ICOS/ICOSLG and PD-1 Co-Expression is Associated with the Progression of Colorectal Precancerous- Carcinoma Immune Microenvironment.

Purpose: This study aimed to investigate the expression of inducible T-cell co-stimulator (ICOS) and its ligand (ICOSLG), along with their association with clinicopathological features and influence on the immune profile in colorectal cancer (CRC). Patients and Methods: The Cancer Genome Atlas Colorectal Adenocarcinoma cohorts were used. We also analyzed 131 clinical samples of colon lesions, including precancerous lesions (hyperplastic polyps, low-grade dysplasia, and high-grade dysplasia) and CRC tissues. We conducted immunohistochemical (IHC) assays and multiple IHC (mIHC) of CD4+, Foxp3+ tumor-infiltrating lymphocytes (TILs), and PD-1/PD-L1 immune checkpoints in precancerous lesions and CRC samples from our patient subsets to determine changes and correlations in ICOS and ICOSLG expression during progression through the adenoma-carcinoma pathway. Results: High expression of ICOS and ICOSLG was a significant factor in CRC in multiple analyses and was positively correlated with CD4+/Foxp3+ TIL density and PD-1/PD-L1 expression, which increased with the sequential progression of lesions from precancerous tissues to carcinoma. Multivariable logistic regression analysis suggested that the location and expression level of ICOS/ICOSLG may be involved in precancerous-carcinoma progression. The co-expression status of PD-1 and ICOS/ ICOSLG could stratify patients with colorectal lesions into three groups of low, moderate, and high risk of progression. According to this classification and mIHC assays, we found a strong correlation between increased PD-1+ICOS+ or PD-1+ICOSLG+ co-expression and CRC, which might be deemed an independent factor in carcinogenesis. Conclusion: Increased ICOS/ICOSLG expression may be associated with the progressive formation of Foxp3+ TILs in the immune microenvironment and may further promote the development of the abnormal cytology of colorectal lesions from precancerous neoplasia to CRC. Our findings support the interpretation that enhanced co-expression of PD-1+ICOS+ or PD-1+ICOSLG+ contributes to the immune-active microenvironment of the colorectal adenoma-carcinoma sequence.

Multicolor fluorescence encoding of different microRNAs in lung cancer tissues at the single-molecule level.

The simultaneous detection of multiple microRNAs (miRNAs) will facilitate early clinical diagnosis. Herein, we demonstrate the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection for simultaneous measurement of different miRNAs in lung cancer tissues. This assay involves two linear templates and two circular templates without the requirement of any fluorescent-labeled probes. The binding of target miRNAs to their corresponding linear templates initiates the cyclic strand displacement amplification, generating many triggers which can specifically hybridize with the corresponding biotin-labeled AP probes to initiate the apurinic/apyrimidic endonuclease 1-assisted cyclic cleavage reaction for the production of more biotin-labeled primers for each miRNA. The resultant two primers can react with their corresponding circular templates to initiate rolling circle amplification which enables the incorporation of Cy5-dCTP/Cy3-dGTP nucleotides, resulting in the simultaneous production of abundant biotin-/multiple Cy5/Cy3-labeled DNA products. After magnetic separation and exonuclease cleavage, the amplified products release abundant Cy5 and Cy3 fluorescent molecules which can be simply monitored by single-molecule detection, with Cy3 indicating miR-21 and Cy5 indicating miR-155. This assay involves three consecutive amplification reactions, enabling the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophore-encoded DNA products which can release abundant fluorescent molecules for the generation of amplified signals. This assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and in lung cancer tissues, and it can distinguish the expression of miRNAs between non-small cell lung cancer patients and healthy persons. The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction.

Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level.

DNA repair enzymes (e.g., DNA glycosylases) play a critical role in the repair of DNA lesions, and their aberrant levels are associated with various diseases. Herein, we develop a sensitive method for simultaneous detection of multiple DNA repair enzymes based on the integration of single-molecule detection with rolling circle amplification (RCA)-driven encoding of different fluorescent molecules. We use human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) as the target analytes. We design a bifunctional double-stranded DNA (dsDNA) substrate with a hypoxanthine base (I) in one strand for hAAG recognition and an uracil (U) base in the other strand for UDG recognition, whose cleavage by APE1 generates two corresponding primers. The resultant two primers can hybridize with their respective circular templates to initiate RCA, resulting in the incorporation of multiple Cy3-dCTP and Cy5-dGTP nucleotides into the amplified products. After magnetic separation and exonuclease cleavage, the Cy3 and Cy5 fluorescent molecules in the amplified products are released into the solution and subsequently quantified by total internal reflection fluorescence (TIRF)-based single-molecule detection, with Cy3 indicating the presence of hAAG and Cy5 indicating the presence of UDG. This strategy greatly increases the number of fluorescent molecules per concatemer through the introduction of RCA-driven encoding of different fluorescent molecules, without the requirement of any specially labeled detection probes for simultaneous detection. Due to the high amplification efficiency of RCA and the high signal-to-ratio of single-molecule detection, this method can achieve a detection limit of 6.10 × 10-9 U mL-1 for hAAG and 1.54 × 10-9 U mL-1 for UDG. It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and the screening of DNA glycosylase inhibitors, holding great potential in early clinical diagnosis and drug discovery.

Diagnostic value of liquid-based cytology and smear cytology in pancreatic endoscopic ultrasound-guided fine needle aspiration: A meta-analysis.

BACKGROUND: Smear cytology (SC) using endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is the established and traditional choice for diagnosing pancreatic lesions. Liquid-based cytology (LBC) is a novel alternative cytological method, however, the comparative diagnostic efficacy of LBC remains inconclusive. AIM: To examine the diagnostic efficacy of LBC and SC for pancreatic specimens obtained through EUS-FNA via a systematic review and meta-analysis. METHODS: A systematic literature search was performed using PubMed, EMBASE, the Cochrane Library, and Web of Science. The numbers of true positives, false positives, true negatives, and false negatives for each cytological test (LBC and CS) were extracted from the included studies. The pooled sensitivity and specificity and the area under the summary receiver operating characteristic curve (AUC) were calculated, and the AUC was compared by Tukey's multiple comparisons test. The quality of the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies II tool. RESULTS: A total of 1656 patients in eight studies were included. The pooled sensitivity and specificity and the AUC for LBC were 0.76 (95%CI: 0.72-0.79), 1.00 (95%CI: 0.98-1.00), and 0.9174, respectively, for diagnosing pancreatic lesions. The pooled estimates for SC were as follows: Sensitivity, 0.68 (95%CI: 0.64-0.71); specificity, 0.99 (95%CI: 0.96-100.00); and AUC, 0.9714. Similarly, the corresponding values for LBC combined with SC were 0.87 (95%CI: 0.84-0.90), 0.99 (95%CI: 0.96-1.00), and 0.9894. Tukey's multiple comparisons test was used to compare the sensitivities and AUCs of the three diagnostic methods; statistically significant differences were found between the three methods, and LBC combined with SC was superior to both LBC (P < 0.05) and SC (P < 0.05). The pooled sensitivity and AUC did not change significantly in the sensitivity analysis. CONCLUSION: LBC may be sensitive than SC in the cytological diagnosis of pancreatic lesions, however, the superior diagnostic performance of their combination emphasizes their integrated usage in the clinical evaluation of pancreatic lesions.

Diagnostic efficacy of the Japan Narrow-band-imaging Expert Team and Pit pattern classifications for colorectal lesions: A meta-analysis.

BACKGROUND: Pit pattern classification using magnifying chromoendoscopy is the established method for diagnosing colorectal lesions. The Japan Narrow-band-imaging (NBI) Expert Team (JNET) classification is a novel NBI magnifying endoscopic classification that focuses on the vessel, and surface patterns. AIM: To determine the diagnostic efficacy of each category of the JNET and Pit pattern classifications for colorectal lesions. METHODS: A systematic literature search was performed using PubMed, Embase, the Cochrane Library, and Web of Science databases. The pooled sensitivity, specificity, diagnostic odds ratio, and area under the summary receiver operating characteristic curve of each category of the JNET and Pit pattern classifications were calculated. RESULTS: A total of 19227 colorectal lesions in 31 studies were included. The diagnostic performance of the JNET classification was equivalent to the Pit pattern classification in each corresponding category. The pooled sensitivity, specificity, and area under the curve (AUC) for each category of the JNET classification were as follows: 0.73 (95%CI: 0.55-0.85), 0.99 (95%CI: 0.97-1.00), and 0.97 (95%CI: 0.95-0.98), respectively, for Type 1; 0.88 (95%CI: 0.78-0.94), 0.72 (95%CI: 0.64-0.79), and 0.84 (95%CI: 0.81-0.87), respectively, for Type 2A; 0.56 (95%CI: 0.47-0.64), 0.91 (95%CI: 0.79-0.96), and 0.72 (95%CI: 0.68-0.76), respectively, for Type 2B; 0.51 (95%CI: 0.42-0.61), 1.00 (95%CI: 1.00-1.00), and 0.90 (95%CI: 0.87-0.93), respectively, for Type 3. CONCLUSION: This meta-analysis suggests that the diagnostic efficacy of the JNET classification may be equivalent to that of the Pit pattern classification. However, due to its simpler and clearer clinical application, the JNET classification should be promoted for the classification of colorectal lesions, and to guide the treatment strategy.

[Species diversity of Brasenia schreberi community in different habitats].

By using Braun-Blanquet Cover Abundance Scale method, the species diversity of Brasenia schreberi community in Dongshan Town (Suzhou City, Jiangsu Province) and Zhuantang Town (Hangzhou City, Zhejiang Province) was studied, and the results showed that in these two towns, B. schreberi community was of simple species composition and relatively low species diversity. The Shannon-Wiener diversity index, Simpson ecological dominance index, and community evenness index all increased obviously when the habitat shifted from pond center to bank nearby. B. schreberi community was similar in the same type habitats of these two towns, and of much more similarity in pond center than at bank nearby. Compared with Zhuantang Town, Dongshan Town had a greater difference of B. schreberi community between the two habitats.