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Introduction Triple negative breast cancer (TNBC) is a heterogeneous breast cancer subtype which continues to portend a particularly poor prognosis compared to other breast cancer subtypes. Immune checkpoint inhibitor (ICI) therapies have emerged as promising options for locally advanced and metastatic TNBC. However, clinical trials have demonstrated mixed results with respect to ICI response. Given varied outcomes and the potential for immune-related adverse events associated with ICIs, there is critical need for identification of accurate biomarkers of response and improved strategies to counteract ICI resistance and/or toxicity. Methods Advanced immunoprofiling of peripheral blood mononuclear cells (PBMCs) and plasma from 7 metastatic TNBC patients with variable responses to anti-PD1 or anti-PDL1 therapy was performed. Samples were analyzed from blood draws obtained: (i) prior to first administration, (ii) while receiving ICI treatment, and (iii) at the time of conformed clinical progression or response to therapy. Response was determined by standard radiological assessment. Immunoprofiling included high parameter flow cytometry, single cell transcriptomics (10x genomics) and secretome analysis (Isoplexis). Single cell RNA profiles from 63,984 cells were analyzed. Results High parameter flow cytometry identified higher circulating levels of a subpopulation of activated CD4+ T cells with a phenotype of CXCR3low CD62Llow CD45RAhigh and CD57high expression in responders versus non-responders. Higher effector function of CD4+ T cells was corroborated by significantly elevated plasma concentrations of IL-2 (p=0.02) and IL-5 (p< 0.0001) among responders. Single cell transcriptomic analysis revealed clusters of B and T cells with distinct activation patterns that were associated with radiographic response. Strikingly, genes involved in B cell activation and T cell-B cell conjugation such as CD81 were found to be highly upregulated among CD4+ T cells from responders. Conclusions Our results are consistent with previous reports describing an association of increased B cell activity in TNBC with improved overall survival. We identified a subpopulation of CD4+ T cells with effector functions consistent with type 2 helper T cells that may not only target cancer cells by direct cytotoxic function, but also promote increased B cell anti-tumor activity. Our study provides insight into specific mechanisms of immune cell interplay that may drive response to ICI therapy. These cell populations and their associated pathways may represent potential biomarkers of response and/or targets for resistance reversal. Citation Format: Avia D. Wilkerson, Patricia A. Rayman, Paul G. Pavicic Jr, Hana Husic, Vladimir Makarov, Ivan Juric, Timothy Chan, Alberto J. Montero, Marcela Diaz-Montero. A multiomic approach to the identification of immune signatures of anti-PD1/PDL1 therapy responders in metastatic triple negative breast cancer: new implications in the role of helper T-cell and B-cell interplay [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-04.

Disputes relating to cryptoassets have proliferated in recent years, along with the rise of the cryptoasset market. Some of these can be resolved using traditional principles of contract, tort or trust law, but proprietary issues raise particular conundrums. While cryptoassets have generally been accepted to be property, that is merely the starting point. To properly resolve proprietary disputes, it is necessary to provide a reasoned and robust explanation for why particular rules of title originally developed in the context of tangible property should apply. In turn, two foundational questions must be answered. First, what is the subject-matter of the property right in cryptoassets? And secondly, what is the proprietary effect of a blockchain transaction? These issues have received relatively little attention in the literature, which has focused on whether cryptoassets are property at all, and existing contributions (including the Law Commission's recent Consultation Paper on Digital Assets) which do engage these issues are far from reaching consensus. This paper critically examines those views and puts forward its own reasoned approach for the application of traditional rules of title to cryptoassets.

Supp. Fig.1. CONSORT Flow Diagram Supp. Fig.2. MRI Perfusion Parameters Supp. Fig. 3. Immunohistochemistry Supp. Fig. 4. Gene Expression Profile Supp. Table 1. Adverse Events Supp. Table 2. Pharmacokinetic Data Supp. Table 3. Molar Drug Levels Supplemental Methods

EIF1AX mutations in fatal non-anaplastic thyroid cancer.

List of 410 genes in IMPACT sequencing platform

Supplementary legend

All IMPACT mutation calls. Tumors previously published by Landa et al (1) are highlighted in blue.

Purpose: High-grade gliomas are associated with a dismal prognosis. Notch inhibition via the gamma-secretase inhibitor RO4929097 has emerged as a potential therapeutic option based on modulation of the cancer-initiating cell (CIS) population and a presumed antiangiogenic role.Experimental Design: In this phase 0/I trial, 21 patients with newly diagnosed glioblastoma or anaplastic astrocytoma received RO4929097 combined with temozolomide and radiotherapy. In addition to establishing the MTD, the study design enabled exploratory studies evaluating tumor and brain drug penetration and neuroimaging parameters. We also determined functional effects on the Notch pathway and targeting of CISs through analysis of tumor tissue sampled from areas with and without blood–brain barrier disruption. Finally, recurrent tumors were also sampled and assessed for Notch pathway responses while on treatment.Results: Treatment was well tolerated and no dose-limiting toxicities were observed. IHC of treated tumors showed a significant decrease in proliferation and in the expression of the Notch intracellular domain (NICD) by tumor cells and blood vessels. Patient-specific organotypic tumor explants cultures revealed a specific decrease in the CD133+ CIS population upon treatment. Perfusion MRI demonstrated a significant decrease in relative plasma volume after drug exposure. Gene expression data in recurrent tumors suggested low Notch signaling activity, the upregulation of key mesenchymal genes, and an increase in VEGF-dependent angiogenic factors.Conclusions: The addition of RO4929097 to temozolomide and radiotherapy was well tolerated; the drug has a variable blood–brain barrier penetration. Evidence of target modulation was observed, but recurrence occurred, associated with alterations in angiogenesis signaling pathways. Clin Cancer Res; 22(19); 4786–96. ©2016 AACR.

Purpose: Patients with anaplastic thyroid cancer (ATC) have a very high death rate. In contrast, deaths from non-anaplastic thyroid (NAT) cancer are much less common. The genetic alterations in fatal NAT cancers have not been reported.Experimental Design: We performed next-generation sequencing of 410 cancer genes from 57 fatal NAT primary cancers. Results were compared with The Cancer Genome Atlas study (TCGA study) of papillary thyroid cancers (PTCs) and to the genomic changes reported in ATC.Results: There was a very high prevalence of TERT promoter mutations, comparable with that of ATC, and these co-occurred with BRAF and RAS mutations. A high incidence of chromosome 1q gain was seen highlighting its importance in tumor aggressiveness. Two novel fusion genes DLG5–RET and OSBPL1A–BRAF were identified. There was a high frequency of mutations in MED12 and these were mutually exclusive to TERT promoter mutations and also to BRAF and RAS mutations. In addition, a high frequency of mutations in RBM10 was identified and these co-occurred with RAS mutations and PIK3CA mutations. Compared with the PTCs in TCGA, there were higher frequencies of mutations in TP53, POLE, PI3K/AKT/mTOR pathway effectors, SWI/SNF subunits, and histone methyltransferases.Conclusions: These data support a model, whereby fatal NAT cancers arise from well-differentiated tumors through the accumulation of key additional genetic abnormalities. The high rate of TERT promoter mutations, MED12 mutations, RBM10 mutations, and chromosome 1q gain highlight their likely association with tumor virulence. Clin Cancer Res; 23(19); 5970–80. ©2017 AACR.

Supplementary Methods from Genomic Deregulation during Metastasis of Renal Cell Carcinoma Implements a Myofibroblast-Like Program of Gene Expression

Supplementary Figure 8 from Genomic Deregulation during Metastasis of Renal Cell Carcinoma Implements a Myofibroblast-Like Program of Gene Expression

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Supplementary Figure 5 from Genomic Deregulation during Metastasis of Renal Cell Carcinoma Implements a Myofibroblast-Like Program of Gene Expression

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Supplementary Figure 4 from Genomic Deregulation during Metastasis of Renal Cell Carcinoma Implements a Myofibroblast-Like Program of Gene Expression

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Supplementary Figure 2 from Genomic Deregulation during Metastasis of Renal Cell Carcinoma Implements a Myofibroblast-Like Program of Gene Expression

SummaryGlioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hiTregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence.

PURPOSE Immune checkpoint blockade (ICB) in conjunction with chemotherapy is approved for the treatment of extensive-stage small-cell lung cancer (SCLC). Although specific genomic abnormalities such as KEAP1 and STK11 gene mutations are associated with resistance to ICB in non-SCLC, no genomic abnormality has been found in association with resistance to ICB in SCLC. MATERIALS AND METHODS We first analyzed a retrospective cohort of 42 patients with SCLC treated with single-agent ICB or ICB combination (data set A). We then validated our results in a large prospective clinical trial of 460 patients (CheckMate 032, data set B). DNA and RNA sequencing were performed. RESULTS In data set A, patients treated with ICB with RB1 wild-type (WT) had a median overall survival (OS) of 23.1 months (95% CI, 9 to 37.5), whereas the RB1 mutant OS was 5 months (95% CI, 2.5 to 26; P = .04). Differentially expressed gene analysis between RB1 mutant and RB1 WT samples indicated the enrichment of downregulated immune-related genes and an immune exclusion phenotype among RB1 mutant but not in the RB1 WT tumor samples. We then assessed results from 460 patients enrolled in CheckMate 032, a trial of nivolumab (NIVO) or NIVO + ipilimumab only in SCLC. In this large cohort, RB1 WT patients had significantly improved outcome with NIVO therapy compared with mutant patients (hazard ratio, 1.41; 95% CI, 1.02 to 2.01; P = .041). High RB1 loss-of-function (LOF) signature scores significantly associated with neuroendocrine subtypes (ASCL1 and NeuroD1). However, neuroendocrine subtypes did not associate with OS. Remarkably, patients with lower RB1 LOF scores had longer OS following treatment with NIVO. CONCLUSION SCLC patients with RB1 WT status or lower RB1 LOF signature scores by transcriptomics have better outcomes with ICB monotherapy.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUTMPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUTMPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUTneoepitopes with high affinity are underrepresented in patients with CALRMUTMPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUTMPN would not efficiently respond to a CALRMUTfragment cancer vaccine but would when immunized with a modified CALRMUTheteroclitic peptide vaccine approach. We found that heteroclitic CALRMUTpeptides specifically designed for the MHC-I alleles of patients with CALRMUTMPN efficiently elicited a CALRMUTcross-reactive CD8+T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUTnative peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+T cell response to the CALRMUTfragment upon immunization with a CALRMUTheteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALRMUTMPN.

Germline pathogenic variants (gPV) in cancer susceptibility genes are present in roughly 15% of all melanoma diagnoses. Prior studies note improvements in checkpoint immunotherapy responsiveness in patients with a gPV, which is associated with an increased neo-antigen load in gPV patients. We have also previously reported improvements in melanoma specific survival (MSS) in gPV patients vs non-gPV. No studies to date have examined the immune environment in gPV patients which may account for improvements in survival. In our study, melanoma patients with a personal or family history of multiple cancers were offered germline testing utilizing a commercial germline cancer sequencing panel. Enrolled patients also received a blood draw with subsequent flow cytometry to analyze the immune phenotype. In a cohort of 540 melanoma patients, 80 had a gPV. Multivariable linear regression indicated that significantly fewer polymorphonuclear myeloid derived suppressor cells (PMN-MDSC’s) were noted relative to non-gPV patients when adjusting for melanoma stage at germline testing [β = -.45, 95% CI (-.76, -.15), p = .0036]. This difference was most significant in cutaneous, later stage (IIB-IV) patients, primarily stage III. We also interrogated patterns in gene signature between gPV and non-gPV patients in the TCGA PanCancer cutaneous melanoma cohort. We identified reductions in CCL27 (p = .007) and CCL1 (p = .06), genes associated with MDSC recruitment, across all stages in gPV patients, as well as reductions in ARG1 (p= .05) and S100A7 (p = .08), genes associated with PMN-MDSC’s, in later stage gPV patients. Pathway differences were analyzed using gene set enrichment analysis (GSEA). Analysis of Hallmark gene sets identified significantly upregulated signatures in inflammatory response, TNF-α signaling, IFN-γ signaling, and IL-2 STAT5 signaling in late stage gPV patients. KEGG pathway analysis further identified NK cell mediated cytotoxicity and TCR signaling as significantly upregulated in late stage gPV patients. Notably, fatty acid metabolism pathways, which have been implicated in immunosuppression through MDSC suppressive function, were downregulated in gPV patients. These results suggest that gPV melanoma is associated with both a depletion of PMN-MDSC’s and an upregulation of inflammatory microenvironmental genes in later stages which may explain improved outcomes. Citation Format: Alan Shen, Roshan Lodha, Brandon Bungo, Michelle Arbesman, Nikhil Pramod, Jennifer Ko, Brian Gastman, C. Marcela Diaz, Timothy Chan, Joshua Arbesman, Pauline Funchain. Germline variant positive melanoma patients exhibit an enhanced inflammatory microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5963.

(1) Background: EWS fusion genes are associated with Ewing sarcoma and other Ewing family tumors including desmoplastic small round tumor, DSRCT. We utilize a clinical genomics workflow to reveal real-world frequencies of EWS fusion events, cataloging events that are similar, or divergent at the EWS breakpoint. (2) Methods: EWS fusion events from our next-generation sequencing panel (NGS) samples were first sorted by breakpoint or fusion junctions to map out the frequency of breakpoints. Fusion results were illustrated as in-frame fusion peptides involving EWS and a partner gene. (3) Results: From 2471 patient pool samples for fusion analysis at the Cleveland Clinic Molecular Pathology Laboratory, we identified 182 fusion samples evolved with the EWS gene. They are clustered in several breakpoints: chr22:29683123 (65.9%), and chr22:29688595 (2.7%). About 3/4 of Ewing sarcoma and DSRCT tumors have an identical EWS breakpoint motif at Exon 7 (SQQSSSYGQQ-) fused to a specific part of FLI1 (NPSYDSVRRG or-SSLLAYNTSS), ERG (NLPYEPPRRS), FEV (NPVGDGLFKD) or WT1 (SEKPYQCDFK). Our method also worked with Caris transcriptome data, too. Our primary clinical utility is to use this information to identify neoantigens for therapeutic purposes. (4) Conclusions and future perspectives: our method allows interpretation of what peptides result from the in-frame translation of EWS fusion junctions. These sequences, coupled with HLA-peptide binding data, are used to identify potential sequences of cancer-specific immunogenic peptides for Ewing sarcoma or DSRCT patients. This information may also be useful for immune monitoring (e.g., circulating T-cells with fusion-peptide specificity) to detect vaccine candidates, responses, or residual disease.

Background: While pediatric out-of-hospital cardiac arrests (OHCAs) are relatively uncommon, they have a much higher number of potential years of life lost per event. School-located public access automated external defibrillators (AED) may be beneficial to school-aged OHCAs, but also other OHCAs within the school and in the surrounding community. We sought to identify the incidence of OHCAs within and nearby schools in British Columbia (BC), to estimate the number that may benefit from school-located AEDs. Methods: We used prospectively-collected data from the BC OHCA Registry from 2013 to 2018. We examined the addresses of all OHCAs to determine those occurring in public primary and secondary schools. We geo-plotted all OHCAs to identify the number of OHCAs within walking distance of a school. Assuming an average pedestrian speed for AED retrieval of 1.8 m/second, we calculated the number of school-vicinity OHCAs for which a bystander could retrieve an AED prior to a 6.5 minute emergency medical system response interval, assuming that AEDs would be located on the exterior of a school building. Results: There were a total of 401,423 children enrolled at 824 schools annually in the study footprint. Of a total of 12,480 EMS-treated OHCAs (220 aged < 18 years), 20 were in in schools, of which 4 were Conclusion: Outcomes of school-located OHCAs are encouraging, especially those with AED application. While the incidence of school-located OHCAs is low, a substantial proportion of OHCAs occur within a retrievable distance to a school, and thus accessible external school-located AEDs may improve overall OHCA outcomes of a community.

Introduction: Drone-delivered defibrillators may improve response for out-of-hospital cardiac arrest (OHCA). Prior studies have assumed that drones may be stationed at any police, fire, or paramedic station; however, cross-service implementation may not be logistically feasible. We sought to compare estimated response times by drone base location type. Methods: We included OHCAs (Jan. 2014 to Dec. 2020) in southern Vancouver Island, British Columbia, Canada where OHCA response includes fire and paramedic services. We created four models with candidate drone base locations at: police stations, fire stations, paramedic stations, and on a grid with 1 km sides as an optimistic model. We used mathematical optimization to select 1-5 drone bases for each model. Assuming a drone system had been in place during the study period and accounting for drone availability, we estimated 9-1-1 call-to-defibrillator intervals (measured to either drone, paramedic, or fire arrival) and calculated the proportion of OHCAs where a drone would arrive prior to fire and paramedic for each model. Median response times were compared to historical response using one-sided sign tests. Results: We included 1,610 OHCAs with a median historical response time of 6.4 mins (IQR 5.0-8.6). We identified 21 police stations, 59 fire stations, 21 paramedic stations, and 7,008 grid locations in the study area. Median 9-1-1 call-to-defibrillator intervals ranged from 4.3-5.3 mins for police, 4.3-5.3 mins for fire, 4.5-5.4 mins for paramedic, and 4.2-5.4 mins for grid locations (all P Conclusion: Locating drone bases at different types of emergency service stations significantly decreases 9-1-1 call-to-defibrillator intervals, while resulting in similar response intervals to those achieved using optimistic grid-optimal locations.

Introduction: Use of bystander-administered naloxone may lead to improved likelihood of recovery from opioid overdose. We sought to determine the accessibility of public access naloxone kits on nearby opioid overdose incidents if placed at public transit stops, compared to placing kits outside pharmacies or with existing public access automated external defibrillators (PADs). Methods: We included all incidents in Metro Vancouver, British Columbia responded to by British Columbia Emergency Health Services coded as a drug overdose with naloxone administered on-scene (Dec. 2014 to Aug. 2020). We geo-coded all public transit bus stops and used a mathematical optimization model to select bus stops where publicly accessible naloxone kits could be placed to maximize accessibility (defined as ≤100 m walking distance) to opioid overdoses. We evaluated accessibility on out-of-sample OHCAs using five-fold cross validation and compared against two baseline policies: placing publicly accessible naloxone kits at all pharmacies identified by the College of Pharmacists of British Columbia, and placing kits at all PADs identified by the British Columbia AED Registry. Statistical analysis was conducted using McNemar’s test. Results: We identified 14,318 opioid overdoses, 8,972 bus stops, 736 pharmacies, and 425 PADs. Accessibility of public naloxone kits for opioid overdose locations was 5.1% when placed at all pharmacies and 3.5% when placed with all existing PADs. Optimized naloxone kit placement using bus stops as candidate locations resulted in significantly higher accessibility than both pharmacy and PAD-based placement at 14.8% with 10 optimized locations (P Conclusion: Optimizing placement of public access naloxone kits at select public transit locations can provide significantly higher accessibility to opioid overdose locations compared to placement at pharmacies or at existing PAD locations.

KLHL40-related nemaline myopathy is a severe autosomal recessive muscle disorder. The current study describes 4 cases of KLHL40-related nemaline myopathy in Hong Kong ethnic Chinese presenting within 3 years, which are confirmed with clinicopathologic features and genetic studies. The incidence is estimated to be at least 1 in 45 226 livebirths (at least 1 in 41 608 among ethnic Chinese livebirths) in Hong Kong. Hyponatremia appears to be another common feature in these patients. Salient histological features include nemaline bodies ranging from 200 to 500 nm in diameters on ultrastructural examination as well as negative KLHL40 immunohistochemistry; type II fiber predominance is obvious in 2 cases. We demonstrate the founder effect associated with genetic variant c.1516A>C (p.Thr506Pro) by polymorphic marker analysis, which revealed a 0.56–0.75-Mb or 0.41–0.78-cM shared haplotype encompassing the disease allele. The mutation is believed to have occurred around 412 generations ago or 6220 BCE, as estimated using DMLE+ and a formula described by Boehnke. We believe the founder variant might possibly underlie a sizable portion of nemaline myopathy in ethnic Chinese. Analysis of the KLHL40 gene may be considered as the first-tier testing of congenital myopathy in this ethnic group.