SLKB: synthetic lethality knowledge base.

Emerging CRISPR-Cas9 technology permits synthetic lethality (SL) screening of large number of gene pairs from gene combination double knockout (CDKO) experiments. However, the poor integration and annotation of CDKO SL data in current SL databases limit their utility, and diverse methods of calculating SL scores prohibit their comparison. To overcome these shortcomings, we have developed SL knowledge base (SLKB) that incorporates data of 11 CDKO experiments in 22 cell lines, 16,059 SL gene pairs and 264,424 non-SL gene pairs. Additionally, within SLKB, we have implemented five SL calculation methods: median score with and without background control normalization (Median-B/NB), sgRNA-derived score (sgRNA-B/NB), Horlbeck score, GEMINI score and MAGeCK score. The five scores have demonstrated a mere 1.21% overlap among their top 10% SL gene pairs, reflecting high diversity. Users can browse SL networks and assess the impact of scoring methods using Venn diagrams. The SL network generated from all data in SLKB shows a greater likelihood of SL gene pair connectivity with other SL gene pairs than non-SL pairs. Comparison of SL networks between two cell lines demonstrated greater likelihood to share SL hub genes than SL gene pairs. SLKB website and pipeline can be freely accessed at https://slkb.osubmi.org and https://slkb.docs.osubmi.org/, respectively.

An HTLV-1 envelope mRNA vaccine is immunogenic and protective in New Zealand rabbits.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission.

HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo.

Comparing Accuracy of Helicobacter pylori Identification Using Traditional Hematoxylin and Eosin-Stained Glass Slides With Digital Whole Slide Imaging.

With advancements in the field of digital pathology, there has been a growing need to compare the diagnostic abilities of pathologists using digitized whole slide images against those when using traditional hematoxylin and eosin (H&E)-stained glass slides for primary diagnosis. One of the most common specimens received in pathology practices is an endoscopic gastric biopsy with a request to rule out Helicobacter pylori (H. pylori) infection. The current standard of care is the identification of the organisms on H&E-stained slides. Immunohistochemical or histochemical stains are used selectively. However, due to their small size (2-4 µm in length by 0.5-1 µm in width), visualization of the organisms can present a diagnostic challenge. The goal of the study was to compare the ability of pathologists to identify H. pylori on H&E slides using a digital platform against the gold standard of H&E glass slides using routine light microscopy. Diagnostic accuracy rates using glass slides vs digital slides were 81% vs 72% (P = .0142) based on H&E slides alone. When H. pylori immunohistochemical slides were provided, the diagnostic accuracy was significantly improved to comparable rates (96% glass vs 99% digital, P = 0.2199). Furthermore, differences in practice settings (academic/subspecialized vs community/general) and the duration of sign-out experience did not significantly impact the accuracy of detecting H. pylori on digital slides. We concluded that digital whole slide images, although amenable in different practice settings and teaching environments, does present some shortcomings in accuracy and precision, especially in certain circumstances and thus is not yet fully capable of completely replacing glass slide review for identification of H. pylori. We specifically recommend reviewing glass slides and/or performing ancillary stains, especially when there is a discrepancy between the degree of inflammation and the presence of microorganisms on digital images.

An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer.

OBJECTIVES: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

Portable Mid-Infrared Spectroscopy Combined with Chemometrics to Diagnose Fibromyalgia and Other Rheumatologic Syndromes Using Rapid Volumetric Absorptive Microsampling.

The diagnostic criteria for fibromyalgia (FM) have relied heavily on subjective reports of experienced symptoms coupled with examination-based evidence of diffuse tenderness due to the lack of reliable biomarkers. Rheumatic disorders that are common causes of chronic pain such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and chronic low back pain are frequently found to be comorbid with FM. As a result, this can make the diagnosis of FM more challenging. We aim to develop a reliable classification algorithm using unique spectral profiles of portable FT-MIR that can be used as a real-time point-of-care device for the screening of FM. A novel volumetric absorptive microsampling (VAMS) technique ensured sample volume accuracies and minimized the variation introduced due to hematocrit-based bias. Blood samples from 337 subjects with different disorders (179 FM, 158 non-FM) collected with VAMS were analyzed. A semi-permeable membrane filtration approach was used to extract the blood samples, and spectral data were collected using a portable FT-MIR spectrometer. The OPLS-DA algorithm enabled the classification of the spectra into their corresponding classes with 84% accuracy, 83% sensitivity, and 85% specificity. The OPLS-DA regression plot indicated that spectral regions associated with amide bands and amino acids were responsible for discrimination patterns and can be potentially used as spectral biomarkers to differentiate FM and other rheumatic diseases.

CEDA: integrating gene expression data with CRISPR-pooled screen data identifies essential genes with higher expression.

MOTIVATION: Clustered regularly interspaced short palindromic repeats (CRISPR)-based genetic perturbation screen is a powerful tool to probe gene function. However, experimental noises, especially for the lowly expressed genes, need to be accounted for to maintain proper control of false positive rate. METHODS: We develop a statistical method, named CRISPR screen with Expression Data Analysis (CEDA), to integrate gene expression profiles and CRISPR screen data for identifying essential genes. CEDA stratifies genes based on expression level and adopts a three-component mixture model for the log-fold change of single-guide RNAs (sgRNAs). Empirical Bayesian prior and expectation-maximization algorithm are used for parameter estimation and false discovery rate inference. RESULTS: Taking advantage of gene expression data, CEDA identifies essential genes with higher expression. Compared to existing methods, CEDA shows comparable reliability but higher sensitivity in detecting essential genes with moderate sgRNA fold change. Therefore, using the same CRISPR data, CEDA generates an additional hit gene list. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm-Controlled Gene Expression in Mice.

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

Molecular profiling of kidney compartments from serial biopsies differentiate treatment responders from non-responders in lupus nephritis.

The immune pathways that define treatment response and non-response in lupus nephritis (LN) are unknown. To characterize these intra-kidney pathways, transcriptomic analysis was done on protocol kidney biopsies obtained at flare (initial biopsy (Bx1)) and after treatment (second biopsy (Bx2)) in 58 patients with LN. Glomeruli and tubulointerstitial compartments were isolated using laser microdissection. RNA was extracted and analyzed by nanostring technology with transcript expression from clinically complete responders, partial responders and non-responders compared at Bx1 and Bx2 and to the healthy controls. Top transcripts that differentiate clinically complete responders from non-responders were validated at the protein level by confocal microscopy and urine ELISA. At Bx1, cluster analysis determined that glomerular integrin, neutrophil, chemokines/cytokines and tubulointerstitial chemokines, T cell and leukocyte adhesion genes were able to differentiate non-responders from clinically complete responders. At Bx2, glomerular monocyte, extracellular matrix, and interferon, and tubulointerstitial interferon, complement, and T cell transcripts differentiated non-responders from clinically complete responders. Protein analysis identified several protein products of overexpressed glomerular and tubulointerstitial transcripts at LN flare, recapitulating top transcript findings. Urine complement component 5a and fibronectin-1 protein levels reflected complement and fibronectin expression at flare and after treatment. Thus, transcript analysis of serial LN kidney biopsies demonstrated how gene expression in the kidney changes with clinically successful and unsuccessful therapy. Hence, these insights into the molecular landscape of response and non-response may help align LN management with the pathogenesis of kidney injury.

Novel Automation of an Enzyme-Linked Immunosorbent Spot Assay Testing Method: Comparable Diagnostic Performance of the T-SPOT.TB Test Using Manual Density Gradient Cell Isolation versus Automated Positive Selection with the T-Cell Select Kit.

The diagnosis of latent tuberculosis (TB) infection (LTBI) is critical to improve TB treatment and control, and the T-SPOT.TB test is a commercial enzyme-linked immunosorbent spot assay used for this purpose. The objective of the study was to increase automation and extend the time between blood collection and processing for the T-SPOT.TB test from 0 to 8 h to 0 to 54 h. The previous maximum time between blood collection and processing for the T-SPOT.TB test is 32 h using T-Cell Xtend. For this, we compared the T-SPOT.TB test using manual peripheral blood mononuclear cell (PBMC) isolation by density gradient separation at 0 to 8 h (reference method, control arm) to an automated PBMC isolation method using magnetic beads (T-Cell Select kit) at 0 to 55 h postcollection. A total of 620 subjects were enrolled from 4 study sites, and blood samples were collected from each volunteer, comprising 1,850 paired samples in total. Overall agreement between both methods was 96.8% (confidence interval [CI], 95.9 to 97.6%), with 95.8% (CI, 93.5 to 97.5%) positive and 97.1% negative agreement (CI, 96.1 to 97.9%). In summary, there was a strong overall agreement between the automated and manual T-SPOT.TB test processing methods. The results suggest that the T-SPOT.TB test can be processed using automated positive selection with magnetic beads using T-Cell Select to decrease hands-on time. Also, this cell isolation method allowed for the time between blood collection and processing to range from 0 to 55 h. Additional studies in larger and diverse patient populations including immunocompromised and pediatric patients are needed.

Variants in LRRC34 reveal distinct mechanisms for predisposition to papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) demonstrates high heritability and a low somatic mutation burden relative to other cancers. Therefore, the genetic risk predisposing to PTC is likely due to a combination of low penetrance variants. A recent genome-wide association study revealed the association of PTC with a missense variant, rs6793295, at 3q26 in a gene called Leucine Repeat Rich Containing 34 (LRRC34). METHODS: We report the mechanisms of PTC risk at 3q26 using a combination of overexpression, mass spectroscopy, knockdown, transcriptome profiling, migration assays and genetic analysis. RESULTS: We observed differential binding of wild-type and missense LRRC34 to RANBP1. Overexpression of missense LRRC34 reduced RanGTP levels and increased apoptosis. We also identified a second linkage disequilibrium (LD) block upstream of LRRC34 containing regulatory variants with allele-specific expression. Transcriptome profiling of LRRC34 knockdown cells showed changes in genes involved with cellular movement. LRRC34 knockdown reduced the migration of thyroid cancer cell lines. Lastly, we assessed the relative contribution of PTC risk from each locus using haplotype analysis. CONCLUSIONS: Our study demonstrates two separate mechanisms, one in G protein signalling and the other in transcriptional control, dictating PTC risk at 3q26 using both biochemical and genetic techniques.

Power analysis for RNA-Seq differential expression studies using generalized linear mixed effects models.

BACKGROUND: Power analysis becomes an inevitable step in experimental design of current biomedical research. Complex designs allowing diverse correlation structures are commonly used in RNA-Seq experiments. However, the field currently lacks statistical methods to calculate sample size and estimate power for RNA-Seq differential expression studies using such designs. To fill the gap, simulation based methods have a great advantage by providing numerical solutions, since theoretical distributions of test statistics are typically unavailable for such designs. RESULTS: In this paper, we propose a novel simulation based procedure for power estimation of differential expression with the employment of generalized linear mixed effects models for correlated expression data. We also propose a new procedure for power estimation of differential expression with the use of a bivariate negative binomial distribution for paired designs. We compare the performance of both the likelihood ratio test and Wald test under a variety of simulation scenarios with the proposed procedures. The simulated distribution was used to estimate the null distribution of test statistics in order to achieve the desired false positive control and was compared to the asymptotic Chi-square distribution. In addition, we applied the procedure for paired designs to the TCGA breast cancer data set. CONCLUSIONS: In summary, we provide a framework for power estimation of RNA-Seq differential expression under complex experimental designs. Simulation results demonstrate that both the proposed procedures properly control the false positive rate at the nominal level.

Metabolic fingerprinting for diagnosis of fibromyalgia and other rheumatologic disorders.

Diagnosis and treatment of fibromyalgia (FM) remains a challenge owing to the lack of reliable biomarkers. Our objective was to develop a rapid biomarker-based method for diagnosing FM by using vibrational spectroscopy to differentiate patients with FM from those with rheumatoid arthritis (RA), osteoarthritis (OA), or systemic lupus erythematosus (SLE) and to identify metabolites associated with these differences. Blood samples were collected from patients with a diagnosis of FM (n = 50), RA (n = 29), OA (n = 19), or SLE (n = 23). Bloodspot samples were prepared, and spectra collected with portable FT-IR and FT-Raman microspectroscopy and subjected to metabolomics analysis by ultra-HPLC (uHPLC), coupled to a photodiode array (PDA) and tandem MS/MS. Unique IR and Raman spectral signatures were identified by pattern recognition analysis and clustered all study participants into classes (FM, RA, and SLE) with no misclassifications (p < 0.05, and interclass distances > 2.5). Furthermore, the spectra correlated (r = 0.95 and 0.83 for IR and Raman, respectively) with FM pain severity measured with fibromyalgia impact questionnaire revised version (FIQR) assessments. Protein backbones and pyridine-carboxylic acids dominated this discrimination and might serve as biomarkers for syndromes such as FM. uHPLC-PDA-MS/MS provided insights into metabolites significantly differing among the disease groups, not only in molecular m/z+ and m/z- values but also in UV-visible chromatograms. We conclude that vibrational spectroscopy may provide a reliable diagnostic test for differentiating FM from other disorders and for establishing serologic biomarkers of FM-associated pain.

Exploratory analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients receiving neo-adjuvant chemotherapy for operable breast cancer.

BACKGROUND: While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC. METHODS: Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student's t-test. RESULTS: This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression. CONCLUSIONS: This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients. TRIAL REGISTRATION: NCT04022616.

MYH9 binds to lncRNA gene PTCSC2 and regulates FOXE1 in the 9q22 thyroid cancer risk locus.

A locus on chromosome 9q22 harbors a SNP (rs965513) firmly associated with risk of papillary thyroid carcinoma (PTC). The locus also comprises the forkhead box E1 (FOXE1) gene, which is implicated in thyroid development, and a long noncoding RNA (lncRNA) gene, papillary thyroid cancer susceptibility candidate 2 (PTCSC2). How these might interact is not known. Here we report that PTCSC2 binds myosin-9 (MYH9). In a bidirectional promoter shared by FOXE1 and PTCSC2, MYH9 inhibits the promoter activity in both directions. This inhibition can be reversed by PTCSC2, which acts as a suppressor. RNA knockdown of FOXE1 in primary thyroid cells profoundly interferes with the p53 pathway. We propose that the interaction between the lncRNA, its binding protein MYH9, and the coding gene FOXE1 underlies the predisposition to PTC triggered by rs965513.

Fully moderated t-statistic in linear modeling of mixed effects for differential expression analysis.

BACKGROUND: Gene expression profiling experiments with few replicates lead to great variability in the estimates of gene variances. Toward this end, several moderated t-test methods have been developed to reduce this variability and to increase power for testing differential expression. Most of these moderated methods are based on linear models with fixed effects where residual variances are smoothed under a hierarchical Bayes framework. However, they are inadequate for designs with complex correlation structures, therefore application of moderated methods to linear models with mixed effects are needed for differential expression analysis. RESULTS: We demonstrated the implementation of the fully moderated t-statistic method for linear models with mixed effects, where both residual variances and variance estimates of random effects are smoothed under a hierarchical Bayes framework. We compared the proposed method with two current moderated methods and show that the proposed method can control the expected number of false positives at the nominal level, while the two current moderated methods fail. CONCLUSIONS: We proposed an approach for testing differential expression under complex correlation structures while providing variance shrinkage. The proposed method is able to improve power by moderation and controls the expected number of false positives properly at the nominal level.

An IL-15-based superagonist ALT-803 enhances the NK cell response to cetuximab-treated squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide and epidermal growth factor receptor (EGFR) is overexpressed in greater than 90% of patient tumors. Cetuximab is a monoclonal antibody that binds to EGFR and can activate immune cells, such as natural killer (NK) cells, that express receptors for the Fc (constant region) of immunoglobulin G. IL-15 (interleukin-15) is a critical factor for the development, proliferation and activation of effector NK cells. A novel IL-15 compound known as ALT-803 that consists of genetically modified IL-15 plus the IL-15 receptor alpha protein (IL15Rα) fused to the Fc portion of IgG1 has recently been developed. We hypothesized that treatment with ALT-803 would increase NK cell-mediated cytotoxicity of cetuximab-coated head and neck squamous cells. CD56+ NK cells from normal healthy donors were treated overnight with ALT-803 and tested for their ability to lyse cetuximab-coated tumor cells. Cytotoxicity was greater following NK cell ALT-803 activation, as compared to controls. ALT-803-treated NK cells secreted significantly higher levels of IFN-γ than control conditions. Additionally, NK cells showed increased levels of phospho-ERK and phospho-STAT5 when co-cultured with cetuximab-coated tumors and ALT-803. Administration of both cetuximab and ALT-803 to mice harboring Cal27 SCCHN tumors resulted in significantly decreased tumor volume when compared to controls and compared to single-agent treatment alone. Overall, the present data suggest that cetuximab treatment in combination with ALT-803 in patients with EGFR-positive SCCHN may result in significant NK cell activation and have important anti-tumor activity.