Research progress of CTC, ctDNA, and EVs in cancer liquid biopsy.

Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vehicles (EVs) have received significant attention in recent times as emerging biomarkers and subjects of transformational studies. The three main branches of liquid biopsy have evolved from the three primary tumor liquid biopsy detection targets-CTC, ctDNA, and EVs-each with distinct benefits. CTCs are derived from circulating cancer cells from the original tumor or metastases and may display global features of the tumor. ctDNA has been extensively analyzed and has been used to aid in the diagnosis, treatment, and prognosis of neoplastic diseases. EVs contain tumor-derived material such as DNA, RNA, proteins, lipids, sugar structures, and metabolites. The three provide different detection contents but have strong complementarity to a certain extent. Even though they have already been employed in several clinical trials, the clinical utility of three biomarkers is still being studied, with promising initial findings. This review thoroughly overviews established and emerging technologies for the isolation, characterization, and content detection of CTC, ctDNA, and EVs. Also discussed were the most recent developments in the study of potential liquid biopsy biomarkers for cancer diagnosis, therapeutic monitoring, and prognosis prediction. These included CTC, ctDNA, and EVs. Finally, the potential and challenges of employing liquid biopsy based on CTC, ctDNA, and EVs for precision medicine were evaluated.

Two cases of placental trisomy 21 mosaicism causing false-negative NIPT results.

BACKGROUND: Non-invasive prenatal testing (NIPT) using cell-free DNA has been widely used for prenatal screening to detect the common fetal aneuploidies (such as trisomy 21, 18, and 13). NIPT has been shown to be highly sensitive and specific in previous studies, but false positives (FPs) and false negatives (FNs) occur. Although the prevalence of FN NIPT results for Down syndrome is rare, the impact on families and society is significant. CASE PRESENTATION: This article described two cases of foetuses that tested "negative" for trisomy 21 by NIPT technology using the semiconductor sequencing platform. However, the fetal karyotypes of amniotic fluid were 46,XY, + 21 der(21;21)(q10;q10) and 47,XY, + 21 karyotypes, respectively. Placental biopsies confirmed that, in the first case, the chromosome 21 placenta chimerism ratio ranged from 13 to 88% with the 46,XX, + 21,der(21;21)(q10;q10)[86]/46,XX[14] karyotype of placental chorionic cells (middle of fetal-side placental tissue). However, in the second case, of all the placental biopsies, percentage of total chimerism was less than 30%; and placental biopsies taken at the middle of maternal side and middle of fetal side, also had variable trisomy 2 mosaicism levels of 10% and 8%, respectively. Ultimately, the pregnancies were interrupted at 30 gestational age (GA) and 27GA, respectively. CONCLUSIONS: In this study, we present two cases of FN NIPT results that might have been caused by biological mechanisms, as opposed to poor quality, technical errors, or negligence. Clinical geneticists and their patients must understand that NIPT is a screening procedure.

Application of extracellular vesicles proteins in cancer diagnosis.

Early tumor diagnosis is crucial for its treatment and reduction of death, with effective tumor biomarkers being important tools. Extracellular vesicles (EVs) are small vesicles secreted by cells with various biomolecules, including proteins, nucleic acids, and lipids. They harbor a double membrane structure. Previous studies on EVs in cancer diagnosis and therapy focused on miRNAs. Nonetheless, EVs contain proteins that represent physiological and pathological state of their parental cells. EVs proteins can reflect the pathological state of some diseases, which provides a basis for diagnosis and treatment. This study describes the role of EVs in cancer and summarizes the use of EVs proteins as diagnostic markers in different cancer types. Specifically, we discuss the potential and shortcomings of EVs as tumor biomarkers.

Comprehensive Pan-Cancer Analysis of Senescence With Cancer Prognosis and Immunotherapy.

Senescence is a double-edged sword in tumorigenesis and affects the immunotherapy response through the modulation of the host's immune system. However, there is currently a lack of comprehensive analysis of the senescence-related genes (SRGs) in human cancers, and the predictive role of senescence in cancer immunotherapy response has not been explored. The multi-omics approaches were performed in this article to conduct a systematic pan-cancer genomic analysis of SRGs in cancer. In addition, we calculated the generic senescence score (SS) to quantify the senescence levels in cancers and explored the correlations of SS with cancer prognosis, biological processes, and tumor microenvironment (TME). The gene signatures were deregulated in multiple cancers and indicated a context-dependent correlation with prognosis, tumor-immune evasion, and response to therapy across various tumor types. Further analysis disclosed that SS was positively associated with the infiltration levels of immune suppressive cells, including induced Tregs (iTregs), central memory Ts (Tcms), and natural Tregs (nTregs), and negatively associated with immune killer cells, including natural killers (NKs) and mucosal-associated invariant Ts (MAITs). Moreover, the SS was significantly correlated with tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), immune-related genes, and immune checkpoints and had a predictive value of immunotherapy response. Thus, the expression of SRGs was involved in resistance to several anticancer drugs. Our work illustrates the characterization of senescence across various malignancies and highlights the potential of senescence as a biomarker of the response to immunotherapy.

Rapid diagnosis of seven high-risk human papillomavirus subtypes by a novel loop-mediated isothermal amplification method.

Current human papillomavirus (HPV) detection methods require complex instruments, skilled staff and have a high cost. Therefore, novel testing approaches are needed which are easy to implement, highly sensitive, and low cost. Loop-mediated isothermal amplification (LAMP) is an isothermal amplification technique. In this study, according to the conditions in China, a novel LAMP method for detecting seven high-risk HPV subtypes (16, 18, 33, 39, 45, 52, and 58) was designed and evaluated. The DNA from plasmid and cervical specimens was extracted using Chelex 100 and measured by qPCR and LAMP assay. LAMP products were observed under ultraviolet light. HPV sequences were successfully amplified and a plateau time of 19-75 min was maintained. The concentration of positive reactions ranged between 20 copies/µL and 200000 copies/µL. Additionally, there was no cross-reactivity between HPV16, 18, 33, 39, 45, 52, 58, 31, 35, 45, 51, 56, 59, 66, or 68. For clinical samples, the LAMP assay had high sensitivity and specificity for HPV16, 18, 33, 39, 45, 52, and 58. However, 5% (72/1447) of the samples tested yielded false-positive results. In conclusion, the novel LAMP assay for HPV16, 18, 33, 39, 45, 52, and 58 has high sensitivity and specificity, a low cost, and is simple and rapid to perform. The LAMP assay can improve HPV detection in resource-limited settings, especially in primary care hospitals and rural areas.

Bioinformatics analysis shows that TOP2A functions as a key candidate gene in the progression of cervical cancer.

Cervical cancer (CC) is one of the most prevalent types of cancer affecting females worldwide. However, the molecular mechanisms underlying the development and progression of CC remains to be elucidated. Taking the high incidence and mortality rates amongst women into consideration, the identification of novel biomarkers to prevent CC is of great significance and required to improve diagnosis. Using three raw microarray datasets from the Gene Expression Omnibus database, 188 differentially expressed genes (DEGs) were identified. Gene Ontology and pathway analyses were performed on the DEGs. Through protein-protein interaction network construction and module analysis, eight hub genes [cell division cycle 6, cyclin-dependent kinase 1 (CDK1), cell division control protein 45, budding uninhibited by benzimidazoles 1 (BUB1), DNA topoisomerase II α (TOP2A) and minichromosome maintenance complex component 4, CCNB2 and CCNB1] were identified, but only TOP2A was considered a prognostic factor in survival analysis. There were strong positive correlations between TOP2A and BUB1 (P<0.0001, rs=0.635), CDK1 (P<0.0001, rs=0.511), centromere protein F (CENPF) (P<0.0001, rs=0.677), Rac GTPase activating protein 1 (RACGAP1) (P<0.0001, rs=0.612), F-box protein 5 (FBXO5) (P<0.0001, rs=0.585) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) (P<0.0001, rs=0.584). Additionally, BUB1, CDK1, CENPF, RACGAP1, FBXO5 and BUB1B are all potentially suitable candidate targets for the diagnosis and treatment of CC. In conclusion, the present study identified TOP2A as a potential tumor oncogene and a biomarker for the prognosis of CC.

Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes.

PURPOSE: Currently, thalassemia is commonly detected using gap-polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) reverse dot blot, which have high requirements of space, instruments, and personnel. Therefore, it is necessary to develop a new method for thalassemia detection with high sensitivity, low cost, and simple and fast operation. In this study, we aimed to design and evaluate a new method for detecting three α-thalassemia genes including -Southeast Asian (SEA), -α3.7, and -α4.2 and five ß-thalassemia genes including 654M, 41/42M, -28M, 17M, and 27/28M based on loop-mediated isothermal amplification (LAMP). METHODS: Primer sequences were designed using Primer Explorer V4 software. Blood samples (5 mL) were collected from all participants in EDTA. DNA was extracted using Chelex 100 and was subjected to LAMP. LAMP products were detected by fluorescence development in ultraviolet light. RESULTS: We found that LAMP assays for positive samples of thalassemia reached a plateau before 60 minutes, whereas the negative control samples entered the plateau after 70 minutes or showed no amplification. The concentration range of positive reactions was between 20-60 pg/µL and 20-60 ng/µL. Additionally, there were no cross-reactivities among 8 thalassemia subtypes. For clinical samples, the positive sample tube showed strong green fluorescence, whereas the negative tube showed light green fluorescence. According to these results, the LAMP method has high sensitivity for detecting thalassemia (252/254). However, 43 false-positive results were obtained in the LAMP test. The LAMP assay was also of low cost and with simple and fast operation. CONCLUSION: The novel LAMP assay can be completed within 60 min using a heating block or a water bath, and the result can be read visually based on color change to detect thalassemia. The LAMP assay fulfills the requirements of field application and resource-limited areas, especially those with primary hospitals and rural areas.