pVACview: an interactive visualization tool for efficient neoantigen prioritization and selection.

Background: Neoantigen targeting therapies including personalized vaccines have shown promise in the treatment of cancers, particularly when used in combination with checkpoint blockade therapy. At least 100 clinical trials involving these therapies are underway globally. Accurate identification and prioritization of neoantigens is highly relevant to designing these trials, predicting treatment response, and understanding mechanisms of resistance. With the advent of massively parallel DNA and RNA sequencing technologies, it is now possible to computationally predict neoantigens based on patient-specific variant information. However, numerous factors must be considered when prioritizing neoantigens for use in personalized therapies. Complexities such as alternative transcript annotations, various binding, presentation and immunogenicity prediction algorithms, and variable peptide lengths/registers all potentially impact the neoantigen selection process. There has been a rapid development of computational tools that attempt to account for these complexities. While these tools generate numerous algorithmic predictions for neoantigen characterization, results from these pipelines are difficult to navigate and require extensive knowledge of the underlying tools for accurate interpretation. This often leads to over-simplification of pipeline outputs to make them tractable, for example limiting prediction to a single RNA isoform or only summarizing the top ranked of many possible peptide candidates. In addition to variant detection, gene expression and predicted peptide binding affinities, recent studies have also demonstrated the importance of mutation location, allele-specific anchor locations, and variation of T-cell response to long versus short peptides. Due to the intricate nature and number of salient neoantigen features, presenting all relevant information to facilitate candidate selection for downstream applications is a difficult challenge that current tools fail to address. Results: We have created pVACview, the first interactive tool designed to aid in the prioritization and selection of neoantigen candidates for personalized neoantigen therapies including cancer vaccines. pVACview has a user-friendly and intuitive interface where users can upload, explore, select and export their neoantigen candidates. The tool allows users to visualize candidates across three different levels, including variant, transcript and peptide information. Conclusions: pVACview will allow researchers to analyze and prioritize neoantigen candidates with greater efficiency and accuracy in basic and translational settings The application is available as part of the pVACtools pipeline at pvactools.org and as an online server at pvacview.org.

Dendritic cell subsets and implications for cancer immunotherapy.

Dendritic cells (DCs) play a central role in the orchestration of effective T cell responses against tumors. However, their functional behavior is context-dependent. DC type, transcriptional program, location, intratumoral factors, and inflammatory milieu all impact DCs with regard to promoting or inhibiting tumor immunity. The following review introduces important facets of DC function, and how subset and phenotype can affect the interplay of DCs with other factors in the tumor microenvironment. It will also discuss how current cancer treatment relies on DC function, and survey the myriad ways with which immune therapy can more directly harness DCs to enact antitumor cytotoxicity.

Overexpression of human DNA polymerase theta is a biomarker of aggressive and DNA repair-deficient papillary thyroid cancers.

BACKGROUND: DNA polymerase theta (POLQ) is an enzyme that repairs double-strand DNA breaks. POLQ is overexpressed in several cancer types, and increased expression is associated with a poor prognosis. Ablating POLQ function in vitro increases drug sensitivity to agents that cause double-strand DNA breaks, including chemotherapies and ionizing radiation. POLQ's role in thyroid cancer remains poorly understood. METHODS: Expression of POLQ and other genes of interest were analyzed in 513 papillary thyroid cancers (505 primary tumors and 8 metastatic lesions) and 59 normal thyroid samples available in the Cancer Genome Atlas. The Cancer Genome Atlas RNA and DNA sequencing data were queried with the Xena platform. The Recombination Proficiency Score was calculated to assess DNA repair efficiency. Other signaling events associated with thyroid tumorigenesis and clinical outcomes were analyzed. Univariate and multivariate analyses were performed. Treatment with the POLQ inhibitors ART558 and Novobiocin tested the effect of POLQ inhibition on in vitro thyroid cancer growth. RESULTS: POLQ expression was increased in papillary thyroid cancers compared to normal thyroid tissue (P < .05). POLQ expression levels were inversely correlated with Recombination Proficiency Score levels (P < .05). POLQ expression was highest in tall cell papillary thyroid cancers and in metastases. Higher POLQ expression was also associated with dedifferentiation, BRAF signaling, and shorter progression-free intervals (P < .05). Treatment with POLQ inhibitors decreased in vitro thyroid cancer growth (P < .05). CONCLUSION: These findings suggest that increased POLQ expression could serve as a valuable clinical marker and a potential therapeutic target in the treatment of thyroid cancer.

Transcription factor C/EBPα is required for the development of Ly6Chi monocytes but not Ly6Clo monocytes.

Monocytes comprise two major subsets, Ly6Chi classical monocytes and Ly6Clo nonclassical monocytes. Notch2 signaling in Ly6Chi monocytes triggers transition to Ly6Clo monocytes, which require Nr4a1, Bcl6, Irf2, and Cebpb. By comparison, less is known about transcriptional requirements for Ly6Chi monocytes. We find transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) is highly expressed in Ly6Chi monocytes, but down-regulated in Ly6Clo monocytes. A few previous studies described the requirement of C/EBPα in the development of neutrophils and eosinophils. However, the role of C/EBPα for in vivo monocyte development has not been understood. We deleted the Cebpa +37 kb enhancer in mice, eliminating hematopoietic expression of C/EBPα, reproducing the expected neutrophil defect. Surprisingly, we also found a severe and selective loss of Ly6Chi monocytes, while preserving Ly6Clo monocytes. We find that BM progenitors from Cebpa +37-/- mice rapidly progress through the monocyte progenitor stage to develop directly into Ly6Clo monocytes even in the absence of Notch2 signaling. These results identify a previously unrecognized role for C/EBPα in maintaining Ly6Chi monocyte identity.

Cost-Effectiveness of Parathyroid Cryopreservation and Autotransplantation.

Importance: Delayed autotransplantation of cryopreserved parathyroid tissue (DACP) is the only surgical treatment for permanent postoperative hypoparathyroidism. Studies suggest that only a small minority of cryopreserved samples are ultimately autotransplanted with highly variable outcomes. For these reasons, many have questioned the economic utility of the process, although, to the authors' knowledge, this has never been formally studied. Objective: To report the clinical outcomes of parathyroid cryopreservation and DACP at a large academic institution and to determine the cost-effectiveness of this treatment. Design, Setting, and Participants: An institutional review board-approved, retrospective review of patients at a single institution who underwent DACP over a 17-year period was conducted with a median follow-up of 48.2 months. A forward-looking cost-utility analysis was then performed to determine the economic utility of cryopreservation/DACP vs usual care (monitoring and supplementation). Patients who had parathyroid tissue in cryopreserved storage between August 2005 to September 2022 at a single-center, academic, quaternary care center were identified. Exposure: Parathyroid cryopreservation and DACP. Main Outcomes and Measures: Graft functionality, clinical outcomes, and cost utility using a willingness-to-pay threshold of $100 000 per quality-adjusted life-year (QALY). Results: A total of 591 patients underwent cryopreservation. Of these, 10 patients (1.7%; mean [SD] age, 45.6 [17.9] years; 6 male [60%]) underwent DACP. A minority of autografts (2 [20%]) were subsequently fully functional, one-half (5 [50%]) were partially functional, and 3 (30%) were not functional. The cost-utility model estimated that at a large academic center over 10 years, the additional cost of 591 patients undergoing cryopreservation and 10 patients undergoing autotransplantation would be $618 791.64 (2022 dollars) and would add 8.75 QALYs, resulting in a cost per marginal QALY of $70 719.04, which is less than the common willingness-to-pay threshold of $100 000/QALY. Conclusions and Relevance: The reimplantation rate of cryopreserved tissue was low (<2%), but when implanted, autografts were at least partially functional 70% of the time. In the first-ever, to the authors' knowledge, formal cost analysis for this treatment, results of the current model suggest that cryopreservation and autotransplantation were cost-effective compared with the usual care for hypoparathyroidism at a large, academic institution. It is recommended that each surgical center consider whether the economic and logistical commitments necessary for cryopreservation are worthwhile for their individual needs.

Molecular characteristics of isthmus papillary thyroid cancers: Supporting evidence for unfavorable clinical behavior.

BACKGROUND: Previous studies demonstrate isthmus thyroid nodules are more likely to be malignant than lobar nodules. Additional data suggest that isthmus papillary thyroid cancers (PTCs) are more aggressive than lobar PTCs. We hypothesize that isthmus PTCs have a more unfavorable molecular profile. METHODS: The Cancer Genome Atlas (TCGA) database was queried to analyze clinical, mutation and gene expression data of isthmus PTCs compared to non-isthmus PTCs. RESULTS: We analyzed characteristics of 472 â€‹PTCs, including 19 isthmus PTCs. There were no significant differences between isthmus and non-isthmus PTC demographic and clinical variables or the frequency of RAS family, fusion driver, TERT, and tumor suppressor gene mutations. There was a trend towards increased BRAF mutations (68% vs 55%, p â€‹= â€‹0.28). A more aggressive gene expression profile was observed in isthmus PTC compared to lobar/multifocal PTC with differences in ERK score (19.4 vs 7.71, p â€‹< â€‹0.05) and TDS score (-0.58 vs 0.02, p â€‹< â€‹0.05). CONCLUSIONS: These results provide a possible molecular explanation for the more aggressive behavior reported in isthmus PTCs.

Transforming the Future of Surgeon-Scientists.

OBJECTIVE: To create a blueprint for surgical department leaders, academic institutions, and funding agencies to optimally support surgeon-scientists. BACKGROUND: Scientific contributions by surgeons have been transformative across many medical disciplines. Surgeon-scientists provide a distinct approach and mindset toward key scientific questions. However, lack of institutional support, pressure for increased clinical productivity, and growing administrative burden are major challenges for the surgeon-scientist, as is the time-consuming nature of surgical training and practice. METHODS: An American Surgical Association Research Sustainability Task Force was created to outline a blueprint for sustainable science in surgery. Leaders from top NIH-sponsored departments of surgery engaged in video and in-person meetings between January and April 2023. A strength, weakness, opportunities, threats analysis was performed, and workgroups focused on the roles of surgeons, the department and institutions, and funding agencies. RESULTS: Taskforce recommendations: (1) SURGEONS: Growth mindset : identifying research focus, long-term planning, patience/tenacity, team science, collaborations with disparate experts; Skill set : align skills and research, fill critical skill gaps, develop team leadership skills; DEPARTMENT OF SURGERY (DOS): (2) MENTORSHIP: Chair : mentor-mentee matching/regular meetings/accountability, review of junior faculty progress, mentorship training requirement, recognition of mentorship (eg, relative value unit equivalent, awards; Mentor: dedicated time, relevant scientific expertise, extramural funding, experience and/or trained as mentor, trusted advisor; Mentee : enthusiastic/eager, proactive, open to feedback, clear about goals; (3) FINANCIAL SUSTAINABILITY: diversification of research portfolio, identification of matching funding sources, departmental resource awards (eg, T-/P-grants), leveraging of institutional resources, negotiation of formalized/formulaic funds flow investment from academic medical center toward science, philanthropy; (4) STRUCTURAL/STRATEGIC SUPPORT: Structural: grants administrative support, biostats/bioinformatics support, clinical trial and research support, regulatory support, shared departmental laboratory space/equipment; Strategic: hiring diverse surgeon-scientist/scientists faculty across DOS, strategic faculty retention/ recruitment, philanthropy, career development support, progress tracking, grant writing support, DOS-wide research meetings, regular DOS strategic research planning; (5) COMMUNITY AND CULTURE: Community: right mix of faculty, connection surgeon with broad scientific community; Culture: building research infrastructure, financial support for research, projecting importance of research (awards, grand rounds, shoutouts); (6) THE ROLE OF INSTITUTIONS: Foundation: research space co-location, flexible start-up packages, courses/mock study section, awards, diverse institutional mentorship teams; Nurture: institutional infrastructure, funding (eg, endowed chairs), promotion friendly toward surgeon-scientists, surgeon-scientists in institutional leadership positions; Expectations: RVU target relief, salary gap funding, competitive starting salaries, longitudinal salary strategy; (7) THE ROLE OF FUNDING AGENCIES: change surgeon research training paradigm, offer alternate awards to K-awards, increasing salary cap to reflect market reality, time extension for surgeon early-stage investigator status, surgeon representation on study section, focused award strategies for professional societies/foundations. CONCLUSIONS: Authentic recommitment from surgeon leaders with intentional and ambitious actions from institutions, corporations, funders, and society is essential in order to reap the essential benefits of surgeon-scientists toward advancements of science.

Epigenetic regulation during cancer transitions across 11 tumour types.

Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis1-4. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of ABCC1 and VEGFA; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of FGF19, ASAP2 and EN1, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial-mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.

Engineering a Novel Modular Adenoviral mRNA Delivery Platform Based on Tag/Catcher Bioconjugation.

mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, the mucosal administration of mRNA based on lipid nanoparticle technology faces challenges in clinical translation. In contrast, adenovirus-based vaccines induce strong T cell responses and have been approved for intranasal delivery. To leverage the inherent strengths of both the mRNA and adenovirus platforms, we developed a novel modular adenoviral mRNA delivery platform based on Tag/Catcher bioconjugation. Specifically, we engineered adenoviral vectors integrating Tag/Catcher proteins at specific locales on the Ad capsid proteins, allowing us to anchor mRNA to the surface of engineered Ad viruses. In proof-of-concept studies, the Ad-mRNA platform successfully mediated mRNA delivery and could be optimized via the highly flexible modular design of both the Ad-mRNA and protein bioconjugation systems.

Magnitude of parathyroid hormone elevation in primary hyperparathyroidism: Does time of day matter?

BACKGROUND: Parathyroid hormone demonstrates a circadian rhythm in nondiseased patients, but it is unclear if this diurnal variation persists in the context of primary hyperparathyroidism. We anecdotally noticed that parathyroid hormone levels drawn early on the morning of parathyroid surgery (preincision parathyroid hormone), were of lower magnitude than values obtained at later times in the day. If present, a time-of-day based variation in parathyroid hormone could have important clinical implications on intraoperative surgical decision making. METHODS: We performed an Institutional Review Board-approved, retrospective chart review of patients undergoing parathyroidectomy for primary hyperparathyroidism between October 2019 and February 2022 at a quaternary care referral center. Demographic, laboratory, imaging, and operative parameters were extracted. Analysis was performed using mixed models for repeated measures with a first order autoregression correlation structure. Parathyroid hormone values were compared before and after hourly intervals between 6:00 A.M. and 12:00 P.M. RESULTS: Of 418 patients, the mean age was 61 years old, 80% of patients were female, and two-thirds had single-gland disease. A total of 933 parathyroid hormone levels were included in the analysis and median parathyroid hormone was 97.3 pg/mL. Parathyroid hormone levels were noted to be significantly lower if they were drawn before 7:00 A.M. This diurnal variation persisted in patients with single-gland and advanced hyperparathyroidism but was abrogated in multi-gland and low-baseline-parathyroid hormone disease. CONCLUSION: In patients with primary hyperparathyroidism, parathyroid hormone levels were significantly lower in the early morning hours, especially in patients with single-gland and high-baseline-parathyroid hormone hyperparathyroidism. This may have implications for intraoperative decision making when utilizing an early morning, preincision parathyroid hormone value.

Enhanced in vitro type 1 conventional dendritic cell generation via the recruitment of hematopoietic stem cells and early progenitors by Kit ligand.

In vitro culture of bone marrow (BM) with Fms-like tyrosine kinase 3 ligand (Flt3L) is widely used to study development and function of type 1 conventional dendritic cells (cDC1). Hematopoietic stem cells (HSCs) and many progenitor populations that possess cDC1 potential in vivo do not express Flt3 and thus may not contribute to Flt3L-mediated cDC1 production in vitro. Here, we present a KitL/Flt3L protocol that recruits such HSCs and progenitors into the production of cDC1. Kit ligand (KitL) is used to expand HSCs and early progenitors lacking Flt3 expression into later stage where Flt3 is expressed. Following this initial KitL phase, a second Flt3L phase is used to support the final production of DCs. With this two-stage culture, we achieved approximately tenfold increased production of both cDC1 and cDC2 compared to Flt3L culture. cDC1 derived from this culture are similar to in vivo cDC1 in their dependence on IRF8, ability to produce IL-12, and induction of tumor regression in cDC1-deficient tumor-bearing mice. This KitL/Flt3L system for cDC1 production will be useful in further analysis of cDC1 that rely on in vitro generation from BM.

IL-6 selectively suppresses cDC1 specification via C/EBPß.

Cytokines produced in association with tumors can impair antitumor immune responses by reducing the abundance of type 1 conventional dendritic cells (cDC1), but the mechanism remains unclear. Here, we show that tumor-derived IL-6 generally reduces cDC development but selectively impairs cDC1 development in both murine and human systems through the induction of C/EBPß in the common dendritic cell progenitor (CDP). C/EBPß and NFIL3 compete for binding to sites in the Zeb2 -165 kb enhancer and support or repress Zeb2 expression, respectively. At homeostasis, pre-cDC1 specification occurs upon Nfil3 induction and consequent Zeb2 suppression. However, IL-6 strongly induces C/EBPß expression in CDPs. Importantly, the ability of IL-6 to impair cDC development is dependent on the presence of C/EBPß binding sites in the Zeb2 -165 kb enhancer, as this effect is lost in Δ1+2+3 mutant mice in which these binding sites are mutated. These results explain how tumor-associated IL-6 suppresses cDC1 development and suggest therapeutic approaches preventing abnormal C/EBPß induction in CDPs may help reestablish cDC1 development to enhance antitumor immunity.

Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology.

Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

Cytosolic EpCAM cooperates with H-Ras to regulate epithelial to mesenchymal transition through ZEB1.

Next generation sequencing of human cancer mutations has identified novel therapeutic targets. Activating Ras oncogene mutations play a central role in oncogenesis, and Ras-driven tumorigenesis upregulates an array of genes and signaling cascades that can transform normal cells into tumor cells. In this study, we investigated the role of altered localization of epithelial cell adhesion molecule (EpCAM) in Ras-expressing cells. Analysis of microarray data demonstrated that Ras expression induced EpCAM expression in normal breast epithelial cells. Fluorescent and confocal microscopy showed that H-Ras mediated transformation also promoted epithelial-to-mesenchymal transition (EMT) together with EpCAM. To consistently localize EpCAM in the cytosol, we generated a cancer-associated EpCAM mutant (EpCAM-L240A) that is retained in the cytosol compartment. Normal MCF-10A cells were transduced with H-Ras together with EpCAM wild-type (WT) or EpCAM-L240A. WT-EpCAM marginally effected invasion, proliferation, and soft agar growth. EpCAM-L240A, however, markedly altered cells and transformed to mesenchymal phenotype. Ras-EpCAM-L240A expression also promoted expression of EMT factors FRA1, ZEB1 with inflammatory cytokines IL-6, IL-8, and IL1. This altered morphology was reversed using MEK-specific inhibitors and to some extent JNK inhibition. Furthermore, these transformed cells were sensitized to apoptosis using paclitaxel and quercetin, but not other therapies. For the first time, we have demonstrated that EpCAM mutations can cooperate with H-Ras and promote EMT. Collectively, our results highlight future therapeutic opportunities in EpCAM and Ras mutated cancers.

Computational prediction of MHC anchor locations guides neoantigen identification and prioritization.

Neoantigens are tumor-specific peptide sequences resulting from sources such as somatic DNA mutations. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. Because most somatic mutations are single-nucleotide variants, changes between wild-type and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient's specific MHC molecules. Whereas a subset of peptide positions are presented to the T cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T cell responses. We computationally predicted anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6 to 38% of neoantigen candidates are potentially misclassified and can be rescued using allele-specific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline, and improve the identification process for relevant clinical studies.

Analysis of Preoperative Predictors of Single and Multigland Primary Hyperparathyroidism.

INTRODUCTION: Preoperative differentiation of single-gland (SG) versus multigland (MG) primary hyperparathyroidism (PHPT) can assist with surgical planning, treatment prognostication, and patient counseling. The aim of this study was to identify preoperative predictors of SG-PHPT. METHODS: Retrospective analysis of 408 patients with PHPT who underwent parathyroidectomy at a tertiary referral center. Comprehensive preoperative parameters, including demographic, laboratory, clinical, and imaging results were analyzed. Univariate analysis and binary logistic regression identified preoperative predictors of SG-PHPT. Receiver operator curves were used to analyze the predictive values of existing and novel preoperative predictive models. RESULTS: Elevated parathyroid hormone (PTH) (99.1 pg/mL in SG versus 93.0 pg/mL in MG), elevated calcium (10.8 mg/dL in SG versus 10.6 mg/dL in MG), lower phosphate levels (2.80 mg/dL in SG versus 2.95 mg/dL in MG), and positive imaging (ultrasound 75.6% in SG versus 56.5% in MG; sestamibi 70.8% in SG versus 45.5% in MG) were significantly associated with SG-PHPT. The Washington University Score (a predictive scoring system made from calcium, PTH, phosphate, ultrasound, and sestamibi) and the Washington University Index ([calcium × PTH]/phosphate) were comparable to previous scoring systems used to predict SG versus MG-PHPT. CONCLUSIONS: The association of lower phosphate with SG-PHPT is a novel finding. Previously identified predictors of SG-PHPT, including elevated PTH and positive imaging were confirmed. The Washington University Score and Index are comparable to previously described models and can be used to help surgeons predict if a patient may have SG versus MG-PHPT.

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.

Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Background: Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. Methods: We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Results: Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Conclusions: Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

A randomized phase 2 study of neoadjuvant carboplatin and paclitaxel with or without atezolizumab in triple negative breast cancer (TNBC) - NCI 10013.

Atezolizumab with chemotherapy has shown improved progression-free and overall survival in patients with metastatic PD-L1 positive triple negative breast cancer (TNBC). Atezolizumab with anthracycline- and taxane-based neoadjuvant chemotherapy has also shown increased pathological complete response (pCR) rates in early TNBC. This trial evaluated neoadjuvant carboplatin and paclitaxel with or without atezolizumab in patients with clinical stages II-III TNBC. The co-primary objectives were to evaluate if chemotherapy and atezolizumab increase pCR rate and tumor infiltrating lymphocyte (TIL) percentage compared to chemotherapy alone in the mITT population. Sixty-seven patients (ages 25-78 years; median, 52 years) were randomly assigned - 22 patients to Arm A, and 45 to Arm B. Median follow up was 6.6 months. In the modified intent to treat population (all patients evaluable for the primary endpoints who received at least one dose of combination therapy), the pCR rate was 18.8% (95% CI 4.0-45.6%) in Arm A, and 55.6% (95% CI 40.0-70.4%) in Arm B (estimated treatment difference: 36.8%, 95% CI 8.5-56.6%; p = 0.018). Grade 3 or higher treatment-related adverse events occurred in 62.5% of patients in Arm A, and 57.8% of patients in Arm B. One patient in Arm B died from recurrent disease during the follow-up period. TIL percentage increased slightly from baseline to cycle 1 in both Arm A (mean ± SD: 0.6% ± 21.0%) and Arm B (5.7% ± 15.8%) (p = 0.36). Patients with pCR had higher median TIL percentages (24.8%) than those with non-pCR (14.2%) (p = 0.02). Although subgroup analyses were limited by the small sample size, PD-L1-positive patients treated with chemotherapy and atezolizumab had a pCR rate of 75% (12/16). The addition of atezolizumab to neoadjuvant carboplatin and paclitaxel resulted in a statistically significant and clinically relevant increased pCR rate in patients with clinical stages II and III TNBC. (Funded by National Cancer Institute).