Outcomes for potentially Resectable patients undergoing primary chemoradiation treatment for T1-T2 HPV Negative oropharyngeal squamous cell carcinoma.

BACKGROUND: Transoral surgical resectability (TOS) is a prognostic factor for patients with HPV+ T1-2 oropharyngeal squamous cell carcinoma (OPSCC) disease undergoing radiotherapy (RT), but it is unclear whether this holds for HPV-negative (HPV-) patients. We aimed to compare outcomes of potential TOS-candidates vs. non-TOS candidates, among patients who underwent RT/CRT for early T-stage HPV- OPSCC. METHODS: For patients treated with RT/CRT for early T-stage HPV-negative OPSCC between 2014 and 2021, pretreatment imaging was reviewed by four head-and-neck surgeons, masked to clinical outcomes, to assess primary-site suitability for TOS. Extracapsular extension (ECE) was assessed by a head-and-neck neuroradiologist. We compared outcomes based on surgical resectability relating to: (1) the primary site tumor alone, and (2) the primary site plus the absence/presence of ECE (overall assessment). Kaplan-Meier curves for overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS) were compared using the log-rank test. RESULTS: Seventy patients were included in the analysis. The primary site was TOS-favorable in 46/70 (66%). Based on the overall assessment, 41/70 (58.6%) were TOS-favorable. The 3-year OS, DSS and PFS for primary site TOS-favorable versus unfavorable were OS: 76.9% versus 37.4%; DSS: 78.1% versus 46.2%, PFS: 69.9% versus 41.3%, (log-rank test = 0.01, 0.03, 0.04; respectively). Additionally, patients with an overall assessment of TOS favorability demonstrated better survival outcomes compared with TOS-unfavorable patients (OS: 77.3% vs. 46.2%; DSS: 78.2% vs. 56.5%, PFS: 72.3% vs. 42.1%, log-rank test = 0.01, 0.04, 0.01; respectively). CONCLUSION: Patients with TOS-favorable HPV-negative early T-stage OPSCC have superior survival outcomes than TOS-unfavorable patients.

Metabolic pathway-based subtypes associate glycan biosynthesis and treatment response in head and neck cancer.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous malignancy that remains a significant challenge in clinical management due to frequent treatment failures and pronounced therapy resistance. While metabolic dysregulation appears to be a critical factor in this scenario, comprehensive analyses of the metabolic HNSCC landscape and its impact on clinical outcomes are lacking. This study utilized transcriptomic data from four independent clinical cohorts to investigate metabolic heterogeneity in HNSCC and define metabolic pathway-based subtypes (MPS). In HPV-negative HNSCCs, MPS1 and MPS2 were identified, while MPS3 was enriched in HPV-positive cases. MPS classification was associated with clinical outcome post adjuvant radio(chemo)therapy, with MPS1 consistently exhibiting the highest risk of therapeutic failure. MPS1 was uniquely characterized by upregulation of glycan (particularly chondroitin/dermatan sulfate) metabolism genes. Immunohistochemistry and pilot mass spectrometry imaging analyses confirmed this at metabolite level. The histological context and single-cell RNA sequencing data identified the malignant cells as key contributors. Globally, MPS1 was distinguished by a unique transcriptomic landscape associated with increased disease aggressiveness, featuring motifs related to epithelial-mesenchymal transition, immune signaling, cancer stemness, tumor microenvironment assembly, and oncogenic signaling. This translated into a distinct histological appearance marked by extensive extracellular matrix remodeling, abundant spindle-shaped cancer-associated fibroblasts, and intimately intertwined populations of malignant and stromal cells. Proof-of-concept data from orthotopic xenotransplants replicated the MPS phenotypes on the histological and transcriptome levels. In summary, this study introduces a metabolic pathway-based classification of HNSCC, pinpointing glycan metabolism-enriched MPS1 as the most challenging subgroup that necessitates alternative therapeutic strategies.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

The viral etiology of EBV-associated gastric cancers contributes to their unique pathology, clinical outcomes, treatment responses and immune landscape.

Epstein-Barr virus (EBV) is a pathogen known to cause a number of malignancies, often taking years for them to develop after primary infection. EBV-associated gastric cancer (EBVaGC) is one such malignancy, and is an immunologically, molecularly and pathologically distinct entity from EBV-negative gastric cancer (EBVnGC). In comparison with EBVnGCs, EBVaGCs overexpress a number of immune regulatory genes to help form an immunosuppressive tumor microenvironment (TME), have improved prognosis, and overall have an "immune-hot" phenotype. This review provides an overview of the histopathology, clinical features and clinical outcomes of EBVaGCs. We also summarize the differences between the TMEs of EBVaGCs and EBVnGCs, which includes significant differences in cell composition and immune infiltration. A list of available EBVaGC and EBVnGC gene expression datasets and computational tools are also provided within this review. Finally, an overview is provided of the various chemo- and immuno-therapeutics available in treating gastric cancers (GCs), with a focus on EBVaGCs.

HPV-negative head and neck cancers with adverse pathological features carry specific molecular changes that are associated with survival.

BACKGROUND: Adverse pathological features following surgery in head and neck squamous cell carcinoma (HNSCC) are strongly associated with survival and guide adjuvant therapy. We investigated molecular changes associated with these features. METHODS: We downloaded data from the Cancer Genome Atlas and Cancer Proteome Atlas HNSCC cohorts. We compared tumors positive versus negative for perineural invasion (PNI), lymphovascular invasion (LVI), extracapsular spread (ECS), and positive margins (PSM), with multivariable analysis. RESULTS: All pathological features were associated with poor survival, as were the following molecular changes: low cyclin E1 (HR = 1.7) and high PKC-alpha (HR = 1.8) in tumors with PNI; six of 13 protein abundance changes with LVI; greater tumor hypoxia and high Raptor (HR = 2.0) and Rictor (HR = 1.6) with ECS; and low p38 (HR = 2.3), high fibronectin (HR = 1.6), low annexin A1 (HR = 3.1), and high caspase-9 (HR = 1.6) abundances with PSM. CONCLUSIONS: Pathological features in HNSCC carry specific molecular changes that may explain their poor prognostic associations.

HPV16 Intratypic Variants in Head and Neck Cancers: A North American Perspective.

Human papillomavirus (HPV) is the major causative agent for cervical and many head and neck cancers (HNCs). HPVs randomly acquire single nucleotide polymorphisms (SNPs) that may become established via positive selection. Within an HPV type, viral isolates differing by <2% in the L1 region are termed "variants" and classified based on combinations of SNPs. Studies in cervical cancer demonstrate clear differences between HPV16 intratypic variants in terms of persistence of infection, tumor histology, cancer risk, and death. Much less is known about the frequency of HPV16 variants in HNC, and their effects on clinical outcomes. We combined HPV16 positive (HPV16+) HNC samples from a local Southwestern Ontario, Canada cohort with those from the Cancer Genome Atlas to create a larger North American cohort of 149 cases with clinical data and determined the distribution of intratypic variants and their impact on clinical outcomes. Most isolates were lineage A, sublineage A1, or A2, with roughly half exhibiting the T350G polymorphism in E6. Univariable analysis identified significant differences between 350T and 350G intratypic variants in clinical T, N, and O staging, as well as disease-free survival. Multivariable analysis failed to identify any clinical factor as a statistically significant covariate for disease-free survival differences between 350T and 350G. Significant differences in several measures of B-cell mediated immune response were also observed between 350T and 350G intratypic variants. We suggest that HPV genetic variation may be associated with HNC clinical characteristics and may have prognostic value.

Exploiting the endogenous yeast nuclear proteome to identify short linear motifs in vivo.

Peptide-domain interactions mediated by short linear motifs (SLiMs) play crucial roles in cellular biology. The simplicity of SLiMs poses challenges in their computational identification. Existing high-throughput methods for discovering SLiMs lack cellular context as they are typically performed in vitro. We developed a functional selection method using yeast to identify peptides that interact with the endogenous yeast nuclear proteome. Remarkably, peptides selected for in yeast also mediated nuclear import in human cells. Notably, the identified peptides did not resemble classical nuclear localization sequences. This platform has the potential to identify and investigate motifs that interact with the nuclear proteome of yeast and human and to aid in the identification and understanding of alternative protein nuclear import mechanisms.

Loss of LRP1B expression drives acquired chemo and radio-resistance in HPV-positive head and neck cancer.

OBJECTIVES: Although human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients typically experience excellent survival, 15-20 % of patients recur after treatment with chemotherapy and radiation. Therefore, there is a need for biomarkers of treatment failure to guide treatment intensity. MATERIALS AND METHODS: Whole genome sequencing was carried out on HPV+OPSCC patients who were primarily treated with concurrent chemotherapy (cisplatin) and radiation. We then explored whether the loss of LRP1Bwas sufficient to drive an aggressive phenotype, and promote a resistance to cisplatin and radiation therapy both in vitro using HPV+ cell lines (93VU147T, UMSCC47, UWO37 and UWO23) and in vivo. RESULTS: Through integrative genomic analysis of three HPV+OPSCC tumour datasets, we identified that deletion of LRP1B was enriched in samples that recurred following chemo-radiation. Knockdown using siRNA in four HPV+ cell lines (UWO23, UWO37, UMSCC47 and 93VU147T) resulted in increased proliferation of all cases. CRISPR/Cas9 deletion of LRP1B in the same cell line panel demonstrated increased proliferation, clonogenic growth and migration, as well as resistance to both cisplatin and radiation in LRP1B deleted cells compared to their respective non-targeting control cells. Cell line derived xenograft studies indicated that the LRP1B knockout tumours were more resistant to cisplatin and radiation therapy compared to their controls invivo. CONCLUSION: Taken together, our work implicates LRP1B deletion as a potential biomarker for identifying treatment resistant HPV+ OPSCC cases.

Differences in the Tumor Microenvironment of EBV-Associated Gastric Cancers Revealed Using Single-Cell Transcriptome Analysis.

Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with nearly 10% of gastric cancers (GCs). These EBV-associated GCs (EBVaGCs) are molecularly, histopathologically, and clinically distinct from EBV-negative GCs (EBVnGCs). While viral genes in EBVaGCs contribute to the carcinogenesis process, viral proteins also represent foreign antigens that could trigger enhanced immune responses compared to EBVnGCs. Despite prior investigations of the EBVaGC tumor microenvironment (TME), the cellular composition has not been thoroughly explored. In this study, cellular subpopulations overrepresented in EBVaGCs were identified and molecularly characterized. Genes consistently expressed across both bulk tumor and single-cell RNA sequencing data were highlighted, with the expression across the identified cellular subpopulations analyzed. As expected, based on existing histopathological analysis, EBVaGC is characterized by abundant lymphocytic infiltration of the stroma. Our molecular analysis identified three unique immune cell subpopulations in EBVaGC: T and B cells expressing high levels of proliferation markers and B cells expressing T cell features. The proliferating T cell cluster also expressed markers of follicular T helper cells. Overall, EBVaGC also exhibited unique features indicative of a higher inflammatory response. These substantial differences within the TME suggest that further detailed exploration of the cellular composition of EBVaGCs is needed, which may identify cellular subpopulations and phenotypes associated with patient outcomes.

The EBV Gastric Cancer Resource (EBV-GCR): A Suite of Tools for Investigating EBV-Associated Human Gastric Carcinogenesis.

Epstein-Barr virus (EBV) causes lifelong infection in over 90% of the world's population. EBV infection leads to several types of B cell and epithelial cancers due to the viral reprogramming of host-cell growth and gene expression. EBV is associated with 10% of stomach/gastric adenocarcinomas (EBVaGCs), which have distinct molecular, pathological, and immunological characteristics compared to EBV-negative gastric adenocarcinomas (EBVnGCs). Publicly available datasets, such as The Cancer Genome Atlas (TCGA), contain comprehensive transcriptomic, genomic, and epigenomic data for thousands of primary human cancer samples, including EBVaGCs. Additionally, single-cell RNA-sequencing data are becoming available for EBVaGCs. These resources provide a unique opportunity to explore the role of EBV in human carcinogenesis, as well as differences between EBVaGCs and their EBVnGC counterparts. We have constructed a suite of web-based tools called the EBV Gastric Cancer Resource (EBV-GCR), which utilizes TCGA and single-cell RNA-seq data and can be used for research related to EBVaGCs. These web-based tools allow investigators to gain in-depth biological and clinical insights by exploring the effects of EBV on cellular gene expression, associations with patient outcomes, immune landscape features, and differential gene methylation, featuring both whole-tissue and single-cell analyses.

Tumor-Infiltrating T Cells in EBV-Associated Gastric Carcinomas Exhibit High Levels of Multiple Markers of Activation, Effector Gene Expression, and Exhaustion.

Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with 10% of all gastric cancers (GCs) and 1.5% of all human cancers. EBV-associated GCs (EBVaGCs) are pathologically and clinically distinct entities from EBV-negative GCs (EBVnGCs), with EBVaGCs exhibiting differential molecular pathology, treatment response, and patient prognosis. However, the tumor immune landscape of EBVaGC has not been well explored. In this study, a systemic and comprehensive analysis of gene expression and immune landscape features was performed for both EBVaGC and EBVnGC. EBVaGCs exhibited many aspects of a T cell-inflamed phenotype, with greater T and NK cell infiltration, increased expression of immune checkpoint markers (BTLA, CD96, CTLA4, LAG3, PD1, TIGIT, and TIM3), and multiple T cell effector molecules in comparison with EBVnGCs. EBVaGCs also displayed a higher expression of anti-tumor immunity factors (PDL1, CD155, CEACAM1, galectin-9, and IDO1). Six EBV-encoded miRNAs (miR-BARTs 8-3p, 9-5p, 10-3p, 22, 5-5p, and 14-3p) were strongly negatively correlated with the expression of immune checkpoint receptors and multiple markers of anti-tumor immunity. These profound differences in the tumor immune landscape between EBVaGCs and EBVnGCs may help explain some of the observed differences in pathological and clinical outcomes, with an EBV-positive status possibly being a potential biomarker for the application of immunotherapy in GC.

Adenoviruses in medicine: innocuous pathogen, predator, or partner.

The consequences of human adenovirus (HAdV) infections are generally mild. However, despite the perception that HAdVs are harmless, infections can cause severe disease in certain individuals, including newborns, the immunocompromised, and those with pre-existing conditions, including respiratory or cardiac disease. In addition, HAdV outbreaks remain relatively common events and the recent emergence of more pathogenic genomic variants of various genotypes has been well documented. Coupled with evidence of zoonotic transmission, interspecies recombination, and the lack of approved AdV antivirals or widely available vaccines, HAdVs remain a threat to public health. At the same time, the detailed understanding of AdV biology garnered over nearly 7 decades of study has made this group of viruses a molecular workhorse for vaccine and gene therapy applications.

Reduced MHC Class I and II Expression in HPV-Negative vs. HPV-Positive Cervical Cancers.

Cervical cancer (CC) is the second most common cancer in women worldwide and the fourth leading cause of cancer-associated death in women. Although human papillomavirus (HPV) infection is associated with nearly all CC, it has recently become clear that HPV-negative (HPV-) CC represents a distinct disease phenotype with increased mortality. HPV-positive (HPV+) and HPV- CC demonstrate different molecular pathology, prognosis, and response to treatment. Furthermore, CC caused by HPV α9 types (HPV16-like) often have better outcomes than those caused by HPV α7 types (HPV18-like). This study systematically and comprehensively compared the expression of genes involved in major histocompatibility complex (MHC) class I and II presentation within CC caused by HPV α9 types, HPV α7 types, and HPV- CC. We observed increased expression of MHC class I and II classical and non-classical genes in HPV+ CC and overall higher expression of genes involved in their antigen loading and presentation apparatus as well as transcriptional regulation. Increased expression of MHC I-related genes differs from previous studies using cell culture models. These findings identify crucial differences between antigen presentation within the tumor immune microenvironments of HPV+ and HPV- CC, as well as modest differences between HPV α9 and α7 CC. These differences may contribute to the altered patient outcomes and responses to immunotherapy observed between these distinct cancers.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

HPV-Positive and -Negative Cervical Cancers Are Immunologically Distinct.

Although infection with human papillomavirus (HPV) is associated with nearly all cervical cancers (CC), a small proportion are HPV-negative. Recently, it has become clear that HPV-negative CC represent a distinct disease phenotype compared to HPV-positive disease and exhibit increased mortality. In addition, variations between different HPV types associated with CC have been linked to altered molecular pathology and prognosis. We compared the immune microenvironments of CC caused by HPV α9 species (HPV16-like), HPV α7 species (HPV18-like) and HPV-negative disease. HPV-negative CC appeared distinct from other subtypes, with greatly reduced levels of lymphocyte infiltration compared to either HPV α9 or α7 CC. Besides reduced levels of markers indicative of B, T, and NK lymphocytes, the expression of T-cell effector molecules, activation/exhaustion markers, and T-cell receptor diversity were also significantly lower in HPV-negative CC. Interestingly, HPV-negative CC expressed much higher levels of potential neoantigens than HPV-positive CC. These results identify profound differences between the immune landscape of HPV-positive and HPV-negative CC as well as modest differences between HPV α9 and α7 CC. These differences may contribute to altered patient outcomes between HPV-negative and HPV-positive CC and potentially between CC associated with different HPV types.

The HPV Induced Cancer Resource (THInCR): a Suite of Tools for Investigating HPV-Dependent Human Carcinogenesis.

Human papillomaviruses (HPVs) are highly infectious and cause the most common sexually transmitted viral infections. They induce hyperproliferation of squamous epithelial tissue, often forming warts. Virally encoded proteins reprogram gene expression and cell growth to create an optimal environment for viral replication. In addition to their normal roles in infection, functional alterations induced by viral proteins establish conditions that frequently contribute to human carcinogenesis. In fact, ~5% of human cancers are caused by HPVs, with virtually all cervical squamous cell carcinomas (CESC) and an increasing number of head and neck squamous cell carcinomas (HNSC) attributed to HPV infection. The Cancer Genome Atlas (TCGA) molecularly characterized thousands of primary human cancer samples in many cancer types, including CESC and HNSC, and created a comprehensive atlas of genomic, epigenomic, and transcriptomic data. This publicly available genome-wide information provides an unprecedented opportunity to expand the knowledge of the role that HPV plays in human carcinogenesis. While many tools exist to mine these data, few, if any, focus on the comparison of HPV-positive cancers with their HPV-negative counterparts or adjacent normal control tissue. We have constructed a suite of web-based tools, The HPV Induced Cancer Resource (THInCR), to utilize TCGA data for research related to HPV-induced CESC and HNSC. These tools allow investigators to gain greater biological and medical insights by exploring the impacts of HPV on cellular gene expression (mRNA and microRNA), altered gene methylation, and associations with patient survival and immune landscape features. These tools are accessible at https://thincr.ca/. IMPORTANCE The suite of analytical tools of THInCR provides the opportunity to investigate the roles that candidate target genes identified in cell lines or other model systems contribute to in actual HPV-dependent human cancers and is based on large-scale TCGA data sets. Expression of target genes, including both mRNA and microRNA, can be correlated with HPV gene expression, epigenetic changes in DNA methylation, patient survival, and numerous immune features, like leukocyte infiltration, interferon gamma response, T cell response, etc. Data from these analyses may immediately provide evidence to validate in vitro observations, reveal insights into mechanisms of virus-mediated alterations in cell growth, behavior, gene expression, and innate and adaptive immunity and may help hypothesis generation for further investigations.

Special Issue "Human Papillomavirus Clinical Research: From Infection to Cancer".

Papillomaviruses (PVs) are ubiquitous intracellular pathogens that have co-evolved with many different species [...].

The Impact of Surgical Resectability on Outcomes for Patients Undergoing Primary Radiation Treatment for Human Papillomavirus-Related Oropharyngeal Cancer.

PURPOSE: Primary radiation therapy with or without chemotherapy (RT/CRT) is the most common treatment for oropharyngeal squamous cell carcinomas (OPSCC), but there has been an increase in transoral surgery (TOS) for T1-2 tumors. Because only a subset of T1-2 tumors are TOS-favorable, nonrandomized comparisons between RT/CRT and TOS could be confounded by indication. We aimed to compare outcomes of potential TOS-candidates versus non-TOS candidates, among patients who underwent RT/CRT for early T-stage OPSCC. METHODS AND MATERIALS: For patients treated with RT/CRT for early-stage human papilloma virus positive OPSCC between 2014 and 2018, pretreatment imaging was reviewed by 3 head and neck surgeons, blinded to outcomes, to assess primary-site appropriateness for TOS, and extracapsular extension (ECE) was scored by a head and neck neuroradiologist. We compared outcomes based on surgical favorability pertaining to (1) the primary site tumor alone and (2) the primary site and an absence of ECE. Kaplan-Meier estimates for overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS) were compared using the log-rank test, with Cox regression used for multivariable modeling. RESULTS: One hundred and forty-three patients were evaluated, of which 121 were male (84.6%), the median age was 59.4 years, and all of them were p16 positive (100%). The primary site was TOS-favorable in 115 of 143 (80.4%). Patients with TOS-favorable primary site experienced superior 5-year OS (89.8% vs 71.2%, P = .017), DSS (90.4% vs 63.4%, P = .022), and RFS (83% vs 49.4%, P = .04) compared with TOS-unfavorable patients. Similarly, patients with a TOS-favorable primary site and no ECE on imaging 101 of 143 (70.6%), had improved OS, DSS, and RFS (P < .05) compared with TOS-unfavorable patients. CONCLUSIONS: In this first study to assess surgical favorability as a prognostic factor among patients with T1/2 p16+ OPSCC, patients with TOS-favorable early-stage OPSCC have better outcomes than TOS-unfavorable patients. This provides valuable prognostic information for patients, and also suggests the risk of confounding by indication in nonrandomized comparisons of treatment modalities.

Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo.

The Argonaute proteins are present in all three domains of life, which are archaea, bacteria, and eukarya. Unlike the eukaryotic Argonaute proteins, which use small RNA guides to target mRNAs, some prokaryotic Argonaute proteins (pAgos) use a small DNA guide to interfere with DNA and/or RNA targets. However, the mechanisms of pAgo natural function remain unknown. Here, we investigate the mechanism by which pAgo from Natronobacterium gregoryi (NgAgo) targets plasmid and bacteriophage T7 DNA using a heterologous Escherichia coli-based model system. We show that NgAgo expressed from a plasmid linearizes its expression vector. Cotransformation assays demonstrate that NgAgo requires an RNA in trans that is transcribed from the bacteriophage T7 promoter to activate cleavage of a cotransformed plasmid, reminiscent of the trans-RNA function in CRISPR/Cas9. We propose a mechanism to explain how NgAgo eliminates invading foreign DNA and bacteriophage. By leveraging this discovery, we show that NgAgo can be programmed to target a plasmid or a chromosome locus. IMPORTANCE We revealed the mechanism that explains how the NgAgo eliminates the invading foreign DNA and bacteriophage in bacterial cells at 37°C, and by leveraging this discovery, NgAgo can be programmed to target a plasmid or a chromosome locus.

Tumor molecular differences associated with outcome disparities of Black patients with head and neck cancer.

BACKGROUND: Numerous studies of head and neck squamous cell carcinoma (HNSCC) have demonstrated disparate outcomes by race and ethnicity. Beyond known associations with socioeconomic variables, whether these are also associated with differences in tumor molecular composition has thus far been poorly explored. METHODS: We downloaded clinical and multiplatform molecular data from The Cancer Genome Atlas and other published studies. These were compared between non-Hispanic Black (n = 43) and White (n = 354) patients with non-HPV-related tumors, using multivariable models. Publicly available validation cohorts were used. RESULTS: Black patients had poorer progression-free survival than White patients. Tumors of Black patients had greater copy number aberrations, and increased SFRP1 methylation and miRNA-mediated PRG4 silencing associated with poor survival. PI3K/AkT/mTOR pathway proteins were differentially expressed. CONCLUSIONS: There are molecular differences between tumors of Black and White patients that may partially account for differences in survival. These may inform targeted treatment decisions to achieve equitable outcomes.