Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain.

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

Polygenic Risk Score Modifies Prostate Cancer Risk of Pathogenic Variants in Men of African Ancestry.

Prostate cancer risk is influenced by rare and common germline variants. We examined the aggregate association of rare germline pathogenic/likely pathogenic/deleterious (P/LP/D) variants in ATM, BRCA2, PALB2, and NBN with a polygenic risk score (PRS) on prostate cancer risk among 1,796 prostate cancer cases (222 metastatic) and 1,424 controls of African ancestry. Relative to P/LP/D non-carriers at average genetic risk (33%-66% of PRS), men with low (0%-33%) and high (66%-100%) PRS had Odds Ratios (ORs) for overall prostate cancer of 2.08 [95% confidence interval (CI) = 0.58-7.49] and 18.06 (95% CI = 4.24-76.84) among P/LP/D carriers and 0.57 (95% CI = 0.46-0.71) and 3.02 (95% CI = 2.53-3.60) among non-carriers, respectively. The OR for metastatic prostate cancer was 2.73 (95% CI = 0.24-30.54) and 28.99 (95% CI = 4.39-191.43) among P/LP/D carriers and 0.54 (95% CI = 0.31-0.95) and 3.22 (95% CI = 2.20-4.73) among non-carriers, for men with low and high PRS, respectively. Lifetime absolute risks of overall prostate cancer increased with PRS (low to high) from 9.8% to 51.5% in P/LP/D carriers and 5.5% to 23.9% in non-carriers. Lifetime absolute risks of metastatic prostate cancer increased with PRS from 1.9% to 18.1% in P/LP/D carriers and 0.3% to 2.2% in non-carriers These findings suggest that assessment of prostate cancer risk for rare variant carriers should include PRS status. SIGNIFICANCE: These findings highlight the importance of considering rare and common variants to comprehensively assess prostate cancer risk in men of African ancestry.

Germline Mutations in 12 Genes and Risk of Ovarian Cancer in Three Population-Based Cohorts.

BACKGROUND: With the widespread use of multigene panel genetic testing, population-based studies are necessary to accurately assess penetrance in unselected individuals. We evaluated the prevalence of germline pathogenic or likely pathogenic variants (mutations) in 12 cancer-predisposition genes and associations with ovarian cancer risk in three population-based prospective studies [Nurses' Health Study (NHS), NHSII, Cancer Prevention Study II]. METHODS: We included women with epithelial ovarian or peritoneal cancer (n = 776) and controls who were alive and had at least one intact ovary at the time of the matched case diagnosis (n = 1,509). Germline DNA was sequenced for mutations in 12 genes. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) for ovarian cancer risk by mutation status. RESULTS: The mutation frequency across all 12 genes was 11.2% in cases and 3.3% in controls (P < 0.0001). BRCA1 and BRCA2 were the most frequently mutated (3.5% and 3.8% of cases and 0.3% and 0.5% of controls, respectively) and were associated with increased ovarian cancer risk [OR, BRCA1 = 12.38; 95% confidence interval (CI) = 4.72-32.45; OR, BRCA2 = 9.18; 95% CI = 3.98-21.15]. Mutation frequencies for the other genes were ≤1.0% and only PALB2 was significantly associated with risk (OR = 5.79; 95% CI = 1.09-30.83). There was no difference in survival for women with a BRCA germline mutation versus no mutation. CONCLUSIONS: Further research is needed to better understand the role of other mutations in ovarian cancer among unselected populations. IMPACT: Our data support guidelines for germline genetic testing for BRCA1 and BRCA2 among women diagnosed with epithelial ovarian cancer; testing for PALB2 may be warranted.

ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk.

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

Gene regulatory network inference based on a nonhomogeneous dynamic Bayesian network model with an improved Markov Monte Carlo sampling.

A nonhomogeneous dynamic Bayesian network model, which combines the dynamic Bayesian network and the multi-change point process, solves the limitations of the dynamic Bayesian network in modeling non-stationary gene expression data to a certain extent. However, certain problems persist, such as the low network reconstruction accuracy and poor model convergence. Therefore, we propose an MD-birth move based on the Manhattan distance of the data points to increase the rationality of the multi-change point process. The underlying concept of the MD-birth move is that the direction of movement of the change point is assumed to have a larger Manhattan distance between the variance and the mean of its left and right data points. Considering the data instability characteristics, we propose a Markov chain Monte Carlo sampling method based on node-dependent particle filtering in addition to the multi-change point process. The candidate parent nodes to be sampled, which are close to the real state, are pushed to the high probability area through the particle filter, and the candidate parent node set to be sampled that is far from the real state is pushed to the low probability area and then sampled. In terms of reconstructing the gene regulatory network, the model proposed in this paper (FC-DBN) has better network reconstruction accuracy and model convergence speed than other corresponding models on the Saccharomyces cerevisiae data and RAF data.

Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. SIGNIFICANCE: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

Screening of anti-liver fibrosis peptides from turtle shell protein using two-enzyme hydrolysis by molecular docking.

Turtle shell as a food residue of Pelodiscus sinensis (a type of edible aquatic animal) is widely used in Traditional Chinese Medicine for hepatic fibrosis therapy. Previous studies have demonstrated that the peptides (<6 kDa) derived from turtle shells are considered effective components. The protein of turtle shells has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. In the present study, the protein of turtle shell was hydrolyzed using a two-enzyme combination. It was found that the hydrolysates obtained by a combination of pepsin and trypsin showed the highest anti-liver fibrosis activity relative to other combinations in a cell viability assay. The hydrolysates were separated and purified by ultra-filtration (<6 kDa), gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC). Subsequently, the sequences of purified peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Molecular docking was used to analyze the interaction of these peptides with the transforming growth factor-ß1 (TGF-ß1) receptor. Two (GPPGVPGPGPL, TSLPVPAPV) of these novel peptides displayed lower binding energies to the TGF-ß1 receptor (-8.18 kcal mol-1, -8 kcal mol-1). Finally, the above two peptides were synthesized chemically and their in vitro anti-liver fibrosis activity was verified by MTT assay. Among them, GPPGVPGPGPL showed a better in vitro anti-liver fibrosis activity (IC50: 80.13 µM). We established a method to obtain anti-liver fibrosis peptides from turtle shells by using bioactivity-guided isolation with molecular docking. Turtle shell protein is an excellent source of anti-liver fibrosis peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

Contralateral Breast Cancer Risk Among Carriers of Germline Pathogenic Variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2.

PURPOSE: To estimate the risk of contralateral breast cancer (CBC) among women with germline pathogenic variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2. METHODS: The study population included 15,104 prospectively followed women within the CARRIERS study treated with ipsilateral surgery for invasive breast cancer. The risk of CBC was estimated for PV carriers in each gene compared with women without PVs in a multivariate proportional hazard regression analysis accounting for the competing risk of death and adjusting for patient and tumor characteristics. The primary analyses focused on the overall cohort and on women from the general population. Secondary analyses examined associations by race/ethnicity, age at primary breast cancer diagnosis, menopausal status, and tumor estrogen receptor (ER) status. RESULTS: Germline BRCA1, BRCA2, and CHEK2 PV carriers with breast cancer were at significantly elevated risk (hazard ratio > 1.9) of CBC, whereas only the PALB2 PV carriers with ER-negative breast cancer had elevated risks (hazard ratio, 2.9). By contrast, ATM PV carriers did not have significantly increased CBC risks. African American PV carriers had similarly elevated risks of CBC as non-Hispanic White PV carriers. Among premenopausal women, the 10-year cumulative incidence of CBC was estimated to be 33% for BRCA1, 27% for BRCA2, and 13% for CHEK2 PV carriers with breast cancer and 35% for PALB2 PV carriers with ER-negative breast cancer. The 10-year cumulative incidence of CBC among postmenopausal PV carriers was 12% for BRCA1, 9% for BRCA2, and 4% for CHEK2. CONCLUSION: Women diagnosed with breast cancer and known to carry germline PVs in BRCA1, BRCA2, CHEK2, or PALB2 are at substantially increased risk of CBC and may benefit from enhanced surveillance and risk reduction strategies.

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants.

Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants. Summary: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to several types of cancer. However, variants of uncertain significance (VUS) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants to facilitate current and future clinical management of individuals with these variants. Here we show the results from a saturation genome editing (SGE) and functional analysis of all possible single nucleotide variants (SNVs) from exons 15 to 26 that encode the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants. The assay was based on deep sequence analysis of surviving endogenously targeted human haploid HAP1 cells. The assay was calibrated relative to ClinVar known pathogenic and benign missense standards and 95% prevalence thresholds for functionally abnormal and normal variants were identified. Thresholds were validated based on nonsense and synonymous variants. SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer. The functional assay results were integrated into a ClinGen/ACMG/AMP-like model for clinical classification of the majority of BRCA2 SNVs as pathogenic/likely pathogenic or benign/likely benign. The classified variants can be used for improved clinical management of variant carriers.

Establishment and Characterization of a New Human Intrahepatic Cholangiocarcinoma Cell Line LIV27.

Cholangiocarcinoma (CCA) is a highly lethal cancer arising from the biliary tract epithelium. The cancer biology of this neoplasm is not well understood. To date, only a few CCA cell lines have been reported, which were mostly developed from Asian patients. In this study, we report and characterize a new intrahepatic CCA cell line, LIV27, derived from a surgically resected tumor in a 67-year-old Caucasian woman with primary sclerosing cholangitis (PSC). LIV27 cells grow well in collagen-coated flasks or plates with a doubling time of 57.8 h at passage 14. LIV27 cells have high tumorigenicity in nude mice and stain positive for CK7 and CK19, markers that differentiate CCA from hepatocellular carcinoma. Karyotype analysis showed that LIV27 is aneuploid. We established a single-locus short tandem repeat profile for the LIV27 cell line. This newly established cell line will be a useful model for studying the molecular pathogenesis of, and developing novel therapies for, cholangiocarcinoma.

Genetic Risk of Second Primary Cancer in Breast Cancer Survivors: The Multiethnic Cohort Study.

Women who have had breast cancer in the past are at increased risk of developing a second primary cancer (SPC), including second primary breast cancer (SPBC) or a second primary non-breast cancer (SPNBC). In the Multiethnic Cohort (MEC) Study, we conducted a prospective cohort analysis in 3,223 female breast cancer survivors from five racial/ethnic populations (White, African American, Japanese American, Latino, and Native Hawaiian) to assess the association of rare pathogenic variants (PV) in 37 known cancer predisposition genes with risk of SPC. A total of 719 (22.3%) women developed SPC, of which, 323 (10.0%) were SPBC. Germline PVs in BRCA1 (HR, 2.28; 95% CI, 1.11-4.65) and ERCC2 (HR, 3.51; 95% CI, 1.29-9.54) were significantly enriched in women with SPC. In the subtype analysis for SPBC, a significant association of ERCC2 PVs (HR, 5.09; 95% CI, 1.58-16.4) and a suggestive association of BRCA2 PVs (HR, 2.24; 95% CI, 0.91-5.55) were observed. There was also a higher risk of SPNBC in carriers of BRCA1 PVs (HR, 2.98; 95% CI, 1.21-7.36). These results provide evidence that germline PVs in BRCA1, BRCA2, and ERCC2 contribute to the development of SPC in breast cancer survivors. These findings also suggest that compromised DNA repair mechanisms could be a predisposition factor for SPC in patients with breast cancer, supporting the need for closer monitoring of SPC in women carrying PVs in these genes. SIGNIFICANCE: This multiethnic study links germline pathogenic variants in BRCA1, BRCA2, and ERCC2 to the development of second primary cancer in breast cancer survivors, providing biological insights and biomarkers to guide patient monitoring.

Classification of BRCA2 Variants of Uncertain Significance (VUS) Using an ACMG/AMP Model Incorporating a Homology-Directed Repair (HDR) Functional Assay.

PURPOSE: The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer-testing laboratory. EXPERIMENTAL DESIGN: The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2. RESULTS: Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS. CONCLUSIONS: Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.

An integrative model for the comprehensive classification of BRCA1 and BRCA2 variants of uncertain clinical significance.

Loss-of-function variants in the BRCA1 and BRCA2 susceptibility genes predispose carriers to breast and/or ovarian cancer. The use of germline testing panels containing these genes has grown dramatically, but the interpretation of the results has been complicated by the identification of many sequence variants of undefined cancer relevance, termed "Variants of Uncertain Significance (VUS)." We have developed functional assays and a statistical model called VarCall for classifying BRCA1 and BRCA2 VUS. Here we describe a multifactorial extension of VarCall, called VarCall XT, that allows for co-analysis of multiple forms of genetic evidence. We evaluated the accuracy of models defined by the combinations of functional, in silico protein predictors, and family data for VUS classification. VarCall XT classified variants of known pathogenicity status with high sensitivity and specificity, with the functional assays contributing the greatest predictive power. This approach could be used to identify more patients that would benefit from personalized cancer risk assessment and management.

Tanreqing Injection Attenuates Macrophage Activation and the Inflammatory Response via the lncRNA-SNHG1/HMGB1 Axis in Lipopolysaccharide-Induced Acute Lung Injury.

The etiology of acute lung injury (ALI) is not clear, and the treatment of ALI presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of ALI and to define the target gene of Tanreqing (TRQ), which is a traditional Chinese medicine formula composed of five medicines, scutellaria baicalensis, bear bile powder, goat horn powder, honeysuckle and forsythia. Macrophage activation plays a critical role in many pathophysiological processes, such as inflammation. Although the regulation of macrophage activation has been extensively investigated, there is little knowledge of the role of long noncoding RNAs (lncRNAs) in this process. In this study, we found that lncRNA-SNHG1 expression is distinctly regulated in differently activated macrophages in that it is upregulated in LPS. LncRNA-SNHG1 knockdown attenuates LPS-induced M1 macrophage activation. The SNHG1 promoter was bound by NF-κB subunit p65, indicative of SNHG1 being a direct transcriptional target of LPS-induced NF-κB activation. SNHG1 acts as a proinflammatory driver that leads to the production of inflammatory cytokines and the activation of macrophages and cytokine storms by physically interacting with high-mobility group box 1 (HMGB1) in ALI. TRQ inhibited NF-κB signaling activation and binding of NF-κB to the SNHG1 promoter. In conclusion, this study defined TRQ target genes, which can be further elucidated as mechanism(s) of TRQ action, and provides insight into the molecular pathogenesis of ALI. The lncRNA-SNHG1/HMGB1 axis is an ideal therapeutic for ALI treatment.

Identification and Validation of Autophagy-Related Genes in Sepsis-Induced Acute Respiratory Distress Syndrome and Immune Infiltration.

Purpose: Autophagy-related genes (ARGs) play an important role in the pathophysiology processes of sepsis-induced acute respiratory distress syndrome (ARDS). However, expression profiles of ARGs have rarely been used to explore the relationship between autophagy and sepsis-induced ARDS. Therefore, we aim to identify and validate the potential ARGs of sepsis-induced ARDS through bioinformatics analysis and experiment validation. Methods: We downloaded GSE32707 data from the Gene Expression Omnibus (GEO) database. The potential differentially expressed genes (DEGs) and differentially expressed ARGs (DEARGs) of sepsis-induced ARDS were screened by R software. Then, we performed functional enrichment analyses to explore the potential biological functions of DEARGs and constructed protein-protein interaction (PPI) networks. Subsequently, correlation analysis and receiver operating characteristic (ROC) curve were used for the DEARGs. In addition, we estimated the proportions of 22 immune cell subsets by using CIBERSORT algorithm. Finally, RNA expression of seven DEARGs were validated by qRT-PCR in blood samples from sepsis-induced ARDS and healthy controls. Results: We identified 28 DEARGs, including 11 up-regulated genes and 17 down-regulated genes, which were primarily involved in autophagy and apoptosis. Seven genes (BAG3, CTSD, ERBB2, MYC, PEA15, RAB24 and SIRT1) with AUC >0.70 were considered possible to be sepsis-induced ARDS hub genes for ROC curve analysis. CIBERSORT results shown that sepsis-induced ARDS contained a higher proportion of naive CD4+ T cells, gamma delta T cells, monocytes, and neutrophils, and lower levels of CD8+ T cells, memory resting CD4+ T cells, follicular helper T cells were relatively lower. The results of qRT-PCR also demonstrated that the expression levels of BAG3, CTSD, ERBB2, MYC and SIRT1 in sepsis-induced ARDS patients and healthy controls had differences. Conclusion: We identified an association between DEGs and immune infiltration in sepsis-induced ARDS and validated BAG3, CTSD, ERBB2, MYC and SIRT1 that may be have excellent diagnostic performance.

Breast Cancer Screening Strategies for Women With ATM, CHEK2, and PALB2 Pathogenic Variants: A Comparative Modeling Analysis.

IMPORTANCE: Screening mammography and magnetic resonance imaging (MRI) are recommended for women with ATM, CHEK2, and PALB2 pathogenic variants. However, there are few data to guide screening regimens for these women. OBJECTIVE: To estimate the benefits and harms of breast cancer screening strategies using mammography and MRI at various start ages for women with ATM, CHEK2, and PALB2 pathogenic variants. DESIGN, SETTING, AND PARTICIPANTS: This comparative modeling analysis used 2 established breast cancer microsimulation models from the Cancer Intervention and Surveillance Modeling Network (CISNET) to evaluate different screening strategies. Age-specific breast cancer risks were estimated using aggregated data from the Cancer Risk Estimates Related to Susceptibility (CARRIERS) Consortium for 32 247 cases and 32 544 controls in 12 population-based studies. Data on screening performance for mammography and MRI were estimated from published literature. The models simulated US women with ATM, CHEK2, or PALB2 pathogenic variants born in 1985. INTERVENTIONS: Screening strategies with combinations of annual mammography alone and with MRI starting at age 25, 30, 35, or 40 years until age 74 years. MAIN OUTCOMES AND MEASURES: Estimated lifetime breast cancer mortality reduction, life-years gained, breast cancer deaths averted, total screening examinations, false-positive screenings, and benign biopsies per 1000 women screened. Results are reported as model mean values and ranges. RESULTS: The mean model-estimated lifetime breast cancer risk was 20.9% (18.1%-23.7%) for women with ATM pathogenic variants, 27.6% (23.4%-31.7%) for women with CHEK2 pathogenic variants, and 39.5% (35.6%-43.3%) for women with PALB2 pathogenic variants. Across pathogenic variants, annual mammography alone from 40 to 74 years was estimated to reduce breast cancer mortality by 36.4% (34.6%-38.2%) to 38.5% (37.8%-39.2%) compared with no screening. Screening with annual MRI starting at 35 years followed by annual mammography and MRI at 40 years was estimated to reduce breast cancer mortality by 54.4% (54.2%-54.7%) to 57.6% (57.2%-58.0%), with 4661 (4635-4688) to 5001 (4979-5023) false-positive screenings and 1280 (1272-1287) to 1368 (1362-1374) benign biopsies per 1000 women. Annual MRI starting at 30 years followed by mammography and MRI at 40 years was estimated to reduce mortality by 55.4% (55.3%-55.4%) to 59.5% (58.5%-60.4%), with 5075 (5057-5093) to 5415 (5393-5437) false-positive screenings and 1439 (1429-1449) to 1528 (1517-1538) benign biopsies per 1000 women. When starting MRI at 30 years, initiating annual mammography starting at 30 vs 40 years did not meaningfully reduce mean mortality rates (0.1% [0.1%-0.2%] to 0.3% [0.2%-0.3%]) but was estimated to add 649 (602-695) to 650 (603-696) false-positive screenings and 58 (41-76) to 59 (41-76) benign biopsies per 1000 women. CONCLUSIONS AND RELEVANCE: This analysis suggests that annual MRI screening starting at 30 to 35 years followed by annual MRI and mammography at 40 years may reduce breast cancer mortality by more than 50% for women with ATM, CHEK2, and PALB2 pathogenic variants. In the setting of MRI screening, mammography prior to 40 years may offer little additional benefit.

Mapping molecular subtype specific alterations in breast cancer brain metastases identifies clinically relevant vulnerabilities.

The molecular events and transcriptional plasticity driving brain metastasis in clinically relevant breast tumor subtypes has not been determined. Here we comprehensively dissect genomic, transcriptomic and clinical data in patient-matched longitudinal tumor samples, and unravel distinct transcriptional programs enriched in brain metastasis. We report on subtype specific hub genes and functional processes, central to disease-affected networks in brain metastasis. Importantly, in luminal brain metastases we identify homologous recombination deficiency operative in transcriptomic and genomic data with recurrent breast mutational signatures A, F and K, associated with mismatch repair defects, TP53 mutations and homologous recombination deficiency (HRD) respectively. Utilizing PARP inhibition in patient-derived brain metastatic tumor explants we functionally validate HRD as a key vulnerability. Here, we demonstrate a functionally relevant HRD evident at genomic and transcriptomic levels pointing to genomic instability in breast cancer brain metastasis which is of potential translational significance.

Influence of Cancer Susceptibility Gene Mutations and ABO Blood Group of Pancreatic Cancer Probands on Concomitant Risk to First-Degree Relatives.

BACKGROUND: ABO blood group is associated with pancreatic cancer risk. Whether ABO blood group alone or when combined with inherited mutation status of index pancreatic cancer cases (probands) can enhance pancreatic cancer risk estimation in first-degree relatives (FDR) is unclear. We examined FDRs' risk for pancreatic cancer based on probands' ABO blood group and probands' cancer susceptibility gene mutation status. METHODS: Data on 23,739 FDRs, identified through 3,268 pancreatic cancer probands, were analyzed. Probands' ABO blood groups were determined serologically or genetically, and 20 cancer susceptibility genes were used to classify probands as "mutation-positive" or "mutation-negative." SIRs and 95% confidence intervals (CI) were calculated, comparing observed pancreatic cancer cases in the FDRs with the number expected in SEER-21 (reference population). RESULTS: Overall, FDRs had 2-fold risk of pancreatic cancer (SIR = 2.00; 95% CI = 1.79-2.22). Pancreatic cancer risk was higher in FDRs of mutation-positive (SIR = 3.80; 95% CI = 2.81-5.02) than mutation-negative (SIR = 1.79; 95% CI = 1.57-2.04) probands (P < 0.001). The magnitude of risk did not differ by ABO blood group alone (SIRblood-group-O = 1.57; 95% CI = 1.20-2.03, SIRnon-O = 1.83; 95% CI = 1.53-2.17; P = 0.33). Among FDRs of probands with non-O blood group, pancreatic cancer risk was higher in FDRs of mutation-positive (SIR = 3.98; 95% CI = 2.62-5.80) than mutation-negative (SIR = 1.66; 95% CI = 1.35-2.03) probands (P < 0.001), but risk magnitudes were statistically similar when probands had blood group O (SIRmutation-positive = 2.65; 95% CI = 1.09-5.47, SIRmutation-negative = 1.48; 95% CI = 1.06-5.47; P = 0.16). CONCLUSIONS: There is a range of pancreatic cancer risk to FDRs according to probands' germline mutation status and ABO blood group, ranging from 1.48 for FDRs of probands with blood group O and mutation-negative to 3.98 for FDRs of probands with non-O blood group and mutation-positive. IMPACT: Combined ABO blood group and germline mutation status of probands can inform pancreatic cancer risk estimation in FDRs.

Construction and Validation of Prognostic Regulation Network Based on RNA-Binding Protein Genes in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (LUSC) is a common histologic subtype of non-small cell lung cancer with a poor prognosis. RNA-binding proteins (RBPs) are key modulators in the posttranscriptional regulation and RBP alterations are commonly found in various cancer types. However, its roles in predicting the tumorigenesis and prognosis have not been identified in LUSC. To identify the roles of RBPs in the tumorigenesis and prognosis of LUSC, the RNA sequencing data of patients with LUSC were retrieved from The Cancer Genome Atlas (TCGA) databases. The differential expressed genes (DEGs) were evaluated and identified. The intersection of manually curated RBPs and tumorigenesis-related DEGs was filtered to the univariate Cox regression analysis. The intersection genes with prognostic value were defined as prognostic RNA-binding protein genes (PRBPGs). Based on them, the predicted model was constructed. Its accuracy was tested by the area under the curve (AUC) of the receiver operator characteristic curve and the risk score. In addition, to explore the key regulatory network, the relationship among PRBPGs, target RNA, and absolute quantification of 50 hallmarks of cancer was also identified by Pearson correlation analysis. A total of 311 genes were filtered as the intersection of 1542 manually curated RBPs and tumorigenesis-related DEGs and the results revealed 17 PRBPGs. Based on them, we constructed the predict model with a relatively high accuracy (AUC: 0.739). The Kaplan-Meier survival curve showed the significant prognostic value of risk score (p < 0.001). Moreover, we uncovered the regulatory networks of PHF5A-TOMM22-oxidative phosphorylation, TLR3-CTSO inflammation-related pathway, SECISBP2L-targeted RNA (ADGRF5, TGFBR2, CD302, AC096921.2, AHCYL2, RPS6KA2, SLC34A2, and SFTPB) angiogenesis, and SECISBP2L-AKAP13 signaling (DNA repair, MTORC1 signaling, and MYC targets). The regulation mechanisms and cellular location of key PRBPGs were validated by assay for targeting accessible chromatin with high-throughput sequencing and single-cell RNA sequencing. Our study identifies PRBPGs as reliable indexes in predicting the tumorigenesis and prognosis of patients with LUSC and provides a well-applied model for predicting the overall survival for patients with LUSC. Besides, we also identified the regulatory network among PRBPGs, target RNA, and cancer gene sets in mediating the LUSC tumorigenesis.

Integrating Network Pharmacology, Transcriptome and Artificial Intelligence for Investigating Into the Effect and Mechanism of Ning Fei Ping Xue Decoction Against the Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a high-mortality disease and lacks effective pharmacotherapy. A traditional Chinese medicine (TCM) formula, Ning Fei Ping Xue (NFPX) decoction, was demonstrated to play a critical role in alleviating inflammatory responses of the lung. However, its therapeutic effectiveness in ARDS and active compounds, targets, and molecular mechanisms remain to be elucidated. The present study investigates the effects of NFPX decoction on ARDS mice induced by lipopolysaccharides (LPS). The results revealed that NFPX alleviated lung edema evaluated by lung ultrasound, decreased lung wet/Dry ratio, the total cell numbers of bronchoalveolar lavage fluid (BALF), and IL-1ß, IL-6, and TNF-α levels in BALF and serum, and ameliorated lung pathology in a dose-dependent manner. Subsequently, UPLC-HRMS was performed to establish the compounds of NFPX. A total of 150 compounds in NFPX were characterized. Moreover, integrating network pharmacology approach and transcriptional profiling of lung tissues were performed to predict the underlying mechanism. 37 active components and 77 targets were screened out, and a herbs-compounds-targets network was constructed. Differentially expressed genes (DEGs) were identified from LPS-treated mice compared with LPS combined with NFPX mice. GO, KEGG, and artificial intelligence analysis indicated that NFPX might act on various drug targets. At last, potential targets, HRAS, SMAD4, and AMPK, were validated by qRT-PCR in ARDS murine model. In conclusion, we prove the efficacy of NFPX decoction in the treatment of ARDS. Furthermore, integrating network pharmacology, transcriptome, and artificial intelligence analysis contributes to illustrating the molecular mechanism of NFPX decoction on ARDS.