Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers.

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5' unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify a reference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine.

Highly Sensitive Enrichment of Low-Frequency Variants by Hairpin Competition Amplification.

Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and pathological processes. However, genotyping low-copy tumor DNA (ctDNA) in patients is challenging due to abundant wild DNA backgrounds. One novel strategy to enrich rare mutations at low variant allele fractions (VAFs) with quantitative polymerase chain reaction (qPCR) and Sanger sequencing was contrived by introducing artificial hairpins into amplicons to compete with primers, coined as the hairpin competition amplification (HCA) system. The influence imposed by artificial hairpins on primer-binding in a high-temperature PCR system was investigated for the first time in this work, paving the way for the optimization of HCA. HCA differs from the previously reported work in which hairpins are formed to inhibit extension of wild-type DNA using 5-exonuclease-negative polymerase, where the readout is dependent on melting curve analysis after asymmetric PCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enriched mutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstrated advantages in multiplex reaction and temperature robustness. In profiling gene status from 12 lung cancer ctDNA samples and 16 thyroid cancer FNA DNA samples, HCA demonstrated a 100% concordance rate compared to ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing can enrich low-abundance variants with high sensitivity and temperature robustness, presenting a novel and effective tool for precision diagnosis and treatment of rare variant diseases.

Combination of ultrasound-guided percutaneous A1 pulley release and intra-tendon sheath injection improves the therapeutic outcomes in adult trigger finger patients.

AIM: This study aimed to use high-frequency ultrasound guidance to compare the efficacy of percutaneous release combined with intra-tendon sheath injection (PR-ITSI) and percutaneous release only (PR-ONLY) in the treatment of adult trigger finger (TF) patients. MATERIALS AND METHODS: A total of 48 patients were randomly divided into PR-ITSI group and PR-ONLY group. The thickness of the A1 pulley was measured prior to surgery and 1-year after surgery. Visual Analogue Scale (VAS) score and Patient Global Impression of Improvement (PGI-I) scale score of affected fingers were evaluated at 1 day, 1 month, and 1 year after surgery. RESULTS: The overall difference of VAS score between the two groups after treatment was statistically significant (p<0.001), while the VAS scores gradually decreased in both groups at different time-points after treatment. The VAS scores in the PR-ITSI group at 1 day and 1 month after surgery were 1.475 and 0.904 (p<0.001), respectively, which were lower than those in the PR-ONLY group. Different treatment methods had no effect on the VAS score at 1 year after surgery (p=0.055). The thickness of the A1 pulley at 1 year after surgery was lower than that before surgery (p<0.001), whereas there was no significant difference in A1 pulley thickness between the two groups (p=0.095). The rate of PGI-I scale improvement by one grade at 1 day, 1 month, and 1 year after surgery in the PR-ITSI group was 15.322 times (95%CI: 4.466-52.573, p<0.001), 14.807 times (95%CI: 2.931-74.799, p=0.001), and 15.557 times (95%CI: 1.119-216.307, p=0.041), respectively, than that in the PR-ONLY group. CONCLUSION: Ultrasound-guided PR-ITSI is superior to PR-ONLY in the VAS score and PGI-I scale for adult TF patients.

Role of ultrasound-guided technique in the treatment of gynecologic diseases: A systematic review and meta-analysis.

BACKGROUND: The efficacy and safety of ultrasound-guided interventional procedures for gynecologic diseases have not been evaluated comprehensively. Therefore, we aimed to provide an evidence-based medical reference for clinical treatment in this meta-analysis. METHODS: The literature searches were conducted in databases of PubMed, Embase, and Web of Science for eligible studies published from 2002 to May 2022. All literature was searched, screened, and reviewed by 2 researchers separately. RevMan 5.3.0 was used to analyze the relevant data. RESULTS: A total of 12 articles consisting of 2854 patients were included. Compared with the control group, meta-analysis showed that ultrasound-guided technique in the experimental group was associated with a higher effective rate [risk ratio = 0.84, 95% confidence interval (CI) (0.84, 0.84)], a lower probability of recurrence [odds ratio = 0.13, 95% CI (0.13, 0.13), P < .00001], and a greater effective reintervention rate [odds ratio = 3.39, 95% CI (1.29, 8.86), P = .01]. However, it was not significantly associated with fewer adverse reactions. CONCLUSIONS: Ultrasound-guided technique in treating gynecologic diseases exerted a positive effect and had fewer adverse reactions, which could be popularized and applied in clinical practice.

Upregulated Long Noncoding RNA LINC01234 Predicts Unfavorable Prognosis for Colorectal Cancer and Negatively Correlates With KLF6 Expression.

BACKGROUND: LINC01234, a long noncoding RNA (lncRNA), is overexpressed in several cancers, including colorectal cancer (CRC). We investigated the role of LINC01234 in CRC development and confirmed its correlation with Krüppel-like factor 6 (KLF6), a tumor suppressor gene that is dysregulated in CRC. METHODS: We tested mRNA levels using quantitative reverse transcription PCR (qRT-PCR). Tissue samples from patients with CRC, inflammatory bowel disease (IBD), hyperplastic polyp, and adenoma were included. Correlations between clinicopathological parameters, overall survival (OS) rate, and LINC01234 were analyzed using Kruskal-Wallis H test. Additionally, cell proliferation, apoptosis, and tumor formation in nude mice were tested to investigate the mechanism of LINC01234. Western blotting was used to determine protein levels. RESULTS: LINC01234 expression was significantly upregulated in CRC tissues and CRC cell lines than in non-tumor tissues and normal epithelial cells, respectively. LINC01234 was associated with high tumor stage, larger tumor size, and metastasis. Patients with higher LINC01234 expression showed reduced OS. Cell proliferation was inhibited by LINC01234 knockdown, whereas apoptosis was enhanced. Mice injected with SW480 cells with LINC01234 knockdown displayed decreased tumor volume, weight, and Ki-67 levels compared with those injected with control cells. KLF6 was negatively regulated by LINC01234. Overexpression of KLF6 showed effects similar to those observed following LINC01234 knockdown on cell proliferation and apoptosis. CONCLUSIONS: LINC01234 could be a prognostic biomarker in CRC patients. Upregulation of LINC01234 in CRC promotes tumor development through negative regulation of KLF6.

Prognostic significance and molecular mechanisms of adenosine triphosphate-binding cassette subfamily C members in gastric cancer.

Gastric cancer (GC) is one of the major leading causes of tumor-related deaths worldwide. Adenosine triphosphate-binding cassette subfamily C (ABCC) consists of 13 members, ABCC1 to 13, which were examined for their associations with GC.The online Kaplan-Meier Plotter database was used to determine the prognostic significance of ABCC subfamily members in GC. Stratified analyses were performed using gender, disease stage, degree of tumor differentiation, expression of human epidermal growth factor receptor 2 (HER2), and Lauren classification. Molecular mechanisms were examined using the database for annotation, visualization, and integrated discovery database.ABCC1, ABCC3, ABCC7, ABCC8, ABCC9, and ABCC10 expression showed prognostic significance in the whole population and in male and female subpopulations (all P ≤ .05). Furthermore, high expression of most ABCC family members always suggested a poor prognosis, except for ABCC7 (P > .05). Stratified analyses revealed that ABCC1, ABCC3, ABCC7, ABCC8, ABCC9, and ABCC10 expression showed prognostic significance for the whole population, as well as male and female populations. ABCC2 and ABCC9 were significantly correlated with all disease stages, while ABCC2 and ABCC6 were significantly correlated with all Lauren classifications. Expression of ABCC1, ABCC3, ABCC5, ABCC7, ABCC8, ABCC9, and ABCC10 was significantly correlated with either negative or positive of HER2 status (all P ≤ .05). Enrichment analysis indicated that these genes were involved in ATPase activity, transmembrane transport, or were ABC transporters (all P ≤ .05).ABCC1, ABCC3, ABCC7, ABCC8, ABCC9, and ABCC10 may be potential prognosis biomarkers for GC, acting as ABC transporters and via ATPase activity.

Diagnostic accuracy of contrast-enhanced ultrasound in assessing the therapeutic response to radio frequency ablation for liver tumors: systematic review and meta-analysis.

BACKGROUND: To review the diagnostic accuracy of contrast-enhanced ultrasound (CEUS) used to detect residual or recurrent liver tumors after radiofrequency ablation (RFA). This technique uses contrast-enhanced computer tomography or/and contrast-enhanced magnetic resonance imaging as the gold standard of investigation. METHODS: MEDLINE, EMBASE, and COCHRANE were systematically searched for all potentially eligible studies comparing CEUS with the reference standard that follows RFA. Risk of bias and applicability concerns were addressed by adopting the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Pooled point estimates for sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratios (DOR) with 95% CI were computed before plotting the sROC (summary receiver operating characteristic) curve. Meta-regression and subgroup analysis were used to identify the source of the heterogeneity that was detected. Publication bias was evaluated using Deeks' funnel plot asymmetry test. RESULTS: Ten eligible studies on 1162 lesions that occurred between 2001 and 2016 were included in the final analysis. The quality of the included studies assessed by the QUADAS-2 tool was considered reasonable. The pooled sensitivity and specificity of CEUS in detecting residual or recurrent liver tumors had the following values: 0.90 (95% CI 0.85-0.94) and 1.00 (95% CI 0.99-1.00), respectively. Overall DOR was 420.10 (95% CI 142.30-1240.20). The sources of heterogeneity could not be precisely identified by meta-regression or subgroup analysis. No evidence of publication bias was found. CONCLUSION: This study confirmed that CEUS exhibits high sensitivity and specificity in assessing therapeutic responses to RFA for liver tumors.