The landscape of drug sensitivity and resistance in sarcoma.

Sarcomas are rare malignancies with over 100 distinct histological subtypes. Their rarity and heterogeneity pose significant challenges to identifying effective therapies, and approved regimens show varied responses. Novel, personalized approaches to therapy are needed to improve patient outcomes. Patient-derived tumor organoids (PDTOs) model tumor behavior across an array of malignancies. We leverage PDTOs to characterize the landscape of drug resistance and sensitivity in sarcoma, collecting 194 specimens from 126 patients spanning 24 distinct sarcoma subtypes. Our high-throughput organoid screening pipeline tested single agents and combinations, with results available within a week from surgery. Drug sensitivity correlated with clinical features such as tumor subtype, treatment history, and disease trajectory. PDTO screening can facilitate optimal drug selection and mirror patient outcomes in sarcoma. We could identify at least one FDA-approved or NCCN-recommended effective regimen for 59% of the specimens, demonstrating the potential of our pipeline to provide actionable treatment information.

Divergent Evolution in Bilateral Prostate Cancer: a Case Study.

Multifocal prostate cancer is a prevalent phenomenon, with most cases remaining uncharacterized from a genomic perspective. A patient presented with bilateral prostate cancer. On systematic biopsy, two indistinguishable clinicopathologic lesions were detected. Whole-genome sequencing displayed somatically unrelated tumours with distinct driver CNA regions, suggesting independent origins of the two tumors. We demonstrated that similar clinicopathologic multifocal tumours, which might be interpreted as clonal disease, can in fact represent independent cancers. Genetic prognostics can prevent mischaracterization of multifocal disease to enable optimal patient management.

Metapipeline-DNA: A Comprehensive Germline & Somatic Genomics Nextflow Pipeline.

Summary: DNA sequencing is becoming more affordable and faster through advances in high-throughput technologies. This rise in data availability has contributed to the development of novel algorithms to elucidate previously obscure features and led to an increased reliance on complex workflows to integrate such tools into analyses pipelines. To facilitate the analysis of DNA sequencing data, we created metapipeline-DNA, a highly configurable and extensible pipeline. It encompasses a broad range of processing including raw sequencing read alignment and recalibration, variant calling, quality control and subclonal reconstruction. Metapipeline-DNA also contains configuration options to select and tune analyses while being robust to failures. This standardizes and simplifies the ability to analyze large DNA sequencing in both clinical and research settings. Availability: Metapipeline-DNA is an open-source Nextflow pipeline under the GPLv2 license and is freely available at https://github.com/uclahs-cds/metapipeline-DNA.

Upgrading of Grade Group 1 Prostate Cancer at Prostatectomy: Germline Risk Factors in a Prospective Cohort.

BACKGROUND: Localized prostate tumors show significant spatial heterogeneity, with regions of high-grade disease adjacent to lower-grade disease. Consequently, prostate cancer biopsies are prone to sampling bias, potentially leading to underestimation of tumor grade. To study the clinical, epidemiologic and molecular hallmarks of this phenomenon, we conducted a prospective study of grade upgrading: differences in detected prostate cancer grade between biopsy and surgery. METHODS: We established a prospective, multi-institutional cohort of men with Grade Group 1 (GG1) prostate cancer on biopsy who underwent radical prostatectomy. Upgrading was defined as detection of GG2+ in the resected tumor. Germline DNA from 192 subjects was subjected to whole-genome sequencing to quantify ancestry, pathogenic variants in DNA damage response genes and polygenic risk. RESULTS: Of 285 men, 67% upgraded at surgery. PSA density and percent of cancer in pre-prostatectomy positive biopsy cores were significantly associated with upgrading. No assessed genetic risk factor was predictive of upgrading, including polygenic risk scores for prostate cancer diagnosis. CONCLUSIONS: In a cohort of low-grade prostate cancer patients, a majority upgraded at radical prostatectomy. PSA density and percent of cancer in pre-prostatectomy positive biopsy cores portended the presence of higher-grade disease, while germline genetics was not informative in this setting. Patients with low-risk prostate cancer, but elevated PSA density or percent cancer in positive biopsy cores, may benefit from repeat biopsy, additional imaging or other approaches to complement active surveillance. IMPACT: Further risk stratification of patients with low-risk prostate cancer may provide useful context for active surveillance decision-making.

The Proteogenomics of Prostate Cancer Radioresistance.

Prostate cancer is frequently treated with radiotherapy. Unfortunately, aggressive radioresistant relapses can arise, and the molecular underpinnings of radioresistance are unknown. Modern clinical radiotherapy is evolving to deliver higher doses of radiation in fewer fractions (hypofractionation). We therefore analyzed genomic, transcriptomic, and proteomic data to characterize prostate cancer radioresistance in cells treated with both conventionally fractionated and hypofractionated radiotherapy. Independent of fractionation schedule, resistance to radiotherapy involved massive genomic instability and abrogation of DNA mismatch repair. Specific prostate cancer driver genes were modulated at the RNA and protein levels, with distinct protein subcellular responses to radiotherapy. Conventional fractionation led to a far more aggressive biomolecular response than hypofractionation. Testing preclinical candidates identified in cell lines, we revealed POLQ (DNA Polymerase Theta) as a radiosensitizer. POLQ-modulated radioresistance in model systems and was predictive of it in large patient cohorts. The molecular response to radiation is highly multimodal and sheds light on prostate cancer lethality. SIGNIFICANCE: Radiation is standard of care in prostate cancer. Yet, we have little understanding of its failure. We demonstrate a new paradigm that radioresistance is fractionation specific and identified POLQ as a radioresistance modulator.

Hepatic enzyme induction and its potential effect on thyroid hormone metabolism in the metamorphosing tadpole of Xenopus laevis (African clawed frog).

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-ß-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species.

moPepGen: Rapid and Comprehensive Proteoform Identification.

Gene expression is a multi-step transformation of biological information from its storage form (DNA) into functional forms (protein and some RNAs). Regulatory activities at each step of this transformation multiply a single gene into a myriad of proteoforms. Proteogenomics is the study of how genomic and transcriptomic variation creates this proteoform diversity, and is limited by the challenges of modeling the complexities of gene-expression. We therefore created moPepGen, a graph-based algorithm that comprehensively enumerates proteoforms in linear time. moPepGen works with multiple technologies, in multiple species and on all types of genetic and transcriptomic data. In human cancer proteomes, it detects and quantifies previously unobserved noncanonical peptides arising from germline and somatic genomic variants, noncoding open reading frames, RNA fusions and RNA circularization. By enabling efficient identification and quantitation of previously hidden proteins in both existing and new proteomic data, moPepGen facilitates all proteogenomics applications. It is available at: https://github.com/uclahs-cds/package-moPepGen.

NFTest: automated testing of Nextflow pipelines.

MOTIVATION: The ongoing expansion in the volume of biomedical data has contributed to a growing complexity in the tools and technologies used in research with an increased reliance on complex workflows written in orchestration languages such as Nextflow to integrate algorithms into processing pipelines. The growing use of workflows involving various tools and algorithms has led to increased scrutiny of software development practices to avoid errors in individual tools and in the connections between them. RESULTS: To facilitate test-driven development of Nextflow pipelines, we created NFTest, a framework for automated pipeline testing and validation with customizability options for Nextflow features. It is open-source, easy to initialize and use, and customizable to allow for testing of complex workflows with test success configurable through a broad range of assertions. NFTest simplifies the testing burden on developers by automating tests once defined and providing a flexible interface for running tests to validate workflows. This reduces the barrier to rigorous biomedical workflow testing and paves the way toward reducing computational errors in biomedicine. AVAILABILITY AND IMPLEMENTATION: NFTest is an open-source Python framework under the GPLv2 license and is freely available at https://github.com/uclahs-cds/tool-NFTest. The call-sSNV Nextflow pipeline is available at: https://github.com/uclahs-cds/pipeline-call-sSNV.

PipeVal: light-weight extensible tool for file validation.

MOTIVATION: The volume of biomedical data generated each year is growing exponentially as high-throughput molecular, imaging and mHealth technologies expand. This rise in data volume has contributed to an increasing reliance on and demand for computational methods, and consequently to increased attention to software quality and data integrity. RESULTS: To simplify data verification in diverse data-processing pipelines, we created PipeVal, a light-weight, easy-to-use, extensible tool for file validation. It is open-source, easy to integrate with complex workflows, and modularized for extensibility for new file formats. PipeVal can be rapidly inserted into existing methods and pipelines to automatically validate and verify inputs and outputs. This can reduce wasted compute time attributed to file corruption or invalid file paths, and significantly improve the quality of data-intensive software. AVAILABILITY AND IMPLEMENTATION: PipeVal is an open-source Python package under the GPLv2 license and it is freely available at https://github.com/uclahs-cds/package-PipeVal. The docker image is available at: https://github.com/uclahs-cds/package-PipeVal/pkgs/container/pipeval.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Oncolytic DNX-2401 virotherapy plus pembrolizumab in recurrent glioblastoma: a phase 1/2 trial.

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Synchronization of Arabidopsis flowering time and vegetative growth stage via FT overexpression can reveal inherent heterosis due to heterozygosity in intraspecific hybrids.

Intraspecific hybrids of Arabidopsis sometimes display heterosis. However, allelic variation of flowering repressor genes causes late flowering in F1, which might distort the potential heterosis effect due to prolonged vegetative growth. Here, overexpression of flowering gene FT synchronized flowering and eliminated growth differentials between parental and F1. These findings indicate the possibility of quantitatively demonstrating the inherent heterosis caused by heterozygosity.

A polygenic two-hit hypothesis for prostate cancer.

Prostate cancer is one of the most heritable cancers. Hundreds of germline polymorphisms have been linked to prostate cancer diagnosis and prognosis. Polygenic risk scores can predict genetic risk of a prostate cancer diagnosis. Although these scores inform the probability of developing a tumor, it remains unknown how germline risk influences the tumor molecular evolution. We cultivated a cohort of 1250 localized European-descent patients with germline and somatic DNA profiling. Men of European descent with higher genetic risk were diagnosed earlier and had less genomic instability and fewer driver genes mutated. Higher genetic risk was associated with better outcome. These data imply a polygenic "two-hit" model where germline risk reduces the number of somatic alterations required for tumorigenesis. These findings support further clinical studies of polygenic risk scores as inexpensive and minimally invasive adjuncts to standard risk stratification. Further studies are required to interrogate generalizability to more ancestrally and clinically diverse populations.

Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

The telomere length landscape of prostate cancer.

Replicative immortality is a hallmark of cancer, and can be achieved through telomere lengthening and maintenance. Although the role of telomere length in cancer has been well studied, its association to genomic features is less well known. Here, we report the telomere lengths of 392 localized prostate cancer tumours and characterize their relationship to genomic, transcriptomic and proteomic features. Shorter tumour telomere lengths are associated with elevated genomic instability, including single-nucleotide variants, indels and structural variants. Genes involved in cell proliferation and signaling are correlated with tumour telomere length at all levels of the central dogma. Telomere length is also associated with multiple clinical features of a tumour. Longer telomere lengths in non-tumour samples are associated with a lower rate of biochemical relapse. In summary, we describe the multi-level integration of telomere length, genomics, transcriptomics and proteomics in localized prostate cancer.

Qualitative analysis of actual Standard for Exchange of Nonclinical Data (SEND) datasets for Data Domains: Proposition from Japan Pharmaceutical Manufacturers Association SEND Taskforce Team on standardization of nonclinical data.

The Standard for Exchange of Nonclinical Data (SEND) has been adopted by the US FDA, which has required pharmaceutical companies who are developing new drugs for the US market to implement SEND. The Japan Pharmaceutical Manufacturers Association (JPMA) SEND Taskforce Team responded to this situation by starting a project to better understand the contents of SEND datasets. The project focused on domains generally included in the SEND domains for single- and repeat-dose general toxicology studies, and surveyed what kind of information are populated in which domains and in what way. The qualitative analysis of the results indicated that variations exist based on whether or not an individual variable was populated and on how the variable was populated. The Taskforce Team recommends reducing variations not only in the SEND datasets but also in the descriptions in the study protocol and/or final study report. Reduction of such variations should lead to higher quality datasets with powerful and increased searchability so that accumulated SEND datasets should become more valuable. These efforts would provide regulatory agencies with easier review of SEND datasets, which contributes to efficient development of new drug candidates.

Precision Radiotherapy: Reduction in Radiation for Oropharyngeal Cancer in the 30 ROC Trial.

BACKGROUND: Patients with human papillomavirus-related oropharyngeal cancers have excellent outcomes but experience clinically significant toxicities when treated with standard chemoradiotherapy (70 Gy). We hypothesized that functional imaging could identify patients who could be safely deescalated to 30 Gy of radiotherapy. METHODS: In 19 patients, pre- and intratreatment dynamic fluorine-18-labeled fluoromisonidazole positron emission tomography (PET) was used to assess tumor hypoxia. Patients without hypoxia at baseline or intratreatment received 30 Gy; patients with persistent hypoxia received 70 Gy. Neck dissection was performed at 4 months in deescalated patients to assess pathologic response. Magnetic resonance imaging (weekly), circulating plasma cell-free DNA, RNA-sequencing, and whole-genome sequencing (WGS) were performed to identify potential molecular determinants of response. Samples from an independent prospective study were obtained to reproduce molecular findings. All statistical tests were 2-sided. RESULTS: Fifteen of 19 patients had no hypoxia on baseline PET or resolution on intratreatment PET and were deescalated to 30 Gy. Of these 15 patients, 11 had a pathologic complete response. Two-year locoregional control and overall survival were 94.4% (95% confidence interval = 84.4% to 100%) and 94.7% (95% confidence interval = 85.2% to 100%), respectively. No acute grade 3 radiation-related toxicities were observed. Microenvironmental features on serial imaging correlated better with pathologic response than tumor burden metrics or circulating plasma cell-free DNA. A WGS-based DNA repair defect was associated with response (P = .02) and was reproduced in an independent cohort (P = .03). CONCLUSIONS: Deescalation of radiotherapy to 30 Gy on the basis of intratreatment hypoxia imaging was feasible, safe, and associated with minimal toxicity. A DNA repair defect identified by WGS was predictive of response. Intratherapy personalization of chemoradiotherapy may facilitate marked deescalation of radiotherapy.

Noncoding mutations target cis-regulatory elements of the FOXA1 plexus in prostate cancer.

Prostate cancer is the second most commonly diagnosed malignancy among men worldwide. Recurrently mutated in primary and metastatic prostate tumors, FOXA1 encodes a pioneer transcription factor involved in disease onset and progression through both androgen receptor-dependent and androgen receptor-independent mechanisms. Despite its oncogenic properties however, the regulation of FOXA1 expression remains unknown. Here, we identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic single-nucleotide variants in primary prostate tumors. We find that deletion and repression of these cis-regulatory elements significantly decreases FOXA1 expression and prostate cancer cell growth. Six of the ten single-nucleotide variants mapping to FOXA1 regulatory plexus significantly alter the transactivation potential of cis-regulatory elements by modulating the binding of transcription factors. Collectively, our results identify cis-regulatory elements within the FOXA1 plexus mutated in primary prostate tumors as potential targets for therapeutic intervention.

Wrist flexible heart pulse sensor integrated with a soft pump and a pneumatic balloon membrane.

To monitor health and diagnose disease in the early stage, future healthcare standards will likely include the continuous monitoring of various vital data. One approach to collect such information is a wearable and flexible device, which detects information from the skin surface. An important dataset is heart pulse information. Herein a method to monitor the detailed pulse signal from a wrist stably and reliably is proposed. Specifically, a soft pneumatic balloon operated by a soft pump applies the appropriate pressure over a tactile sensor onto the radial artery of the wrist to detect detailed heart pulse waves. The soft pump, pneumatic balloon, and flexible tactile pressure sensor are characterized as a fundamental study. Additionally, a proof-of-concept of this integrated device platform is demonstrated by monitoring the heart pulse from a wrist with and without the soft pump functions.