CASMART, a one-step CRISPR Cas12a-mediated isothermal amplification for rapid and high-resolution digital detection of rare mutant alleles.

Convenient, ultrasensitive, and accurate detection of rare variants is essential for early cancer diagnosis and precision medicine, however, despite years of efforts, tools that have all these qualities remain elusive. Here, we developed a one-step CRISPR/Cas12a-based digital diagnostic platform for accurately quantifying mutant alleles, referred to as the CRISPR ASsoaciated Mutation Allele Rapid Test (CASMART). The platform accurately quantifies the variant allele frequency of EGFR L858R within 1 h at 42 °C and can detect mutant targets as low as 0.3 copies/µL (0.498 aM) in mock multiplex cfDNA samples. We further investigated the applicability of CASMART using human genomic samples with confirmed EGFR L858R mutations previously measured variant allele frequency by next-generation sequencing. Comparison across platforms revealed equivalent detection performance (Pearson's correlation coefficient, R2 = 0.9208) and high quantification accuracy for mutation allele frequency (intraclass correlation coefficient = 0.959). Our one-step approach enables easy and accurate variant allele frequency measurement of rare mutant alleles without PCR instrumentation, while the assay time was reduced by approximately half compared to the digital PCR with the shortest turnaround. The CASMART is an alternative to conventional single nucleotide polymorphism detection methods with great potential as a next-generation biosensor for rapidly quantifying the variant allele fraction, especially in resource-limited settings.

Clinical characteristics associated with primary cardiac angiosarcoma outcomes: a surveillance, epidemiology and end result analysis.

BACKGROUND: Primary cardiac angiosarcoma (PCAS) is a rare type of tumour. Furthermore, descriptions of the demographic features and prognostic factors of PCAS patients have been poorly reported. METHODS: A population cohort study was conducted using retrospectively extracted data from the SEER (Surveillance, Epidemiology and End Results) database for patients with histological diagnoses of PCAS; the extracted information included demographic, treatment and outcome data. RESULTS: A total of 168 cases of PCAS from 1973 to 2013 were included. The mean age at diagnosis was 44.4 ± 15.5 years. PCAS was more prevalent in men than in women. The majority of PCAS patients were white (67.3%), while the incidence of PCAS in black individuals was relatively infrequent (19.0%). In addition, 87 cases were classified as distant stage, 44 as regional stage, and 33 as localized stage. The median disease-specific survival (DSS) was 7.22 months, and the 1-, 2- and 5-year DSS rate for PCAS patients was 34.7%, 14.3% and 10.2%, respectively. Further multivariate analyses showed that an age at (greater than or equal to) 45 years (HR 2.165), no radiotherapy (HR 1.629), tumour size > 5 cm (HR 3.182), and the summary stage was associated with worse PCAS-related survival. Cancer-directed surgery and radiotherapy significantly improved the DSS for patients with PCAS (P < 0.05). The C-index of the nomograms was 0.706 (95% CI 0.654-0.758), and the calibration curves showed good agreement between the nomogram prediction and actual observation. CONCLUSION: PCAS is a rare cancer that is prone to have poor prognoses. To understand PCAS more thoroughly, more cases with adequate information are needed.

An efficient method that combines the ThinPrep cytologic test with E6/E7 mRNA testing for cervical cancer screening.

Background: Cervical cancer is strongly associated with persistent human papillomavirus (HPV) infections. The ThinPrep cytologic test (TCT), HPV DNA detection, and E6/E7 mRNA testing are widely used to screen for cervical abnormalities. Purpose: This study aimed to find a suitable method for cervical cancer diagnosis (but not for cervical cancer distant metastasis), especially among women whose TCT results are atypical squamous cells of undetermined significance (ASCUS) or worse (including ASCUS). Patients and methods: A total of 301 samples from Wenzhou People's Hospital from June 2014 to September 2017 were collected, we conducted comparative analysis of the diagnostic performance of several conventional screening methods both individually and in combination. Results: We compared the sensitivity, specificity, positive predictive value, negative predictive value, and Youden index retrospectively estimated not only by single TCT, HPV DNA detection, or E6/E7 mRNA testing but also by combination methods, such as TCT+HPV DNA, TCT+E6/E7 mRNA, or TCT+HPV DNA+E6/E7 mRNA. Screening under TCT+E6/E7mRNA was confirmed with relatively higher sensitivity of 76.1% (95% CI: 0.659-0.841), specificity of 74.6% (95% CI: 0.681-0.803), and the highest Youden index of 0.507. Conclusion: The joint screening methods showed relatively reliable specificity and sensitivity for cervical disease screening, and detection by TCT+E6/E7 mRNA has the potential to be a widely used clinical method for cervical cancer screening.

Knockdown of lncRNA LEF1-AS1 inhibited the progression of oral squamous cell carcinoma (OSCC) via Hippo signaling pathway.

It is verified that long non-coding RNAs (lncRNAs) play crucial roles in various cancers. LncRNA LEF1-AS1 is a reported oncogene in colorectal cancer and glioblastoma. In this study, we unveiled that LEF1-AS1 markedly increased in oral squamous cell carcinoma (OSCC) tissues and cell lines. Besides, OSCC patients with high levels of LEF1-AS1 were apt to poor prognosis. Functionally, LEF1-AS1 knockdown inhibited cell survival, proliferation and migration, whereas enhanced cell apoptosis and induced G0/G1 cell cycle arrest in vitro. Consistently, LEF1-AS1 silence hindered tumor growth in vivo. Moreover, LEF1-AS1 inhibition stimulated the activation of Hippo signaling pathway through directly interacting with LATS1. Furtherly, we disclosed that LEF1-AS1 silence abolished the interaction of LEF1-AS1 with LATS1 while enhanced the binding of LATS1 to MOB, therefore promoting YAP phosphorylation but impairing YAP1 nuclear translocation. Additionally, we demonstrated that LEF1-AS1 regulated YAP1 translocation via a LATS1-dependent manner. Furthermore, we also uncovered that YAP1 overexpression abolished the suppressive impact of LEF1-AS1 repression on the biological processes of OSCC cells. In a word, we concluded that LEF1-AS1 served an oncogenic part in OSCC through suppressing Hippo signaling pathway by interacting with LATS1, suggesting the therapeutic and prognostic potential of LEF1-AS1 in OSCC.

Prevalence of human papillomavirus among Wenzhou women diagnosed with cervical intraepithelial neoplasia and cervical cancer.

BACKGROUND: Human papillomavirus (HPV) is associated with an increased risk of cervical cancer. Using a vaccine to prevent HPV infections could be a cost-effective strategy to decrease the incidence of cervical cancer. Learning about the characteristics of CIN patients with HPV infection in Wenzhou is a key step in guiding the use of HPV vaccines and screening for cervical cancer. METHODS: We undertook a retrospective analysis including 2612 women who were treated in the Department of Gynecology and Obstetrics from Jan 2016 to Nov 2017. All of the patients were examined by HPV testing and histology. RESULTS: The prevalence of HR-HPV among women with cervical intraepithelial lesions aged 65-69 years (38.8%) was significantly higher than that of the other age groups. The percentage of patients diagnosed with HPV-positive HSIL progressively increased with age to a maximum of 18.0% in the group of 40 to 44 years of age. HPV 16, 52, and 58 were the three most dominant genotypes among these women, and single infections (950, 73.3%) were more common than multiple infections (346, 26.7%). Compared to cervicitis, the odds ratios (ORs) for LSIL associated with HPV 33, 52, 16 and HPV 58 infection were 5.98, 3.91, 3.65, 3.65, and 3.188, respectively; for HSIL associated with HPV 16, 33, 58 and HPV 31 were 9.30, 7.68, 5.97, and 4.21, respectively. In LSIL, the frequencies of HR-HPV 52,16,58,18 were 19.3,18.2,10.9, and 7.8%, respectively. CONCLUSION: Our study provides important data about the HPV genotype distribution and its correlation with cervical intraepithelial lesions in the Wenzhou population.

Novel ELISA for serodiagnosis of nasopharyngeal carcinoma based on a B cell epitope of Epstein-Barr virus latent membrane protein 2.

Epstein-Barr virus (EBV) is widespread and is associated with nasopharyngeal carcinoma (NPC). Serological detection of EBV is commonly used for screening, diagnosis and epidemiological surveys of NPC. In the present study, a novel B cell multi-epitope peptide fusion protein (EBV-LMP2-3B), which is composed of three B cell linear epitopes (RIEDPPFNSLL, TLNLT and KSLSSTEFIPN) of EBV latent membrane protein 2 (LMP2), was expressed in a prokaryotic expression system and purified using Ni2+-nitrilotriacetate-Sepharose. The immunogenicity and binding specificity of EBV-LMP2-3B were evaluated on the basis of antibody responses in immunized BALB/c mice, western blotting and indirect immunofluorescence assay. Evaluation of EBV-LMP2-3B as a serological diagnostic reagent was performed using an indirect ELISA in 198 patients with NPC and 102 healthy adults. These results revealed that EBV-LMP2-3B was able to eliminate the high-titer serum antibody response in BALB/c mice. Western blot analysis and indirect immunofluorescence assay confirmed that the mouse immune sera recognized the native LMP2. Compared with healthy adults, patients with NPC demonstrated significantly greater reactivity to EBV-LMP2-3B (P<0.05). Furthermore, it was possible to effectively detect specific IgG in sera from patients with NPC, with a sensitivity of 91.91% and specificity of 93.14%, representing an improvement over the traditional viral capsid antigen-IgA-based detection method with 59.59% sensitivity and 75.49% specificity. In conclusion, the EBV-LMP2-3B protein may be used as a serological diagnostic reagent to screen for and diagnose NPC.

DNA plasmid vaccine carrying Chlamydia trachomatis (Ct) major outer membrane and human papillomavirus 16L2 proteins for anti-Ct infection.

Chlamydia trachomatis (Ct) is one of the most frequently encountered sexual infection all over the world, yielding tremendous reproductive problems (e.g. infertility and ectopic pregnancy) in the women. This work described the design of a plasmid vaccine that protect mice from Ct infection, and reduce productive tract damage by generating effective antibody and cytotoxic T cell immunity. The vaccine, s was composed of MOMP multi-epitope and HPV16L2 genes carried in pcDNA plasmid (i.e. pcDNA3.1/MOMP/HPV16L). In transfection, the vaccine expressed the chimeric genes (i.e. MOMP and HPV16L2), as demonstrated via western blot, RT-PCR and fluorescence imaging. In vitro, the vaccine transfected COS-7 cells and expressed the proteins corresponding to the genes carried in the vaccine. Through intramuscular immunization in BALB/c mice, the vaccine induced higher levels of anti-Ct IgG titer, anti-HPV16L2 IgG titer in serum and IgA titer in local mucosal secretions, compared to plasmid vaccines that carry only Ct MOMP multi-epitope or HPV16L2 chimeric component only. In mice intravaginally challenged with Ct, the vaccines pcDNA3.1/MOMP/HPV16L2 generated a higher level of genital protection compared to other vaccine formulations. Additionally, histochemical staining indicated that pcDNA3.1/MOMP/HPV16L2 eliminated mouse genital tract tissue pathologies induced by Ct infection. This work demonstrated that pcDNA/MOMP/HPV16L2 vaccine can protect against Ct infection by regulating antibody production, cytotoxic T cell killing functions and reducing pathological damage in mice genital tract. This work can potentially offer us a new vaccine platform against Ct infection.

Generation of affibody molecules specific for HPV16 E7 recognition.

Cervical cancer caused by infection with high-risk human papillomavirus remains to be the most deadly gynecologic malignancy worldwide. It is well documented that persistent expression of two oncogenes (E6/E7) plays the key roles in cervical cancer. Thus, in vivo detection of the oncoproteins is very important for the diagnosis of the cancer. Recently, affibody molecules have been demonstrated to be a powerful targeting probe for tumor-targeted imaging and diagnosis. In this study, four HPV16 E7-binding affibody molecules (Z HPV16 E7127, Z HPV16E7301, Z HPV16E7384 and Z HPV16E7745) were screened from a phage-displayed peptide library and used for molecular imaging in tumor-bearing mice. Biosensor binding analyses showed first that the four affibody molecules bound to HPV16 E7 with very high affinity and specificity. They co-localized with E7 protein only in two HPV16-positive cancer cells (SiHa and CaSki). Furthermore, affibody ZHPV16E7384 was conjugated with Dylight755 and used for in vivo tumor-imaging. Strongly high-contrast tumor retention of this affibody only occurred in HPV16-derived tumors of mice as early as 30 min post-injection, not in HPV-negative and HPV18-derived tumors. The accumulation of Dylight755-conjugated ZHPV16E7384 in tumor was achieved over a longer time period (24 h). The data here provide strong evidence that E7-specific affibody molecules have great potential used for molecular imaging and diagnosis of HPV-induced cancers.