More Than Results: The Clinical and Research Relationship in the Evolving Detection and Surveillance of SARS-CoV-2.

INTRODUCTION: During the coronavirus disease 2019 (COVID-19) pandemic, real-time reverse transcription polymerase chain reaction (RT-PCR) became an essential tool for laboratories to provide high-sensitivity qualitative diagnostic testing for patients and real-time data to public health officials. Here we explore the predictive value of quantitative data from RT-PCR cycle threshold (Ct) values in epidemiological measures, symptom presentation, and variant transition. METHODS: To examine the association with hospitalizations and deaths, data from 74,479 patients referred to the Avera Institute for Human Genetics (AIHG) for COVID-19 testing in 2020 were matched by calendar week to epidemiological data reported by the South Dakota Department of Health. We explored the association between symptom data, patient age, and Ct values for 101 patients. We also explored changes in Ct values during variant transition detected by genomic surveillance sequencing of the AIHG testing population during 2021. RESULTS: Measures from AIHG diagnostic testing strongly explain variance in the South Dakota state positivity percentage (R2 = 0.758), a two-week delay in hospitalizations (R2 = 0.856), and a four-week delay in deaths (R2 = 0.854). Based on factor analysis of patient symptoms, three groups could be distinguished which had different presentations of age, Ct value, and time from collection. Additionally, conflicting Ct value results among SARSCoV- 2 variants during variant transition may reflect the community transmission dynamics. CONCLUSIONS: Measures of Ct value in RT-PCR diagnostic assays combined with routine screening have valuable applications in monitoring the dynamics of SARS-CoV-2 within communities.

B cell-dependent subtypes and treatment-based immune correlates to survival in stage 3 and 4 lung adenocarcinomas.

Lung cancer is the leading cause of cancer-related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non-small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late-stage NSCLC. The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA-Seq) with deconvolution of RNA-Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B-cell rich patient group had increased expression of CXCL13 and greater abundance of PD1+ CD8 T cells. The presence of B cells and PD1+ CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS). We then assessed the predictive and prognostic utility of these cell types and TLS within publicly available stage 3 and 4 lung adenocarcinoma (LUAD) RNA-Seq datasets. As previously described by others, pre-treatment expression of intratumoral 12-chemokine TLS gene signature is associated with progression free survival (PFS) in patients who receive treatment with immune checkpoint inhibitors (ICI). Notably and unexpectedly pre-treatment percentages of intratumoral B cells are associated with PFS in patients who receive surgery, chemotherapy, or radiation. Further studies to confirm these findings would allow for more effective patient selection for both ICI and non-ICI treatments.

A temporal in vivo catalog of chromatin accessibility and expression profiles in pineoblastoma reveals a prevalent role for repressor elements.

Pediatric pineoblastomas (PBs) are rare and aggressive tumors of grade IV histology. Although some oncogenic drivers are characterized, including germline mutations in RB1 and DICER1, the role of epigenetic deregulation and cis-regulatory regions in PB pathogenesis and progression is largely unknown. Here, we generated genome-wide gene expression, chromatin accessibility, and H3K27ac profiles covering key time points of PB initiation and progression from pineal tissues of a mouse model of CCND1-driven PB. We identified PB-specific enhancers and super-enhancers, and found that in some cases, the accessible genome dynamics precede transcriptomic changes, a characteristic that is underexplored in tumor progression. During progression of PB, newly acquired open chromatin regions lacking H3K27ac signal become enriched for repressive state elements and harbor motifs of repressor transcription factors like HINFP, GLI2, and YY1. Copy number variant analysis identified deletion events specific to the tumorigenic stage, affecting, among others, the histone gene cluster and Gas1, the growth arrest specific gene. Gene set enrichment analysis and gene expression signatures positioned the model used here close to human PB samples, showing the potential of our findings for exploring new avenues in PB management and therapy. Overall, this study reports the first temporal and in vivo cis-regulatory, expression, and accessibility maps in PB.

The Prostate Cancer Androgen Receptor Cistrome in African American Men Associates with Upregulation of Lipid Metabolism and Immune Response.

African-American (AA) men are more likely to be diagnosed with and die from prostate cancer than European American (EA) men. Despite the central role of the androgen receptor (AR) transcription factor in prostate cancer, little is known about the contribution of epigenetics to observed racial disparities. We performed AR chromatin immunoprecipitation sequencing on primary prostate tumors from AA and EA men, finding that sites with greater AR binding intensity in AA relative to EA prostate cancer are enriched for lipid metabolism and immune response genes. Integration with transcriptomic and metabolomic data demonstrated coinciding upregulation of lipid metabolism gene expression and increased lipid levels in AA prostate cancer. In a metastatic prostate cancer cohort, upregulated lipid metabolism associated with poor prognosis. These findings offer the first insights into ancestry-specific differences in the prostate cancer AR cistrome. The data suggest a model whereby increased androgen signaling may contribute to higher levels of lipid metabolism, immune response, and cytokine signaling in AA prostate tumors. Given the association of upregulated lipogenesis with prostate cancer progression, our study provides a plausible biological explanation for the higher incidence and aggressiveness of prostate cancer observed in AA men. SIGNIFICANCE: With immunotherapies and inhibitors of metabolic enzymes in clinical development, the altered lipid metabolism and immune response in African-American men provides potential therapeutic opportunities to attenuate racial disparities in prostate cancer.

Inhibition of GATA2 in prostate cancer by a clinically available small molecule.

Castration-resistant prostate cancer (CRPC) remains highly lethal and in need of novel, actionable therapeutic targets. The pioneer factor GATA2 is a significant prostate cancer (PC) driver and is linked to poor prognosis. GATA2 directly promotes androgen receptor (AR) gene expression (both full-length and splice-variant) and facilitates AR binding to chromatin, recruitment of coregulators, and target gene transcription. Unfortunately, there is no clinically applicable GATA2 inhibitor available at the moment. Using a bioinformatics algorithm, we screened in silico 2650 clinically relevant drugs for a potential GATA2 inhibitor. Validation studies used cytotoxicity and proliferation assays, global gene expression analysis, RT-qPCR, reporter assay, reverse phase protein array analysis (RPPA), and immunoblotting. We examined target engagement via cellular thermal shift assay (CETSA), ChIP-qPCR, and GATA2 DNA-binding assay. We identified the vasodilator dilazep as a potential GATA2 inhibitor and confirmed on-target activity via CETSA. Dilazep exerted anticancer activity across a broad panel of GATA2-dependent PC cell lines in vitro and in a PDX model in vivo. Dilazep inhibited GATA2 recruitment to chromatin and suppressed the cell-cycle program, transcriptional programs driven by GATA2, AR, and c-MYC, and the expression of several oncogenic drivers, including AR, c-MYC, FOXM1, CENPF, EZH2, UBE2C, and RRM2, as well as of several mediators of metastasis, DNA damage repair, and stemness. In conclusion, we provide, via an extensive compendium of methodologies, proof-of-principle that a small molecule can inhibit GATA2 function and suppress its downstream AR, c-MYC, and other PC-driving effectors. We propose GATA2 as a therapeutic target in CRPC.

Noninvasive Assessment of Epidermal Genomic Markers of UV Exposure in Skin.

The measurement of UV-induced DNA damage as a dosimeter of exposure and predictor of skin cancer risk has been proposed by multiple groups. Although UV-induced mutations and adducts are present in normal-appearing UV-exposed epidermis, sampling normal nonlesional skin requires noninvasive methods to extract epidermal DNA for analysis. Here, we demonstrate the feasibility of such an approach, termed surfactant-based tissue acquisition for molecular profiling. Sampling in patients was performed using a felt-tip pen soaked in a mixture of surfactants (Brij-30/N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). In mice, we show that the epidermis can be selectively removed without scarring, with complete healing within 2 weeks. We exposed hairless mice to low-dose UV radiation over a period of 3 months and serially sampled them through up to 2 months following the cessation of UV exposure, observing a progressive increase in a UV signature mutational burden. To test whether surfactant-based tissue acquisition for molecular profiling could be applied to human patients, samples were collected from sun-exposed and sun-protected areas, which were then subjected to high-depth targeted exome sequencing. Extensive UV-driven mosaicism and substantially increased mutational loads in sun-exposed versus sun-protected areas were observed, suggesting that genomic measures, as an integrated readout of DNA damage, repair, and clonal expansion, may be informative markers of UV exposure.

Integrative transcriptomic analysis for linking acute stress responses to squamous cell carcinoma development.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer and commonly arises in chronically UV-exposed skin or chronic wounds. Since UV exposure and chronic wounds are the two most prominent environmental factors that lead to cuSCC initiation, we undertook this study to test whether more acute molecular responses to UV and wounding overlapped with molecular signatures of cuSCC. We reasoned that transcriptional signatures in common between acutely UV-exposed skin, wounded skin, and cuSCC tumors, might enable us to identify important pathways contributing to cuSCC. We performed transcriptomic analysis on acutely UV-exposed human skin and integrated those findings with datasets from wounded skin and our transcriptomic data on cuSCC using functional pair analysis, GSEA, and pathway analysis. Integrated analyses revealed significant overlap between these three datasets, thus highlighting deep molecular similarities these biological processes, and we identified Oncostatin M (OSM) as a potential common upstream driver. Expression of OSM and its downstream targets correlated with poorer overall survival in head and neck SCC patients. In vitro, OSM promoted invasiveness of keratinocytes and cuSCC cells and suppressed apoptosis of irradiated keratinocytes. Together, these results support the concept of using an integrated, biologically-informed approach to identify potential promoters of tumorigenesis.

Discovery, Structure-Activity Relationship, and Biological Activity of Histone-Competitive Inhibitors of Histone Acetyltransferases P300/CBP.

Histone acetyltransferase (HAT) p300 and its paralog CBP acetylate histone lysine side chains and play critical roles in regulating gene transcription. The HAT domain of p300/CBP is a potential drug target for cancer. Through compound screening and medicinal chemistry, novel inhibitors of p300/CBP HAT with their IC50 values as low as 620 nM were discovered. The most potent inhibitor is competitive against histone substrates and exhibits a high selectivity for p300/CBP. It inhibited cellular acetylation and had strong activity with EC50 of 1-3 µM against proliferation of several tumor cell lines. Gene expression profiling in estrogen receptor (ER)-positive breast cancer MCF-7 cells showed that inhibitor treatment recapitulated siRNA-mediated p300 knockdown, inhibited ER-mediated gene transcription, and suppressed expression of numerous cancer-related gene signatures. These results demonstrate that the inhibitor is not only a useful probe for biological studies of p300/CBP HAT but also a pharmacological lead for further drug development targeting cancer.

Correction to: The Emerging Roles of Steroid Hormone Receptors in Ductal Carcinoma in Situ (DCIS) of the Breast.

The original version of this article unfortunately contained mistakes.

The Emerging Roles of Steroid Hormone Receptors in Ductal Carcinoma in Situ (DCIS) of the Breast.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to most types of invasive breast cancer (IBC). Although it is estimated only one third of untreated patients with DCIS will progress to IBC, standard of care for treatment is surgery and radiation. This therapeutic approach combined with a lack of reliable biomarker panels to predict DCIS progression is a major clinical problem. DCIS shares the same molecular subtypes as IBC including estrogen receptor (ER) and progesterone receptor (PR) positive luminal subtypes, which encompass the majority (60-70%) of DCIS. Compared to the established roles of ER and PR in luminal IBC, much less is known about the roles and mechanism of action of estrogen (E2) and progesterone (P4) and their cognate receptors in the development and progression of DCIS. This is an underexplored area of research due in part to a paucity of suitable experimental models of ER+/PR + DCIS. This review summarizes information from clinical and observational studies on steroid hormones as breast cancer risk factors and ER and PR as biomarkers in DCIS. Lastly, we discuss emerging experimental models of ER+/PR+ DCIS.

Assessing inter-component heterogeneity of biphasic uterine carcinosarcomas.

OBJECTIVE: Uterine carcinosarcoma (UCS) is a rare and aggressive form of uterine cancer. It is bi-phasic, exhibiting histological features of both malignant epithelial (carcinoma) and mesenchymal (sarcoma) elements, reflected in ambiguity in accepted treatment guidelines. We sought to study the genomic and transcriptomic profiles of these elements individually to gain further insights into the development of these tumors. METHODS: We macro-dissected carcinomatous, sarcomatous, and normal tissues from formalin fixed paraffin embedded uterine samples of 10 UCS patients. Single nucleotide polymorphism microarrays, targeted DNA sequencing and whole-transcriptome RNA-sequencing were performed. Somatic chromosomal alterations (SCAs), point mutation and gene expression profiles were compared between carcinomatous and sarcomatous components. RESULTS: In addition to TP53, other recurrently mutated genes harboring putative driver or loss-of-function mutations included PTEN, FBXW7, FGFR2, KRAS, PIK3CA and CTNNB1, genes known to be involved in UCS. Intra-patient somatic mutation and SCA profiles were highly similar between paired carcinoma and sarcoma samples. An epithelial-mesenchymal transition (EMT) signature tended to differentiate components, with EMT-like status more common in advanced-stage patients exhibiting higher inter-component SCA heterogeneity. CONCLUSIONS: From DNA analysis, our results indicate a monoclonal disease origin for this cohort. Yet expression-derived EMT statuses of the carcinomatous and sarcomatous components were often discrepant, and advanced cases displayed greater genomic heterogeneity. Therefore, separately-profiled components of UCS tumors may better inform disease progression or potential.

APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

ICG-001 Exerts Potent Anticancer Activity Against Uveal Melanoma Cells.

Purpose: Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/ß-catenin-mediated transcriptional program. Methods: We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model. Results: ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice. Conclusions: Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.

Genome-wide identification and differential analysis of translational initiation.

Translation is principally regulated at the initiation stage. The development of the translation initiation (TI) sequencing (TI-seq) technique has enabled the global mapping of TIs and revealed unanticipated complex translational landscapes in metazoans. Despite the wide adoption of TI-seq, there is no computational tool currently available for analyzing TI-seq data. To fill this gap, we develop a comprehensive toolkit named Ribo-TISH, which allows for detecting and quantitatively comparing TIs across conditions from TI-seq data. Ribo-TISH can also predict novel open reading frames (ORFs) from regular ribosome profiling (rRibo-seq) data and outperform several established methods in both computational efficiency and prediction accuracy. Applied to published TI-seq/rRibo-seq data sets, Ribo-TISH uncovers a novel signature of elevated mitochondrial translation during amino-acid deprivation and predicts novel ORFs in 5'UTRs, long noncoding RNAs, and introns. These successful applications demonstrate the power of Ribo-TISH in extracting biological insights from TI-seq/rRibo-seq data.