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Hit screening, which involves the identification of compounds or targets capable of modulating disease-relevant processes, is an important step in drug discovery. Some assays, such as image-based high-content screenings, produce complex multivariate readouts. To fully exploit the richness of such data, advanced analytical methods that go beyond the conventional univariate approaches should be employed. In this work, we tackle the problem of hit identification in multivariate assays. As with univariate assays, a hit from a multivariate assay can be defined as a candidate that yields an assay value sufficiently far away in distance from the mean or central value of inactives. Viewed another way, a hit is an outlier from the distribution of inactives. A method was developed for identifying multivariate hit in high-dimensional data sets based on principal components and robust Mahalanobis distance (the multivariate analogue to the Z- or T-statistic). The proposed method, termed mROUT (multivariate robust outlier detection), demonstrates superior performance over other techniques in the literature in terms of maintaining Type I error, false discovery rate and true discovery rate in simulation studies. The performance of mROUT is also illustrated on a CRISPR knockout data set from in-house phenotypic screening programme., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Leong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The ENIGMA research consortium develops and applies methods to determine clinical significance of variants in hereditary breast and ovarian cancer genes. An ENIGMA BRCA1/2 classification sub-group, formed in 2015 as a ClinGen external expert panel, evolved into a ClinGen internal Variant Curation Expert Panel (VCEP) to align with Food and Drug Administration recognized processes for ClinVar contributions. The VCEP reviewed American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) classification criteria for relevance to interpreting BRCA1 and BRCA2 variants. Statistical methods were used to calibrate evidence strength for different data types. Pilot specifications were tested on 40 variants and documentation revised for clarity and ease of use. The original criterion descriptions for 13 evidence codes were considered non-applicable or overlapping with other criteria. Scenario of use was extended or re-purposed for eight codes. Extensive analysis and/or data review informed specification descriptions and weights for all codes. Specifications were applied to pilot variants with pre-existing ClinVar classification as follows: 13 uncertain significance or conflicting, 14 pathogenic and/or likely pathogenic, and 13 benign and/or likely benign. Review resolved classification for 11/13 uncertain significance or conflicting variants and retained or improved confidence in classification for the remaining variants. Alignment of pre-existing ENIGMA research classification processes with ACMG/AMP classification guidelines highlighted several gaps in the research processes and the baseline ACMG/AMP criteria. Calibration of evidence strength was key to justify utility and strength of different data types for gene-specific application. The gene-specific criteria demonstrated value for improving ACMG/AMP-aligned classification of BRCA1 and BRCA2 variants., Competing Interests: Declaration of interests M.A. was a paid employee of Invitae. R.C.C. and R.O. are paid employees of Color Health. M.E.R., T.P., and R.K. are paid employees of Ambry Genetics. A.R.M. and M.J.V. received funds from AstraZeneca for contribution to sponsored quality assessments and variant interpretation of BRCA1 and BRCA2 VUS (funds paid to the institution)., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear., Methods: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo., Results: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed i plectin ( i  = invadopodial). i Plectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed i plectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency., Conclusions: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and i plectin., Clinical Trial Registration Number: NCT04608357., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure., Methods: We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls., Results: Circulating amyloid beta (Aβ), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235., Discussion: This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools., Highlights: Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype in vitro and in vivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers., Competing Interests: Declaration of interests N.G.I. has/had a consulting or advisory role in PairX Therapeutics, Verimmune therapeutics, and Invivo surgical; received honoraria from Agilent, and research funding from Merck, all of which are outside this submitted work. D.S.W.T. received honoraria from Bristol-Myers Squibb, Takeda Pharmaceuticals, Novartis, Roche, and Pfizer; has consulting or advisory role in Novartis, Merck, Loxo Oncology, AstraZeneca, Roche, and Pfizer and received research funding from Novartis (Inst), GlaxoSmithKline (Inst), and AstraZeneca (Inst), outside this submitted work. N.G.I., D.S.W.T., S.Y.T., H.S.L., F.T.C., and D.M.R.-J. are listed as co-inventors on the patent application entitled “Method of Modulating Sensitivity To Tyrosine Kinase Inhibitor” (International Publication: WO2022045976A1), which describes a significant proportion of the data here., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (Pten cKO ) retinas. Wnt signaling molecules are also enriched in Pten cKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Metastasis remains the leading cause of mortality in prostate cancer patients. The presence of tumor cells in lymph nodes is an established prognostic indicator for several cancer types, such as melanoma, breast, oral, pancreatic, and cervical cancers. Emerging evidence highlights the role of microRNAs enclosed within extracellular vesicles as facilitators of molecular communication between tumors and metastatic sites in the lymph nodes. This study aims to investigate the potential diagnostic utility of EV-derived microRNAs in liquid biopsies for prostate cancer. By employing microarrays on paraffin-embedded samples, we characterized the microRNA expression profiles in metastatic lymph nodes, non-metastatic lymph nodes, and primary tumor tissues of prostate cancer. Differential expression of microRNAs was observed in metastatic lymph nodes compared to prostate tumors and non-metastatic lymph node tissues. Three microRNAs (miR-140-3p, miR-150-5p, and miR-23b-3p) were identified as differentially expressed between tissue and plasma samples. Furthermore, we evaluated the expression of these microRNAs in exosomes derived from prostate cancer cells and plasma samples. Intriguingly, high Gleason score samples exhibited the lowest expression of miR-150-5p compared to control samples. Pathway analysis suggested a potential regulatory role for miR-150-5p in the Wnt pathway and bone metastasis. Our findings suggest EV-derived miR-150-5p as a promising diagnostic marker for identifying patients with high-grade Gleason scores and detecting metastasis at an early stage.

Application of the prostate-specific antigen (PSA) test, which measures PSA levels in blood, is standard in prostate cancer (PCa) screening. However, because PSA levels may be elevated for reasons other than PCa, it leads to high rates of misdiagnosis and overtreatment. Recently, alteration in the N -glycan sialylation of PSA, specifically increased levels of α2-3-linked N -acetylneuraminic acid (α2-3-Neu5Ac or α2-3-sialic acid), was identified as a potential biomarker for clinically significant PCa. Here, we introduce a robust top-down native mass spectrometry (MS) approach, performed using a combination of α2-3-Neu5Ac-specific and nonspecific neuraminidases and employing center-of-mass monitoring (CoMMon), for quantifying the levels of α2-3-Neu5Ac as a fraction of total N -linked Neu5Ac present on PSA extracted from blood serum. To illustrate the potential of the assay for clinical diagnosis and disease staging of PCa, the percentages of α2-3-Neu5Ac on PSA (%α23PSA) in the serum of low-grade (International Society of Urological Pathology Grade Group/GG1), intermediate-grade (GG2), and high-grade (GG3,4,5) PCa individuals were measured. We observed a high sensitivity (85.5%) and specificity (84.6%) for discrimination of GG1 from clinically significant GG2-5 patients when using a %α23PSA test cut-off of 28.0%. Our results establish that the %α23PSA in blood serum PSA, which can be precisely measured in a non-invasive manner with our dual neuraminidase native MS/CoMMon assay, can discriminate between clinically significant PCa (GG2-5) and low-grade PCa (GG1). Such discrimination has not been previously achieved and represents an important clinical need. This assay could greatly improve the standard PSA test and serve as a valuable PCa diagnostic tool.

Background: Germline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications., Methods: CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories., Results: Updated CDH1 -specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing., Conclusions: The development and evolution of CDH1- specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations., Competing Interests: Competing interests: MA, MR, AM, TB, CP, TP, CO, SP and RK are an employee, a trainee or a consultant for a commercial laboratory that offers genetic testing for CDH1. LZ reports that family members hold leadership positions and ownership interests in Decipher Medicine., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Profiling tumors at single-cell resolution provides an opportunity to understand complexities underpinning lymph-node metastases in head and neck squamous-cell carcinoma. Single-cell RNAseq (scRNAseq) analysis of cancer-cell trajectories identifies a subpopulation of pre-metastatic cells, driven by actionable pathways including AXL and AURK. Blocking these two proteins blunts tumor invasion in patient-derived cultures. Furthermore, scRNAseq analyses of tumor-infiltrating CD8 + T-lymphocytes show two distinct trajectories to T-cell dysfunction, corroborated by their clonal architecture based on single-cell T-cell receptor sequencing. By determining key modulators of these trajectories, followed by validation using external datasets and functional experiments, we uncover a role for SOX4 in mediating T-cell exhaustion. Finally, interactome analyses between pre-metastatic tumor cells and CD8 + T-lymphocytes uncover a putative role for the Midkine pathway in immune-modulation and this is confirmed by scRNAseq of tumors from humanized mice. Aside from specific findings, this study demonstrates the importance of tumor heterogeneity analyses in identifying key vulnerabilities during early metastasis., (© 2023. The Author(s).)

Background: Limited graft availability is a constant clinical concern. Hence, the umbilical cord (UC) is an attractive alternative to autologous grafts. The UC is an inexhaustible tissue source, and its removal is harmless and part of standard of care after the birth of the baby. Minimal information exists regarding the immunological profile of a whole UC when it is considered to be used as a tissue graft. We aimed to characterize the localization and levels of class I human leukocyte antigens (HLAs) to understand the allogenicity of the UC. Additionally, HLA-E and HLA-G are putative immunosuppressive antigens that are abundant in placenta, but their profiles in UC whole tissue are unclear., Hypothesis: The UC as a whole expresses a relatively low but ubiquitous level of HLA-ABC and significant levels of HLA-G and HLA-E., Methods: Healthy patients with no known pregnancy-related complications were approached for informed consent. UCs at term and between 12 and 19 weeks were collected to compare HLA profiles by gestational age. Formalin-fixed paraffin-embedded tissues were sectioned to 5 µm and immunohistochemically stained with a pan-HLA-ABC, two HLA-G-specific, or an HLA-E-specific antibody., Results: HLA-ABC was consistently found present in UCs. HLA-ABC was most concentrated in the UC vessel walls and amniotic epithelium but more dispersed in the Wharton's Jelly. HLA-E had a similar localization pattern to HLA-ABC in whole UC tissues at both gestational ages, but its protein level was lower. HLA-G localization and intensity were poor in all UC tissues analyzed, but additional analyses by Western immunoblot and mass spectrometry revealed a low level of HLA-G in the UC., Conclusion: The UC may address limitations of graft availability. Rather than the presence of HLA-G, the immunosuppressive properties of the UC are more likely due to the abundance of HLA-E and the interaction known to occur between HLA-E and HLA-ABC. The co-localization of HLA-E and HLA-ABC suggests that HLA-E is likely presenting HLA-ABC leader peptides to immune cells, which is known to have a primarily inhibitory effect.

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo . The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo , enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Digital camera sensors use color filters on photodiodes to achieve color selectivity. As the color filters and photosensitive silicon layers are separate elements, these sensors suffer from optical cross-talk, which sets limits to the minimum pixel size. Here, we report hybrid silicon-aluminum nanostructures in the extreme limit of zero distance between color filters and sensors. This design could essentially achieve submicrometer pixel dimensions and minimize the optical cross-talk arising from tilt illuminations. The designed hybrid silicon-aluminum nanostructure has dual functionalities. Crucially, it supports a hybrid Mie-plasmon resonance of magnetic dipole to achieve color-selective light absorption, generating electron hole pairs. Simultaneously, the silicon-aluminum interface forms a Schottky barrier for charge separation and photodetection. This design potentially replaces the traditional dye-based filters for camera sensors at ultrahigh pixel densities with advanced functionalities in sensing polarization and directionality, and UV selectivity via interband plasmons of silicon.

Terahertz (THz) spectroscopy technique has been applied in ex vivo biomechanical properties analysis of human corneas. Upon the application of light pressure on the cornea, the photo elastic birefringent effect, anisotropic deformation, thickness changes and hydration levels will contribute to the sudden phase changes of terahertz time domain signal. The shelf lifetime study shows that the phase shift is reduced and cornea loose the biomechanical properties with the increase of hydration level. Mechanical behaviors have been further studied based on the "fresh" cut corneas with the similar hydration levels. THz signal was collected by focusing inside of the cornea to avoid the phase shift due to light stress caused movement of the corneal surface. By this way, the amount of THz signal refractive index variation is correlated to the elastic property of the corneas. The correlation between the THz signal phase shift and refractive index shift due to the corneal strain can be used to derive the elastic Young's modulus. Our results demonstrated the THz spectroscopy, as a non-contact and non-invasive detection method, could be potential for understanding the mechanism of corneal deformation under the action of intraocular pressure in the physiological environment in future., (© 2022. The Author(s).)

Dielectric optical nanoantennas are promising as fundamental building blocks in next generation color displays, metasurface holograms, and wavefront shaping optical devices. Due to the high refractive index of the nanoantenna material, they support geometry-dependent Mie resonances in the visible spectrum. Although phase change materials, such as the germanium-antimony-tellurium alloys, and post-transition metal oxides, such as ITO, have been used to tune antennas in the near-infrared spectrum, reversibly tuning the response of dielectric antennas in the visible spectrum remains challenging. In this paper, we designed and experimentally demonstrated dielectric nanodisc arrays exhibiting reversible tunability of Mie resonances in the visible spectrum. We achieved tunability by exploiting phase transitions in Sb 2 S 3 nanodiscs. Mie resonances within the nanodisc give rise to structural colors in the reflection mode. Crystallization and laser-induced amorphization of these Sb 2 S 3 resonators allow the colors to be switched back and forth. These tunable Sb 2 S 3 nanoantenna arrays could enable the next generation of high-resolution color displays, holographic displays, and miniature LiDAR systems.

Background: Extracellular vesicles (EVs) are cell-derived lipid bilayer enclosed structures shed from the plasma membrane by all cell types. Evidence of EV presence in biological fluids has led to considerable efforts focused on identifying their cargo and determining their utility as a non-invasive diagnostic platform for cancer. In this study, we identify circulating STEAP1 (six-transmembrane epithelial antigen of the prostate 1)-positive EVs in the plasma of healthy males and prostate cancer patients and evaluate its diagnostic and prognostic significance., Methods: STEAP1 was identified on EVs in prostate cancer patient plasma. EVs were validated using electron microscopy, Western blot, nanoparticle tracking analysis, and nanoscale flow cytometry. STEAP1-positive EVs were quantified for 121 males with prostate cancer and 55 healthy age-matched control males. An evaluation of STEAP1 in prostate cancer tissue was also performed using established prostate cancer cohort data (TCGA, MSKCC, and SU2C/PCF Dream Team)., Results: Evaluation of STEAP1-positive EVs by nanoscale flow cytometry identified a significant increase in prostate cancer patient plasma compared to healthy males. However, no association was found between total STEAP1 EV levels and disease recurrence or overall survival. Cohort data from prostate cancer tissue also found STEAP1 to be elevated in prostate cancer while no significant association with recurrence or overall survival was identified., Conclusions: STEAP1 is known to be enriched on the cells of the prostate with potential clinical significance in prostate cancer. Our results identify and quantitate STEAP1-positive EVs in plasma and provide rationale for a STEAP1 EV-based liquid biopsy as a diagnostic strategy in prostate cancer., (© 2021. The Author(s).)

Metastasis is present in approximately 30% of patients diagnosed with renal cell carcinoma (RCC) and is associated with a 5-year survival rate of < 15%. Kidney injury molecule 1 (KIM-1), encoded by the HAVCR1 gene, is a proximal tubule cell-surface glycoprotein and a biomarker for early detection of RCC, but its pathophysiological significance in RCC remains unclear. We generated human and murine RCC cell lines either expressing or lacking KIM-1, respectively, and compared their growth and metastatic properties using validated methods. Surprisingly, KIM-1 expression had no effect on cell proliferation or subcutaneous tumour growth in immune deficient (Rag1 -/- ) Balb/c mice, but inhibited cell invasion and formation of lung metastasis in the same model. Further, we show that the inhibitory effect of KIM-1 on metastases was observed in both immune deficient and immune competent mice. Transcriptomic profiling identified the mRNA for the pro-metastatic GTPase, Rab27b, to be downregulated significantly in KIM-1 expressing human and murine RCC cells. Finally, analysis of The Cancer Genome Atlas (TCGA) data revealed that elevated HAVCR1 mRNA expression in the two most common types of RCC, clear cell and papillary RCC, tumours correlated with significantly improved overall patient survival. Our findings reveal a novel role for KIM-1 in inhibiting metastasis of RCC and suggests that tumour-associated KIM-1 expression may be a favourable prognostic factor.

Wild-type KRAS (KRAS WT ) amplification has been shown to be a secondary means of KRAS activation in cancer and associated with poor survival. Nevertheless, the precise role of KRAS WT overexpression in lung cancer progression is largely unexplored. Here, we identify and characterize a KRAS-responsive lncRNA, KIMAT1 (ENSG00000228709) and show that it correlates with KRAS levels both in cell lines and in lung cancer specimens. Mechanistically, KIMAT1 is a MYC target and drives lung tumorigenesis by promoting the processing of oncogenic microRNAs (miRNAs) through DHX9 and NPM1 stabilization while halting the biogenesis of miRNAs with tumor suppressor function via MYC-dependent silencing of p21, a component of the Microprocessor Complex. KIMAT1 knockdown suppresses not only KRAS expression but also KRAS downstream signaling, thereby arresting lung cancer growth in vitro and in vivo. Taken together, this study uncovers a role for KIMAT1 in maintaining a positive feedback loop that sustains KRAS signaling during lung cancer progression and provides a proof of principle that interfering with KIMAT1 could be a strategy to hamper KRAS-induced tumorigenesis.

Chromosomal instability (CIN) is a driver of clonal diversification and intratumor heterogeneity, providing genetic diversity that contributes to tumor progression. It is estimated that approximately 80% of solid cancers, including non-small cell lung cancer (NSCLC), exhibit features of CIN, which affects tumor growth and response to therapy. However, the molecular mechanisms connecting CIN to tumor progression are still poorly understood. Through an RNAi screen performed on genes involved in CIN and overexpressed in human lung adenocarcinoma samples, we identified the cytoskeleton-associated protein 2-like (CKAP2L) as a potential oncogene that promotes lung cancer proliferation and growth in vitro and in vivo . Mechanistically, CKAP2L directly interacted with RNA Pol II and regulated transcription elongation of key genes involved in spindle assembly checkpoint, chromosome segregation, cell cycle, and E2F signaling. Furthermore, depletion of CKAP2L increased the sensitivity of NSCLC cells to alvocidib, a pan-CDK inhibitor, leading to a significant reduction of cell proliferation and an increase in cell death. Altogether, these findings shed light on the molecular mechanisms through which CKAP2L, a protein involved in CIN, promotes cancer progression and suggest that its inhibition represents a novel therapeutic strategy in NSCLC. SIGNIFICANCE: These findings demonstrate the oncogenic function of CKAP2L through regulation of transcription elongation and suggest that targeting CKAP2L could enhance therapeutic response in patients with NSCLC., (©2021 American Association for Cancer Research.)

Background: Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities., Methods: Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies., Findings: We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival., Interpretation: Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors., Funding: Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI)., Competing Interests: Declaration of Interests DSWT received honoraria from Bristol-Myers Squibb, Takeda Pharmaceuticals, Novartis, Roche, and Pfizer; and has consulting or advisory role in Novartis, Merck, Loxo Oncology, AstraZeneca, Roche, and Pfizer. DSWT also received research funding from Novartis (Inst), GlaxoSmithKline (Inst), and AstraZeneca (Inst), outside this submitted work., (Copyright © 2021. Published by Elsevier B.V.)

Most NUTM1-rearranged neoplasms (NRNs) have fusions between NUTM1 and BRD (bromodomain-containing) family members and are termed NUT carcinomas (NCs) because they show some squamous differentiation. However, some NRNs are associated with fusions between NUTM1 and members of the MAD (MAX dimerization) gene family of MYC antagonists. Here we describe a small round cell malignancy from the gastro-esophageal junction with a previously unreported fusion between NUTM1 and the MAD family member MXI1. In contrast to NCs, the MXI1-NUTM1 tumor did not show squamous differentiation and did not express MYC, TP63 or SOX2, genes known to be targets of BRD-NUTM1 proteins and critical for NC oncogenesis. Transcriptome analysis showed paradoxical enrichment of MYC target genes in the MXI1-NUTM1 tumor despite the lack of MYC expression. When expressed in vitro MXI1-NUTM1 partially phenocopied MYC, enhancing cell proliferation and cooperating with oncogenic HRAS to produce anchorage-independent cell growth. These data provide evidence that MAD family members, which are normally repressors of MYC activity, can be converted into MYC-like mimics by fusion to NUTM1. The pathological features and novel oncogenic mechanism of the MXI1-NUTM1 tumor show that identification of NUTM1 fusion partners can be important for accurate diagnostic classification of some NRN subtypes, and potentially may guide therapeutic options.

Objectives: We have previously identified and validated a panel of molecular prognostic markers (ATP13A3, SSR3, and ANO1) for Head and Neck Squamous Cell Carcinoma (HNSCC). The aim of this study was to investigate the consequence of ATP13A3 dysregulation on signaling pathways, to aid in formulating a therapeutic strategy targeting ATP13A3-overexpressing HNSCC., Materials and Methods: Gene Set Enrichment Analysis (GSEA) was performed on HNSCC microarray expression data (Internal local dataset [n = 92], TCGA [n = 232], EMBL [n = 81]) to identify pathways associated with high expression of ATP13A3. Validation was performed using immunohistochemistry (IHC) on tissue microarrays (TMAs) of head and neck cancers (n = 333), staining for ATP13A3 and phosphorylated Aurora kinase A (phospho-T288). Short interfering RNA was used to knockdown ATP13A3 expression in patient derived HNSCC cell lines. Protein expression of ATP13A3 and Aurora kinase A was then assessed by immunoblotting., Results: GSEA identified Aurora kinase pathway to be associated with high expression of ATP13A3 (p = 0.026). The Aurora kinase pathway was also associated with a trend towards poor prognosis and tumor aggressiveness (p = 0.086, 0.094, respectively). Furthermore, the immunohistochemical staining results revealed a significant association between Aurora kinase activity and high ATP13A3 expression (p < 0.001). Knockdown of ATP13A3 in human head and neck cell lines showed decrease in Aurora kinase A levels., Conclusion: Tumors with high ATP13A3 are associated with high Aurora kinase activity. This suggests a potential therapeutic role of Aurora kinase inhibitors in a subset of poor prognosis HNSCC patients with overexpression of ATP13A3., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features., Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting., Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations., Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271 ., (©2020 American Association for Cancer Research.)

While plasma concentration kinetics has traditionally been the predictor of drug pharmacological effects, it can occasionally fail to represent kinetics at the site of action, particularly for solid tumors. This is especially true in the case of delivery of therapeutic macromolecules (drug-loaded nanomaterials or monoclonal antibodies), which can experience challenges to effective delivery due to particle size-dependent diffusion barriers at the target site. As a result, disparity between therapeutic plasma kinetics and kinetics at the site of action may exist, highlighting the importance of target site concentration kinetics in determining the pharmacodynamic effects of macromolecular therapeutic agents. Assessment of concentration kinetics at the target site has been facilitated by non-invasive in vivo imaging modalities. This allows for visualization and quantification of the whole-body disposition behavior of therapeutics that is essential for a comprehensive understanding of their pharmacokinetics and pharmacodynamics. Quantitative non-invasive imaging can also help guide the development and parameterization of mathematical models for descriptive and predictive purposes. Here, we present a review of the application of state-of-the-art imaging modalities for quantitative pharmacological evaluation of therapeutic nanoparticles and monoclonal antibodies, with a focus on their integration with mathematical models, and identify challenges and opportunities. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Diagnostic Tools > in vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology., (© 2020 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.)

Background: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC., Patients and Methods: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies., Results: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years., Conclusion: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches., Competing Interests: Disclosure BYK served on Invitae Corporation's Advisory Board from 2017 to 2018. IAM has acted on the Advisory Boards for AstraZeneca, Clovis Oncology, Tesaro, Carrick Therapeutics and Takeda. His institution receives funding from AstraZeneca. RG is on the Advisory Boards for AstraZeneca, Tesaro, Clovis and Immunogen and does consultancy work for SOTIO. She has received support to attend conferences from AstraZeneca, Roche and Tesaro. Her institution has received research funding from Boehringer Ingelheim and Lilly/Ignyta and she is the national co-ordinating investigator for the UK for trials sponsored by AstraZeneca and Tesaro and site principal investigator for trials sponsored by AstraZeneca, Tesaro, Immunogen, Pfizer, Lilly and Clovis. PAF has received grants from Novartis, BioNtech and Cepheid as well as personal fees from Novartis, Roche, Pfizer, Celgene, Daiichi-Sankyo, TEVA, Astra Zeneca, Merck Sharp & Dohme, Myelo Therapeutics, MacroGenics, Eisai and Puma during the conduct of the study. JDB has acted on Advisory Boards for AstraZeneca and has received support from GSK to attend conferences. His institution receives funding from AstraZeneca and Aprea. UM has shares in Abcodia Ltd. Sandra Orsulic and Beth Y. Karlan have patents on predictive gene signatures in ovarian cancer (US010253368 and EU2908913). All remaining authors have declared no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gliomas are a diverse group of brain tumors comprised of malignant cells ('tumor' cells) and non-malignant 'normal' cells, including neural (neurons, glia), inflammatory (microglia, macrophage) and vascular cells. Tumor heterogeneity arises in part because, within the glioma mass, both 'tumor' and 'normal' cells secrete factors that form a unique microenvironment to influence tumor progression. Extracellular vesicles (EVs) are critical mediators of intercellular communication between immediate cellular neighbors and distantly located cells in healthy tissues/organs and in tumors, including gliomas. EVs mediate cell-cell signaling as carriers of nucleic acid, lipid and protein cargo, and their content is unique to cell types and physiological states. EVs secreted by non-malignant neural cells have important physiological roles in the healthy brain, which can be altered or co-opted to promote tumor progression and metastasis, acting in combination with glioma-secreted EVs. The cell-type specificity of EV content means that 'vesiculome' data can potentially be used to trace the cell of origin. EVs may also serve as biomarkers to be exploited for disease diagnosis and to assess therapeutic progress. In this review, we discuss how EVs mediate intercellular communication in glioma, and their potential role as biomarkers and readouts of a therapeutic response.

A subset of lung adenocarcinomas is driven by the EML4-ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on-target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4-ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti-apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first-, second- and third-generation ALK inhibitors., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.

Increasing reports of neurological and psychiatric complications due to psychostimulant synthetic cathinones (SCs) have recently raised public concern. However, the precise mechanism of SC toxicity is unclear. This paucity of understanding highlights the need to investigate the in-vitro toxicity and mechanistic pathways of three SCs: butylone, pentylone, and 3,4-Methylenedioxypyrovalerone (MDPV). Human neuronal cells of SH-SY5Y were cultured in supplemented DMEM/F12 media and differentiated to a neuronal phenotype using retinoic acid (10 μM) and 12- O -tetradecanoylphorbol-13-acetate (81 nM). Trypan blue and lactate dehydrogenase assays were utilized to assess the neurotoxicity potential and potency of these three SCs. To investigate the underlying neurotoxicity mechanisms, measurements included markers of oxidative stress, mitochondrial bioenergetics, and intracellular calcium (Ca 2+ ), and cell death pathways were evaluated at two doses (EC 15 and EC 40 ), for each drug tested. Following 24 h of treatment, all three SCs exhibited a dose-dependent neurotoxicity, characterized by a significant ( p < 0.0001 vs. control) production of reactive oxygen species, decreased mitochondrial bioenergetics, and increased intracellular Ca 2+ concentrations. The activation of caspases 3 and 7 implicated the orchestration of mitochondrial-mediated neurotoxicity mechanisms for these SCs. Identifying novel therapeutic agents to enhance an altered mitochondrial function may help in the treatment of acute-neurological complications arising from the illicit use of these SCs., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Introduction: SCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient stratification and response monitoring., Methods: We applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA [the percentage of genomic regions amplified]), Z-score (a measure of standard deviation), and Moran's I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample., Results: Genome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival., Conclusions: We demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Management of locally advanced head and neck squamous cell carcinoma (HNSCC) requires a multi-prong approach comprising surgery, radiation and/or chemotherapy, yet outcomes are limited. This is largely due to a paucity of biomarkers that can predict response to specific treatment modalities. Here, we evaluated TGFβ3 protein levels in extracellular vesicles (EVs) released by HNSCC cells as a predictor for response to chemoradiation therapy (CRT). To this end, specific EV-fractions were isolated from cell lines or HNSCC patient plasma, and TGFβ3 protein was quantified. In patients treated with CRT, TGFβ3 levels were found to be significantly higher in plasma EV-fractions or non-responders compared with responders. High levels of TGFβ3 levels in Annexin V-EVs were associated with the worst progression-free survival. In vitro experiments demonstrated that TGFβ3 silencing sensitized HNSCC cells to cytotoxic therapies, and this phenotype could be rescued by treatment with exogenous. In addition, specific EV-fractions shed by cisplatin-resistant cells were sufficient to transfer the resistant phenotype to sensitive cells through activation of TGFβ-signaling pathway. Therefore, our data show that TGFβ3 transmitted through EV plays a significant role in response to cytotoxic therapy, which can be exploited as a potential biomarker for CRT response in HNSCC patients treated with curative intent., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Caveolin-1 is a transmembrane protein with both tumor promoter and suppressor functions that remain poorly understood. Cav1 phosphorylation by Src kinase on tyrosine 14 is closely associated with focal adhesion dynamics and tumor cell migration, however the role of pCav1 in vivo in tumor progression remains poorly characterized. Herein, we expressed phosphomimetic Y14D, wild type, and non-phosphorylatable Y14F forms of Cav1 in MDA-MB-435 cancer cells. Expression of Cav1Y14D reduced cell proliferation and induced the TP53 tumor suppressor. Ectopic expression in MDA-MB-435 cells of Y14 phosphorylatable Cav1 was required for induction of TP53 in response to oxidative stress. Cav1Y14D promotes an apparent reversal of the Warburg effect and markedly inhibited tumor growth in vivo . However, Cav1 induced pseudopodial recruitment of glycolytic enzymes, and time-lapse intravital imaging showed increased invadopodia protrusion and extravasation into blood vessels for Cav1WT and Y14D but not for Y14F. Our results suggest that Cav1 Y14 phosphorylation levels play a role in the conflicting demands on metabolic resources associated with cancer cell proliferation versus motility., Competing Interests: CONFLICTS OF INTEREST We declare there are no conflicts of interest with respect to the material included in this manuscript.

Background: The development of phenotypic biomarkers to aid the selection of treatment for patients with castrate-resistant prostate cancer (CRPC) is an important priority. Plasma exosomes have excellent potential as real-time biomarkers to characterize the tumor because they are easily accessible in the blood and contain DNA, RNA, and protein from the parent cell. This study aims to investigate the characteristics of putative prostate-specific plasma extracellular vesicle (EV) markers and their relationship with clinical outcomes., Methods and Patients: We investigated plasma EVs in a total of 89 patients with prostate cancer (PCa) at different stages of disease progression. EVs were isolated using both precipitation and ultracentrifugation methods; physical characterization was performed using dynamic light scattering, acetylcholinesterase (AChE) activity, and velocity gradients. An immunocapture method was developed for the evaluation of prostate-specific membrane antigen (PSMA)-positive exosomes. Exosomal messenger RNA (mRNA) was quantified using droplet digital polymerase chain reaction for the expression of KLK3 and androgen receptor splice variant 7 (AR-V7) genes, which code prostate-specific antigen (PSA) and AR-V7, respectively. Serum sex steroids were measured using liquid chromatography-tandem mass spectroscopy., Results: Isolated exosomes from patients with CRPC had a smaller hydrodynamic size than those isolated from localized patients with PCa, while AChE activity showed no difference. Moreover, no differences were observed after initiation of androgen deprivation therapy in serial patient samples. Velocity gradients identified that PSMA-positive exosomes occupied a specific fraction of isolated EVs. A total of 35 patients with CRPC had mRNA analyzed from isolated plasma exosomes. Detectable exosomal KLK3 corresponded with higher concomitant serum PSA measurements, as expected (mean, 112.6 vs 26.61 ng/mL; P = .065). Furthermore, detectable levels of AR-V7 mRNA were associated with a shorter time to progression (median, 16.0 vs 28.0 months; P = .0499). Furthermore, detectable exosomal AR-V7 was significantly associated with testosterone levels below the lower limit of quantification (<0.1 nM)., Conclusions: Our results suggest that exosomal AR-V7 is correlated with lower sex steroid levels in CRPC patients with a poorer prognosis. PSMA immunocapture does not appear sufficient to isolate PCa-specific exosomes., (© 2019 Wiley Periodicals, Inc.)

Purpose: Colorectal cancer is a commonly encountered disease that poses several diagnostic and therapeutic challenges. The inherent heterogeneity of tumor biology and propensity to relapse despite "curative" resection pose significant challenges with regard to response assessment. Although MR imaging already plays a key role in primary staging of patients with rectal carcinoma, its reliability in restaging after neoadjuvant therapy is debatable (Van der broek et al. in Dis Colon Rectum 60(3):274-283, 2017). Therefore, there is significant interest in developing additional methods which may improve diagnostic accuracy. This study aims to evaluate the role of multimodality imaging and liquid biopsy in therapeutic response assessment., Methods: Seventeen patients were enrolled into the study over a span of 24 months. All underwent hybrid PET-MRI and CT-perfusion (CT-P), prior to and following neoadjuvant therapy for locally advanced rectal carcinoma. Twelve of the 17 patients also underwent liquid biopsy, which consisted of blood sampling and analysis of circulating tumor cells (CTCs) and extracellular vesicles (EVs), including cell fragments and microparticles (MPs), using the Cell Search System (Menarini Silicon Biosystems). SUV, DWI, and ADC were calculated during PET-MRI, and several parameters were evaluated during CT-perfusion, including average perfusion, blood flow (BF), blood volume (BV), mean transit time (MTT), permeability-surface area product (PS), contrast extraction efficiency (E), and K-trans (K). Changes observed pre- and post-neoadjuvant therapy in each modality were compared to tumor response at histopathology using a modified Ryan tumor regression grading system., Results: Of the 17 patients included in the study, 14 were classified as non-responders, and 3 were classified as responders as determined by the modified Ryan Tumor Regression Grade (TRG) scoring system (Van der broek et al. in Dis Colon Rectum 60(3):274-283, 2017). When combined, blood markers and CT-P parameters (mean transit time (MTT), K-trans, and permeability-surface area product (PS)) produced the strongest models (p < 0.01). PET (SUV measurement) combined with CT-P-derived K-trans produced a marginally significant (p = 0.057) model for predicting response. MRI-derived ADC value did not provide a significant model for response prediction., Conclusion: A model of CT-P parameters plus liquid biopsy more accurately predicts tumor response than PET-MRI, CT-P alone, or liquid biopsy alone. These results suggest that in the evaluation of treatment response, liquid biopsy could provide additional information to functional imaging modalities such as CT-P and should therefore be explored further in a trial with larger sample size.

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

Objective: To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance., Materials and Methods: Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA)., Results: By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer., Conclusion: The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate., (© 2019 Wiley Periodicals, Inc.)

Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient samples. However, clinical assessment of copy number variations (CNVs) and large-scale structural variations (SVs) is still challenging. While tools exist to estimate both, their results are typically presented separately in tables or static plots which can be difficult to read and are unable to show the context needed for clinical interpretation and reporting. We have addressed this problem with CNspector, a multi-scale interactive browser that shows CNVs in the context of other relevant genomic features to enable fast and effective clinical reporting. We illustrate the utility of CNspector at different genomic scales across a variety of sample types in a range of case studies. We show how CNspector can be used for diagnosis and reporting of exon-level deletions, focal gene-level amplifications, chromosome and chromosome arm level amplifications/deletions and in complex genomic rearrangements. CNspector is a web-based clinical variant browser tailored to the clinical application of next generation sequencing for CNV assessment. We have demonstrated the utility of this interactive software in typical applications across a range of tissue types and disease contexts encountered in the context of diagnostic pathology. CNspector is written in R and the source code is available for download under the GPL3 Licence from https://github.com/PapenfussLab/CNspector . A server running CNspector loaded with the figures from this paper can be accessed at https://shiny.wehi.edu.au/jmarkham/CNspector/index.html .

Background: While prostate cancer can often manifest as an indolent disease, the development of locally-advanced or metastatic disease can cause significant morbidity or mortality. Elucidation of molecular mechanisms contributing to disease progression is crucial for more accurate prognostication and effective treatments. R-Spondin 3 (RSPO3) is a protein previously implicated in the progression of colorectal and lung cancers. However, a role for RSPO3 in prostate cancer prognosis and behaviour has not been explored., Methods: We compare the relative levels of RSPO3 expression between normal prostate tissue and prostate cancer in two independent patient cohorts (Taylor and GSE70768-Cambridge). We also examine the association of biochemical relapse with RSPO3 levels in these cohorts. For elucidation of the biological effect of RSPO3, we use siRNA technology to reduce the levels of RSPO3 in established prostate cancer cell lines, and perform in vitro proliferation, invasion, western blotting for EMT markers and clonogenic survival assays for radiation resistance. Furthermore, we show consequences of RSPO3 knockdown in an established chick chorioallantoic membrane (CAM) assay model of metastasis., Results: RSPO3 levels are lower in prostate cancer than normal prostate, with a tendency for further loss in metastatic disease. Patients with lower RSPO3 expression have lower rates of biochemical relapse-free survival. SiRNA-mediated loss of RSPO3 results in no change to clonogenic survival and a lower proliferative rate, but increased invasiveness in vitro with induction of epithelial-mesenchymal transition (EMT) markers. Consistent with these results, lower RSPO3 expression translates to greater metastatic capacity in the CAM assay. Together, our preclinical findings identify a role of RSPO3 downregulation in prostate cancer invasiveness, and provide a potential explanation for how RSPO3 functions as a positive prognostic marker in prostate cancer.

The concept of vesicles or cell debris released by cancer cells to promote metastasis is not new, but the mechanisms used to currently ascribe their impact in metastasis are of intense debate. A significant increase in reports describing the role of cancer-derived EVs in cancer metastasis has been followed by a growing amount of uncertainty behind these claims. This review will delve into the role of EVs in promoting cancer metastasis by relying on a balanced perspective that looks at challenges faced previously by extracellular vesicle biologists, current technical limitations in the field, and overlooked physiologic mechanisms that may play a confounding role. This review will also discuss how certain experimental approaches are misleading which ultimately lead to overly optimistic mechanisms that have minimally contributed to the pathophysiology of metastasis., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: The prostate cancer (PCa) diagnostic pathway is undergoing a radical change with the introduction of multiparametric magnetic resonance imaging (mpMRI), genomic testing, and different prostate biopsy techniques. It has been proposed that these tests should be used in a sequential manner to optimise risk stratification., Objective: To characterise the genomic, epigenomic, and transcriptomic features of mpMRI-visible and -nonvisible PCa in clinically localised disease., Design, Setting, and Participants: Multicore analysis of fresh prostate tissue sampled immediately after radical prostatectomy was performed for intermediate- to high-risk PCa., Intervention: Low-pass whole-genome, exome, methylation, and transcriptome profiling of patient tissue cores taken from microscopically benign and cancerous areas in the same prostate. Circulating free and germline DNA was assessed from the blood of five patients., Outcome Measurement and Statistical Analysis: Correlations between preoperative mpMRI and genomic characteristics of tumour and benign prostate samples were assessed. Gene profiles for individual tumour cores were correlated with existing genomic classifiers currently used for prognostication., Results and Limitations: A total of 43 prostate cores (22 tumour and 21 benign) were profiled from six whole prostate glands. Of the 22 tumour cores, 16 were tumours visible and six were tumours nonvisible on mpMRI. Intratumour genomic, epigenomic, and transcriptomic heterogeneity was found within mpMRI-visible lesions. This could potentially lead to misclassification of patients using signatures based on copy number or RNA expression. Moreover, three of the six cores obtained from mpMRI-nonvisible tumours harboured one or more genetic alterations commonly observed in metastatic castration-resistant PCa. No circulating free DNA alterations were found. Limitations include the small cohort size and lack of follow-up., Conclusions: Our study supports the continued use of systematic prostate sampling in addition to mpMRI, as avoidance of systematic biopsies in patients with negative mpMRI may mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic PCa are missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied., Patient Summary: Our study shows that tumour heterogeneity within prostate tumours visible on multiparametric magnetic resonance imaging (mpMRI) can lead to misclassification of patients if only one core is used for genomic analysis. In addition, some cancers that were missed by mpMRI had genomic aberrations that are commonly seen in advanced metastatic prostate cancer. Avoiding biopsies in mpMRI-negative cases may mean that such potentially lethal cancers are missed., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that is clinically asymptomatic in its early stages of development. Non-invasive testing for pancreatic cancer biomarkers would significantly improve early detection and patient care. Extracellular vesicles (EVs) are circulating tumor fragments present in the blood and may express cancer specific biomarkers that would enable early detection of pancreatic cancer. We tested the utility of a blood test enumerating EVs positive for the pancreas-specific marker Glycoprotein 2 (GP2) and the putative pancreatic cancer marker Glypican-1 (GPC1) in patients with PDAC. Various levels of GPC1-positive and GP2/GPC1-positive EVs were detected in PDAC patients but were not significantly higher than benign pancreatic disease (BPD) patients. The sensitivity and specificity of the GPC1 EV test was 26.67% and 87.50% respectively, whereas the sensitivity and specificity for the GPC1+GP2 EV test was 23.33% and 90.00% respectively. Immunohistochemistry of GPC1 expression in a tissue microarray of PDAC and various controls also did not demonstrate specificity of GPC1 to PDAC. Hence, enumeration of GPC1-positive EVs, solely or in conjunction with GP2, was unable to effectively distinguish between BPD and pancreatic cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Chemo-resistance is one of the major causes of cancer-related deaths. Here we used single-cell transcriptomics to investigate divergent modes of chemo-resistance in tumor cells. We observed that higher degree of phenotypic intra-tumor heterogeneity (ITH) favors selection of pre-existing drug-resistant cells, whereas phenotypically homogeneous cells engage covert epigenetic mechanisms to trans-differentiate under drug-selection. This adaptation was driven by selection-induced gain of H3K27ac marks on bivalently poised resistance-associated chromatin, and therefore not expressed in the treatment-naïve setting. Mechanistic interrogation of this phenomenon revealed that drug-induced adaptation was acquired upon the loss of stem factor SOX2, and a concomitant gain of SOX9. Strikingly we observed an enrichment of SOX9 at drug-induced H3K27ac sites, suggesting that tumor evolution could be driven by stem cell-switch-mediated epigenetic plasticity. Importantly, JQ1 mediated inhibition of BRD4 could reverse drug-induced adaptation. These results provide mechanistic insights into the modes of therapy-induced cellular plasticity and underscore the use of epigenetic inhibitors in targeting tumor evolution.