benefited from pembrolizumab had decreasing TTMV-HPV DNA scores and a complete responder's TTMV-HPV DNA became undetectable. Long-term pembrolizumab responses were observed in one patient from the trial (5.3 years) and three patients (2.5, 6, and 8 years) from the retrospective cohort. Long-term responders had HPV-positive tumors, lacked liver metastases, and achieved a radiological complete response. CONCLUSIONS: Pembrolizumab has durable efficacy in a rare subset of anal cancers. However, despite persistence of HPV infection, indicated by circulating HPV DNA, most advanced anal cancers have low numbers of tumor-associated CD8+PD-1+ T cells and are resistant to pembrolizumab.

Mechanisms of resistance to oncogenic KRAS inhibition in pancreatic cancer.

KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, and Cdk6/Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies.

Demonstrating the Value of the Clinical Nurse Specialist: A "How-to" Guide.

PURPOSE/OBJECTIVES: The purpose of this article is to provide a guide to identifying measurements of value and how to calculate different types of return on investment (ROI). Exemplars of clinical nurse specialist (CNS) work efforts with ROI will be presented. DESCRIPTION OF THE PROJECT/PROBLEM: The CNS is the oldest advanced practice role; however, the role can be vague, making it difficult to articulate the value of the CNS in the organization. Functioning in 3 spheres of impact, the CNS can easily impact practice; however, demonstrating the value of this work is not always on top of mind and is rarely taught in academic programs. OUTCOME: This article describes the difference between revenue generation, cost savings, and cost avoidance, as well as various structural, process, and outcome measures that can be used to calculate ROI. Resources available for performing ROI calculations will be described and shared. CONCLUSION: Distributing work across the 3 spheres creates various opportunities for the CNS to demonstrate value; however, the CNS must be able to articulate that value to the organization. Developing the skill set to consistently identify metrics can be challenging; however, it is critical to the ongoing success and future of the CNS role. Utilizing these metrics to demonstrate the value and then disseminating the outcomes of these contributions will continue to promote the value of the CNS in the future.