Expression of human dCTP pyrophosphatase 1 (DCTPP1) and its association with cisplatin resistance characteristics in ovarian cancer.

Cisplatin (DDP) resistance is a major challenge in treating ovarian cancer patients. A recently discovered enzyme called dCTP pyrophosphatase 1 (DCTPP1) has been implicated in regulating cancer characteristics, including drug responses. In this study, we aimed to understand the role of DCTPP1 in cancer progression and cisplatin response. Using publicly available databases, we analysed the expression and clinical significance of DCTPP1 in ovarian cancer. Our bioinformatics analysis confirmed that DCTPP1 is significantly overexpressed in ovarian cancer and is closely associated with tumour progression and poor prognosis after cisplatin treatment. We also found that DCTPP1 located in oxidoreductase complex and may be involved in various biological processes related to cisplatin resistance, including pyrimidine nucleotide metabolism, the P53 signalling pathway and cell cycle signalling pathways. We observed higher expression of DCTPP1 in cisplatin-resistant cells (SKOV3/DDP) and samples compared to their sensitive counterparts. Additionally, we found that DCTPP1 expression was only enhanced in SKOV3/S cells when treated with cisplatin, indicating different expression patterns of DCTPP1 in cisplatin-sensitive and cisplatin-resistant cancer cells. Our study further supports the notion that cisplatin induces intracellular reactive oxygen species (ROS) and triggers cancer cell death through excessive oxidative stress. Knocking out DCTPP1 reversed the drug resistance of ovarian cancer cells by enhancing the intracellular antioxidant stress response and accumulating ROS. Based on our research findings, we conclude that DCTPP1 has prognostic value for ovarian cancer patients, and targeting DCTPP1 may be clinically significant in overcoming cisplatin resistance in ovarian cancer.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

A time-resolved fluorescence immunoassay for rapid and precise automatic quality control of human papillomavirus type 68 VLPs in human papillomavirus vaccine.

The effectiveness and necessity of human papillomavirus (HPV) vaccination to prevent HPV infection and cervical cancer are increasingly recognized by people. The 15-valent HPV vaccine, which protects against almost high-risk types of HPV viruses identified by WHO, has attracted much attention. However, as the valence of vaccines increases, quality control in the HPV vaccine production process is facing more challenges. The precise quality control of the HPV type 68 virus-like particles (VLPs), one of the unique components of the 15-valent HPV vaccine that distinguishes it from existing vaccines, is the new requirement for vaccine manufacturers. Here we developed a novel time-resolved fluorescence immunoassay (TRFIA) for rapid and precise automatic quality control of HPV68 VLPs in HPV vaccine. Two murine monoclonal antibodies specifically targeting the HPV68 L1 protein were used to establish a classical sandwich assay. Except for pretreating the vaccine sample, the whole analysis process was performed by a fully automated machine, which saves detection time and gets rid of manual error. Multiple experiments established that the current novel TRFIA can efficiently and reliably analyses HPV68 VLPs. Present novel TRFIA has exhibited merits with speed, robustness, high sensitivity with a minimum detection value of 0.08 ng/mL, considerable accuracy, a wide detection range (up to 1000 ng/mL) and excellent specificity. It is also expected to provide a new detection method for quality control for each HPV type VLPs. To summarize, the novel TRFIA is of great interest for application in HPV vaccine quality control.

A custom-made time-resolved fluoroimmunoassay for the quantitation of the host cell protein of Vero in rabies vaccine.

Host cell proteins (HCPs) are the process-specific and inevitable impurities during the manufacture via a host cell, which affect the safety or efficacy of the bio-product. However, the commercial HCP enzyme-linked immunosorbent assay (ELISA) kits may not apply to specific products such as rabies vaccine from Vero cells. More advanced and process-specific assay methods are needed in the quality control of rabies vaccine throughout the whole manufacturing process. Therefore, a novel time-resolved fluoroimmunoassay (TRFIA) for the detection of process-specific HCP of Vero cells in rabies vaccine was established in this study. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was used during the preparation of HCP antigen. Based on a sandwich-type immunoassay format, analytes in samples were captured by one antibody coating in the wells and "sandwiched" by another antibody labeled with europium chelates. Due to the complex composition of HCP, both the capture and detected antibodies are polyclonal antibodies from the same anti-HCP antibodies pool. Multiple experiments have identified the optimal conditions to allow the valid and reliable detection of HCP in rabies vaccine. The TRFIA had a satisfactory limit of detection value (0.011 µg/ml) under optimal conditions, with the linear range from 0.0375 to 2.4 µg/ml of HCP. The coefficient variations (CVs) were all < 10%, and the recoveries were in the range of 97.00-102.42%. All the test results of Vero cell protein reference substance were included in the expected concentration, which demonstrated that the present method was available for the test of HCP in rabies vaccine. Based on these results, the novel TRFIA to detect HCP appears to be important for application in modern vaccine quality control during the whole manufacturing process.

Development and characterization of reference materials for EGFR, KRAS, NRAS, BRAF, PIK3CA, ALK, and MET genetic testing.

BACKGROUND: Along with the dramatic development of molecular diagnostic testing for the detection of oncogene variations, reference materials (RMs) have become increasingly important in performance evaluation of genetic testing. OBJECTIVE: In this study, we built a set of RMs for genetic testing based on next-generation sequencing (NGS). METHOD: Solid tumor tissues were selected as the samples of RMs for preparation. NGS was used to determine and validate the variants and the mutation frequency in DNA samples. Digital PCR was used to determine the copy numbers of RNA samples. The performance of the RMs was validated by six laboratories. RESULTS: Thirty common genetic alterations were designed based on these RMs. RMs consisted of a positive reference, a limit of detection reference, and a negative reference. The validation results confirmed the performance of the RMs. CONCLUSION: These RMs may be an attractive tool for the development, validation, and quality monitoring of molecular genetic testing.

Noninvasive prediction of axillary lymph node status in breast cancer using promoter profiling of circulating cell-free DNA.

BACKGROUND: Lymph node metastasis (LNM) is one of the most important factors affecting the prognosis of breast cancer. The accurate evaluation of lymph node status is useful to predict the outcomes of patients and guide the choice of cancer treatment. However, there is still lack of a low-cost non-invasive method to assess the status of axillary lymph node (ALN). Gene expression signature has been used to assess lymph node metastasis status of breast cancer. In addition, nucleosome footprint of cell-free DNA (cfDNA) carries gene expression information of its original tissues, so it may be used to evaluate the axillary lymph node status in breast cancer. METHODS: In this study, we found that the cfDNA nucleosome footprints between the ALN-positive patients and ALN-negative patients showed different patterns by implementing whole-genome sequencing (WGS) to detect 15 ALN-positive and 15 ALN-negative patients. In order to further evaluate its potential for assessing ALN status, we developed a classifier with multiple machine learning models by using 330 WGS data of cfDNA from 162 ALN-positive and 168 ALN-negative samples to distinguish these two types of patients. RESULTS: We found that the promoter profiling between the ALN-positive patients and ALN-negative patients showed distinct patterns. In addition, we observed 1071 genes with differential promoter coverage and their functions were closely related to tumorigenesis. We found that the predictive classifier based on promoter profiling with a support vector machine model, named PPCNM, produced the largest area under the curve of 0.897 (95% confidence interval 0.86-0.93). CONCLUSIONS: These results indicate that promoter profiling can be used to distinguish ALN-positive patients from ALN-negative patients, which may be helpful to guide the choice of cancer treatment.

Integrative analyses of maternal plasma cell-free DNA nucleosome footprint differences reveal chromosomal aneuploidy fetuses gene expression profile.

BACKGROUND: Chromosomal aneuploidy is the most common birth defect. However, the developmental mechanism and gene expression profile of fetuses with chromosomal aneuploidy are relatively unknown, and the maternal immune changes induced by fetal aneuploidy remain unclear. The inability to obtain the placenta multiple times in real-time is a bottleneck in research on aneuploid pregnancies. Plasma cell-free DNA (cfDNA) carries the gene expression profile information of its source cells and may be used to evaluate the development of fetuses with aneuploidy and the immune changes induced in the mother owing to fetal aneuploidy. METHODS: Here, we carried out whole-genome sequencing of the plasma cfDNA of 101 pregnant women carrying a fetus with trisomy (trisomy 21, n = 42; trisomy 18, n = 28; trisomy 13, n = 31) based on non-invasive prenatal testing (NIPT) screening and 140 normal pregnant women to identify differential genes according to the cfDNA nucleosome profile in the region around the transcription start sites (TSSs). RESULTS: The plasma cfDNA promoter profiles were found to differ between aneuploid and euploid pregnancies. A total of 158 genes with significant differences were identified, of which 43 genes were upregulated and 98 genes were downregulated. Functional enrichment and signaling pathway analysis were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases found that these signal pathways were mainly related to the coordination of developmental signals during embryonic development, the control of cell growth and development, regulation of neuronal survival, and immune regulation, such as the MAPK, Hippo, TGF-ß, and Rap1 signaling pathways, which play important roles in the development of embryonic tissues and organs. Furthermore, based on the results of differential gene analysis, a total of 14 immune-related genes with significant differences from the ImmPort database were collected and analyzed. These significantly different immune genes were mainly associated with the maintenance of embryonic homeostasis and normal development. CONCLUSIONS: These results suggest that the distribution characteristics of cfDNA nucleosomes in maternal plasma can be used to reflect the status of fetal development and changes of the immune responses in trisomic pregnancies. Overall, our findings may provide research ideas for non-invasive detection of the physiological and pathological states of other diseases.

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform.

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

Ultrabright nanoparticle-labeled lateral flow immunoassay for detection of anti-SARS-CoV-2 neutralizing antibodies in human serum.

The level of anti-SARS-CoV-2 neutralizing antibodies (NAb) is an indispensable reference for evaluating the acquired protective immunity against SARS-CoV-2. Here, we established an ultrabright nanoparticles-based lateral flow immunoassay (LFIA) for one-step rapid semi-quantitative detection of anti-SARS-CoV-2 NAb in vaccinee's serum. Once embedded in polystyrene (PS) nanoparticles, the aggregation-induced emission (AIE) luminogen, AIE490, exhibited ultrabright fluorescence due to the rigidity of PS and severe inhibition of intramolecular motions. The ultrabright AIE490-PS nanoparticle was used as a fluorescent marker of LFIA. Upon optimized conditions including incubation time, concentrations of coated proteins and conjugated nanoparticles, amounts of antigens modified on the surface of nanoparticles, dilution rate of serum samples, and so on, the ultrabright nanoparticles-based LFIA could accurately identify 70 negative samples and 63 positive samples from human serum (p < 0.0001). The intra- and inter-assay precisions of the established method are above 13% and 16%, respectively. The established LFIA has tremendous practical value of generalization as a rapid semi-quantitative detection method of anti-SARS-CoV-2 NAb. Meanwhile, the AIE490-PS nanoparticle is also promising to detect many other analytes by altering the protein on the surface.

Dual-color quantum dot-loaded nanoparticles based lateral flow biosensor for the simultaneous detection of gastric cancer markers in a single test line.

A dual-color quantum dots-loaded nanoparticles (QPs) based lateral flow biosensor with single test line has been developed. Red and green emitted QPs were conjugated with antibodies and served as detecting probes in assays respectively, while the mixture of various antibodies were immobilized on nitrocellulose membranes as one detection line. Benefit from eliminating the heterogeneity caused by different position on the membrane, current biosensor achieved higher accuracy comparing with prevalent multi-lines or multi-strips lateral flow systems, which is of great significance for analyzing ratio-related diagnostics. The capability and reliability of the multiplex biosensor are also demonstrated by utilizing pepsinogen I (PG I) and pepsinogen II (PG II) as the model analytes. Under the optimal conditions, quantitative detection was achieved with ultra-low limits of detection at 6.9 pM (0.29 ng mL-1, PG I) and 15.7 pM (0.66 ng mL-1, PG II) respectively. The spectra crosstalk was negligible and no apparent cross-reaction was found in simultaneous detection. Furthermore, a good linear correlation of the QPs based lateral flow biosensor and commercial time-resolved fluoroimmunoassay was obtained in the detection of clinical samples, indicating the high reliability of the proposed biosensor.

Immunogenicity and safety of an inactivated SARS-CoV-2 vaccine in people living with HIV: A cross-sectional study.

We aimed to analyze the efficacy and safety of an inactivated SARS-CoV-2 vaccine in people living with HIV (PLWH). A total of 143 PLWH and 50 healthy individuals were included in this study. A commercially available magnetic chemiluminescence enzyme immunoassay kit was used to detect serum IgG and IgM antibodies against SARS-CoV-2. Serum levels of SARS-CoV-2-specific IgG were significantly higher in the control group than in the PLWH group (p = 0.001). Overall, 76% of individuals in the control group were detected with seropositivity IgG against SARS-CoV-2 compared to 58% in the PLWH group (p = 0.024). In PLWH with IgG seropositivity, CD4+ T-cell counts before antiretroviral therapy (ART) was higher (p = 0.015). Multivariable analysis indicated that CD4+ T cells at IgG detection (odds ratio [OR] = 1.004, p = 0.006) and time after vaccination (OR = 0.977, p = 0.014) were independently associated with seropositivity IgG against SARS-CoV-2 in PLWH. Neutralizing antibody (nAb) titers in PLWH against wild-type SARS-CoV-2 were similar to those in the control group (p = 0.160). The proportion of seropositive nAbs against wild-type SARS-CoV-2 was also similar (95% in the control group vs. 97% in the PLWH group, p = 0.665). Similar results were obtained when nAb was detected against the delta variants with similar titers (p = 0.355) and a similar proportion of seropositive nAbs were observed (p = 0.588). All the side effects observed in our study were mild and self-limiting. The inactivated COVID-19 vaccine appears to be safe with good immunogenicity in Chinese PLWH.

Simulated confined placental mosaicism proportion (SCPMP) based on cell-free fetal DNA fraction enrichment can reduce false-positive results in non-invasive prenatal testing.

OBJECTIVE: To decrease the false-positive rate of NIPT using cell-free fetal DNA (cffDNA) fraction enrichment and the simulated confined placental mosaicism proportion (SCPMP) threshold application via cffDNA quantification. METHOD: Using a cffDNA enrichment method, 303 plasma samples with positive NIPT results (Z-score > 3.0; 200 true-positive and 103 false-positive cases) were re-sequenced. A method to calculate the SCPMP based on the quantified cffDNA fraction was developed; the SCPMP threshold between true- and false-positive NIPT results was determined and used for re-analyses. RESULTS: With enrichment, the fetal fraction of the 303 samples was 26.9 ± 8.4%, compared to 11.0 ± 3.2% without enrichment. The optimized threshold method with double determination using the Z-value-defined SCPMP can reduce the false-positive rates for trisomies 21, 18, and 13 by 87%, 80%, and 88.9%, respectively. CONCLUSION: Our optimized method can decrease the false-positive rate of NIPT results.

Europium (III) chelate nanoparticle-based lateral flow immunoassay strips for rapid and quantitative detection of cystatin C in serum.

The concentration of Cys C in the patient's serum can reflect the level of glomerular filtration rate and indicate the occurrence of renal failure. The establishment of a simple and rapid analytical method to quantitatively monitor the concentration of Cys C in serum could help timely detection of renal failure. In this study, we have developed an Eu (III) chelate nanoparticles based lateral flow immunoassay to fulfill real-time monitoring of Cys C concentration in serum within 15 min. This method was performed as a sandwich immunoassay with a wide detection range (0.05-10 µg/mL) and a low limit of detection (24.54 ng/mL). The intra and inter-assay coefficients of variation were 8.31-8.61% and 8.92-9.95%, respectively. Furthermore, the application of this method was evaluated by comparing the determined results with those obtained by chemiluminescence immunoassay, exhibiting a satisfactory correlation (R2 = 0.9830). The developed LFIA method with satisfactory analytical performance has great potential for real-time monitoring of renal failure and self-detection for the high-risk population.

ROS-Induced DCTPP1 Upregulation Contributes to Cisplatin Resistance in Ovarian Cancer.

Cisplatin resistance hinders the improvement of the prognosis of patients with ovarian cancer. Cisplatin induces cancer cell apoptosis by inducing reactive oxygen species (ROS). dCTP pyrophosphatase 1 (DCTPP1) is a newly discovered dNTP pyrophosphatase. This study aimed to identify the role of DCTPP1 in oxidative stress and cisplatin response of ovarian cancer. Our results indicates cisplatin-induced ROS generation was responsible for the upregulation of DCTPP1 in ovarian cancer cells, whereas DCTPP1 knockdown significantly enhanced the sensitivity of ovarian cancer cells to cisplatin, reflect in reactive oxygen species (ROS) generation, double-strand DNA breaks, and cell apoptosis. The expression of redox-related genes and the activation of the PI3/Akt signaling pathway were also inhibited by DCTPP1 knockdown. Our data proposes that the development of therapeutic approaches targeting DCTPP1 may be useful in the treatment of ovarian cancer.

Plasma-Derived Extracellular Vesicles Circular RNAs Serve as Biomarkers for Breast Cancer Diagnosis.

Breast cancer is the second cause of cancer-associated death among women and seriously endangers women's health. Therefore, early identification of breast cancer would be beneficial to women's health. At present, circular RNA (circRNA) not only exists in the extracellular vesicles (EVs) in plasma, but also presents distinct patterns under different physiological and pathological conditions. Therefore, we assume that circRNA could be used for early diagnosis of breast cancer. Here, we developed classifiers for breast cancer diagnosis that relied on 259 samples, including 144 breast cancer patients and 115 controls. In the discovery stage, we compared the genome-wide long RNA profiles of EVs in patients with breast cancer (n=14) and benign breast (n=6). To further verify its potential in early diagnosis of breast cancer, we prospectively collected plasma samples from 259 individuals before treatment, including 144 breast cancer patients and 115 controls. Finally, we developed and verified the predictive classifies based on their circRNA expression profiles of plasma EVs by using multiple machine learning models. By comparing their circRNA profiles, we found 439 circRNAs with significantly different levels between cancer patients and controls. Considering the cost and practicability of the test, we selected 20 candidate circRNAs with elevated levels and detected their levels by quantitative real-time polymerase chain reaction. In the training cohort, we found that BCExoC, a nine-circRNA combined classifier with SVM model, achieved the largest AUC of 0.83 [95% CI 0.77-0.88]. In the validation cohort, the predictive efficacy of the classifier achieved 0.80 [0.71-0.89]. Our work reveals the application prospect of circRNAs in plasma EVs as non-invasive liquid biopsies in the diagnosis and management of breast cancer.

A Deep-Learning Pipeline for TSS Coverage Imputation From Shallow Cell-Free DNA Sequencing.

Cell-free DNA (cfDNA) serves as a footprint of the nucleosome occupancy status of transcription start sites (TSSs), and has been subject to wide development for use in noninvasive health monitoring and disease detection. However, the requirement for high sequencing depth limits its clinical use. Here, we introduce a deep-learning pipeline designed for TSS coverage profiles generated from shallow cfDNA sequencing called the Autoencoder of cfDNA TSS (AECT) coverage profile. AECT outperformed existing single-cell sequencing imputation algorithms in terms of improvements to TSS coverage accuracy and the capture of latent biological features that distinguish sex or tumor status. We built classifiers for the detection of breast and rectal cancer using AECT-imputed shallow sequencing data, and their performance was close to that achieved by high-depth sequencing, suggesting that AECT could provide a broadly applicable noninvasive screening approach with high accuracy and at a moderate cost.

Rapid Monitoring of Vancomycin Concentration in Serum Using Europium (III) Chelate Nanoparticle-Based Lateral Flow Immunoassay.

Establishing personalized medication plans for patients to maximize therapeutic efficacy and minimize the toxicity of vancomycin (VAN) requires rapid, simple, and accurate monitoring of VAN concentration in body fluid. In this study, we have developed a simple and rapid analytical method by integrating Eu (III) chelate nanoparticles (CN-EUs) and lateral flow immunoassay (LFIA) to achieve the real-time monitoring of VAN concentration in serum within 15 min. This approach was performed on nitrocellulose (NC) membrane assembled LFIA strips via indirect competitive immunoassay and exhibited a wide linear range of detection (0.1-80 µg*ml-1) with a low limit of detection (69.2 ng*ml-1). The coefficients of variation (CV) of the intra- and inter-assay in the detection of VAN were 7.12-8.53% and 8.46-11.82%, respectively. The dilution test and specificity indicated this method had a stability that was not affected by the serum matrix and some other antibiotics. Furthermore, the applicability of the proposed method was assessed by comparing the determined results with those measured by LC-MS/MS, showing a satisfactory correlation (R 2 = 0.9713). The proposed CN-EUs-based LFIA manifested promising analytical performance, which showed potential value in the real-time monitoring of VAN and could help optimize the clinical use of more antibiotics.

A chemiluminescence immunoassay for precise automatic quality control of glycoprotein in human rabies vaccine.

Currently, quality control of glycoprotein in the human rabies vaccine is based on enzyme-linked immunosorbent assay (ELISA). However, ELISA does not match the needs of a modernised quality control system. For a long time, human rabies virus vaccine manufacturers have been devoted to seeking a detection platform that is sensitive, accurate, automatic, and feasible for practical applications. Therefore, our team invested major efforts into establishing a fully automated micromagnetic particle (MMP)-based chemiluminescence immunoassay (CLIA) platform. For vaccine quality control, MMP-coupled rabies virus glycoprotein monoclonal antibodies (S037) were used to capture the rabies virus. Another rabies virus glycoprotein antibody (S053) labelled with acridinium ester was added as a signal tracer. After pretreating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a limited detection time (only 30 min) and eliminated manual error. Multiple experiments have identified the optimal conditions allowing valid and reliable assessment of vaccine potency. The CLIA platform has exhibited merits in terms of speed, robustness, high sensitivity (with a minimum detection value of 0.45 mIU/mL), considerable accuracy, and a wide linear range of detection (9.4-1200 mIU/mL). Furthermore, the results showed that the CLIA platform is consistent with the National Institutes of Health test and time-resolved fluorescent immunoassay (TRFIA) in quantitative analysis, and had a better analytic performance than TRFIA. Therefore, the CLIA platform presented here may be important for application in modern vaccine quality control.

A plasma-derived extracellular vesicle mRNA classifier for the detection of breast cancer.

BACKGROUND: According to the global cancer burden data released in 2020, breast cancer (BC) has become the most common cancer in the world. Similar to those of other cancers, the present methods used in clinic for diagnosing early BC are invasive, inaccurate, and insensitive. Hence, new non-invasive methods capable of early diagnosis are needed. METHODS: We applied next-generation sequencing and analyzed the messenger RNA (mRNA) profiles of plasma extracellular vesicles (EVs) derived from 14 BC patients and 6 patients with benign breast lesions. We used 3 regression models, namely support vector machine (SVM), linear discriminate analysis (LDA), and logistic regression (LR), to develop classifiers for use in making predictive BC diagnoses; and used 259 plasma samples, including those obtained from 144 patients with BC, 72 patients with benign breast lesions, and 43 healthy women, which were divided into training groups and validation groups to verify their performances as classifiers by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The area under the curve (AUC) and accuracy, sensitivity, and specificity of the classifiers were cross-validated with the leave-1-out cross-validation (LOOCV) method. RESULTS: Among all combinations assessed with the 3 different regression models, an 8-mRNA combination, named EXOBmRNA, exhibited high performance [accuracy =71.9% and AUC =0.718, 95% confidence interval (CI): 0.652 to 0.784] in the training cohort after LOOCV was performed, showing the largest AUC in the SVM model. The mRNAs in EXOBmRNA were HLA-DRB1, HAVCR1, ENPEP, TIMP1, CD36, MARCKS, DAB2, and CXCL14. In the validation cohort, the AUC of EXOBmRNA was 0.737 (95% CI: 0.636 to 0.837). In addition, gene function and pathway analyses revealed that different levels of gene expression were associated with cancer. CONCLUSIONS: We developed a high-performing predictive classifiers including 8 mRNAs from plasma extracellular vesicles for diagnosing breast cancer.

Noninvasive discrimination of benign and malignant breast lesions using genome-wide nucleosome profiles of plasma cell-free DNA.

BACKGROUND: Breast malignancy is the most frequently diagnosed malignancy in women worldwide, and the diagnosis relies on invasive examinations. However, most clinical breast changes in women are benign, and invasive diagnostic approaches cause unnecessary suffering for the patients. Thus, a novel noninvasive approach for discriminating malignant breast lesions from benign lesions is needed. METHODS: We performed cell-free DNA (cfDNA) sequencing on plasma samples from 173 malignant breast lesion patients, 158 benign breast lesion patients, and 102 healthy women. We then analyzed the cfDNA-based nucleosome profiles, which reflect the various tissues of origin and transcription factor activities. Moreover, by using machine learning classifiers along with the cfDNA sequencing data, we built classifiers for discriminating benign from malignant breast lesions. Receiver operating characteristic curve analyses were used to evaluate the performance of the classifiers. RESULTS: cfDNA-based nucleosome profiles reflected the various tissues of origin and transcription factor activities in benign and malignant breast lesions. The cfDNA-based transcription factor activities and breast malignancy-specific transcription factor-binding site accessibility profiles could accurately distinguish benign and malignant breast lesions, with area under the curve values of 0.777 and 0.824, respectively. CONCLUSIONS: Our proof-of-principle study established a methodology for noninvasively discriminating benign from malignant breast lesions.