Point of care testing (POCT) of nucleic acid (NA) contributes to the timely disease diagnosis, like bacteria and virus screening in households or resource-constrained areas, but its development has always been stagnant. Herein, we proposed an exonuclease III cascaded with CRISPR/Cas12a (Exo-III/Cas12a) amplification strategy and constructed a smartphone-based portable fluorescence detector (SPFD) to repurpose the commercial alpha-fetoprotein (AFP) strip for the ultrasensitive and hand-held detection of NA samples. In detail, the target-initiated-Exo-III/Cas12a strategy realizes the signal amplification and liberates AFP from magnetic beads through the trans-cleavages of activated Cas12a toward the AFP aptamer. After magnetic separation and migration, the fluorescence signals of the test (FT) and control (FC) lines on the AFP strip were digitally output by the SPFD, and the FT/FC was employed for the quantitative analysis to minimize external disturbances and improve accuracy. We experimentally assessed the universe applicability of the proposed NA-POCT platform toward miRNA-155, 16S rRNA of Staphylococcus aureus, and ORF1a/b RNA of Covid-19 pseudovirus, achieving favorable detection limits of 42 aM, 18 CFU/mL, and 87 copies/μL, respectively. Moreover, its simplicity, universality, and admirable detection performance demonstrate a great potential in the aspect of rapidly transforming the existing POCT devices for multiple new applications at the time of need.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the deadliest malignant tumors in China. Microvascular invasion (MVI) often indicates poor prognosis and metastasis in HCC patients. 18F-FDG PET-CT is a new imaging method commonly used to screen for tumor occurrence and evaluate tumor stage.
OBJECTIVE: This study attempted to predict the occurrence of MVI in early-stage HCC through 18F-FDG positron emission tomography (PET)/computed tomography (CT) imaging findings and laboratory data.
METHODS: A total of 113 patients who met the inclusion criteria were divided into two groups based on postoperative pathology: the MVI-positive group and MVI-negative group. We retrospectively analyzed the imaging findings and laboratory data of 113 patients. Imaging findings included tumor size, tumor maximum standard uptake value (SUVmaxT), and normal liver maximum standard uptake value (SUVmaxL). The ratios of SUVmaxT to SUVmaxL (SUVmaxT/L) and an SUVmaxT/L > 2 were defined as active tumor metabolism. The tumor size was indicated by the maximum diameter of the tumor, and a diameter greater than 5 cm was defined as a mass lesion. The laboratory data included the alpha-fetoprotein (AFP) level and the HBeAg level. An AFP concentration > 20 ng/mL was defined as a high AFP level. A HBeAg concentration > 0.03 NCU/mL was defined as HB-positive.
RESULTS: The SUVmaxT/L (p = 0.003), AFP level (p = 0.008) and tumor size (p = 0.015) were significantly different between the two groups. Patients with active tumor metabolism, mass lesions and high AFP levels tended to be MVI positive. Binary logistic regression analysis verified that active tumor metabolism (OR = 4.124, 95% CI, 1.566-10.861; p = 0.004) and high AFP levels (OR = 2.702, 95% CI, 1.214-6.021; p = 0.015) were independent risk factors for MVI. The sensitivity of the combination of these two independent risk factors predicting HCC with MVI was 56.9% (29/51), the specificity was 83.9% (52/62) and the accuracy was 71.7% (81/113).
CONCLUSIONS: Active tumor metabolism and high AFP levels can predict the occurrence of MVI in HCC patients.

BACKGROUND: Developing biosensors with antifouling properties is essential for accurately detecting low-concentration biomarkers in complex biological matrix, which is imperative for effective disease diagnosis and treatment. Herein, an antifouling electrochemical aptasensor qualifying for probing targets in human serum was explored based on newly-devised peptides that could form inverted U-shaped structures with long-term stability.
RESULTS: The inverted U-shaped peptides (U-Pep) with two terminals of thiol groups grafted onto the Au-modified electrode showcase superior antifouling properties in terms of high stability against enzymatic hydrolysis and long acting against biofouling in actual biofluids. The construction of the outlined antifouling electrochemical aptasensor just involved the fabrication of Au-deposited poly(3,4 ethylenedioxythiophene) (Au/PEDOT) modified electrode, followed by one-step co-incubation in the peptides and the aptamer probes with the Au/PEDOT electrode. Taking a typical biomarker of alpha-fetoprotein (AFP) for detection, this elegant antifouling aptasenor demonstrated a nice response for probing the target AFP with a low detection limit of 0.27 pg/mL and a wide linear scope of 1.0 pg/mL to 1.0 μg/mL, and furthermore qualified for assaying of AFP in human serum samples with satisfactory accuracy and feasibility.
CONCLUSIONS: This engineering strategy of U-Pep with long-lasting antifouling efficacy opens a new horizon for high-performance antifouling biosensors suitable for detection in complex bifluids, and it could spark more inspiration for a follow-up exploration of other featured antifouling biomaterials.

Alpha-foetoprotein (AFP) is taken as a diagnostic tumor marker for the screening and diagnosis of cancer. Nucleic acid-based isothermal amplification strategies are emerging as a potential technology in early screening and clinical diagnosis of AFP. The leakages between hairpins dramatically increase the background and reduce the sensitivity. Thus, it is necessary to develop some strategies to reduce the leakage for isothermal amplification strategies. A DNAzyme-locked leakless enzyme-free amplification system was developed for AFP detection in liver cancer and breast cancer. AFP could open the apt-hairpin and initiate the catalytic hairpin assembly (CHA) reaction to produce a Y-shaped duplex. Two tails of a Y-shaped duplex cleaved the two kinds of leakless hairpins. Then, the third tail of the Y-shaped duplex catalyzed the second CHA between the cleaved leakless hairpins to recover the fluorescent intensity. The limit of detection reached 5 fg/mL by the two levels of signal amplifications. Importantly, the leakless hairpin design effectively reduced leakage between hairpins and weakened the background. In addition, it also showed a great promising potential for AFP detection in early screening and clinical diagnosis.

OBJECTIVE: To evaluate the association between serum alpha-fetoprotein (AFP) half-life (HL) and prognosis in prepubertal children with elevated AFP values 3 to 4 weeks after surgery for testicular yolk sac tumors (YST).
METHODS: Prepubertal patients with testicular YST treated with radical orchiectomy between January 2016 and December 2022 were retrospectively reviewed. Negative outcomes were defined as relapse, metastasis or death. Univariate and multivariate logistic regression analyses were conducted to select risk factors for negative outcomes.
RESULTS: A total of 42 patients were eventually enrolled into the study. Patients were divided into non-negative and negative outcomes groups, consisting of 35 and 7 patients, respectively. Thirty-five patients were stage I, two cases were stage II, and five cases were stage IV, according to the Children\'s Oncology Group staging system. The overall survival (OS) rate was 100%. Average AFP values significantly decreased after resection (P < 0.001). A significant positive correlation was shown between pre- and postoperative AFP values (r = 0.60, P < 0.001). Long AFP HL was considered as an independent risk factor for negative outcomes in YST patients underwent radical orchiectomy (P = 0.04). The cut-off value for AFP HL was 5.78 days, regardless of age division.
CONCLUSIONS: Testicular YST is a relatively rare disease in children with an OS of 100%, and salvage chemotherapy is effective even in grade IV patients. The postoperative AFP HL was significantly associated with prognosis in prepubertal patients with testicular YST. The cut-off value for AFP HL is 5.78 days regardless of the effect of physiological AFP elevation.

Objective: To investigate the immunophenotypic and molecular biological characteristics of patients with elevated serum alpha-fetoprotein (AFP) and enteroblastic differentiated gastric adenocarcinoma (GAED). Methods: The clinicopathological data of 13 patients with elevated serum AFP and GAED admitted to Shanxi Cancer Hospital from 2018 to 2020 were collected. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were used to analyze the immune markers and molecular biological characteristics of the pathological tissues of the patients. Kaplan-Meier method and log rank test were used for survival analysis. Results: Among the 13 patients with GAED, 12 were male and 1 was female, aged 41-70 years, with a median age of 64 years. The lesions were mainly located in the gastric antrum (5 cases) and gastric body (4 cases). IHC results showed that the tumor embryonic protein (AFP, SALL4, GPC3), intestinal epithelial differentiation protein (CDX-2, CD10), and some original intestinal epithelial phenotype markers (OCT3/4, Claudin6) were expressed in the tumor tissues. Combined application of multiple markers can reduce the rate of missed diagnosis. Among the 13 patients, 12 had at least one mutation (1 mutation: 1 case, 2-5 mutations: 3 cases, 6-15 mutations: 8 cases), and 1 case was not detected. The gene with the highest mutation frequency was TP53 (10 cases), and other mutant genes included EPHB1 (3 cases), ATRX (2 cases), EPHA5 (2 cases), GATA3 (2 cases), LRP1B (2 cases) and MAP2K4 (2 cases) were also detected. Three of the 13 patients had structural variations, which were C14orf177-GNAS, AIM1-FGFR3, and EPHA6-ROS1 gene rearrangements. All 13 patients had copy number variation, and 11 patients had copy number variation of more than 2 genes. The common amplification genes were IRS2 (5 cases), PTEN (5 cases), GNAS (4 cases), CCNE1 (3 cases), CEBPA (3 cases), PCK1 (3 cases) and ERBB2 (2 cases). The common deletion genes were SOX2 (5 cases) and MYC (5 cases). Among the 13 patients, 4 died, and 2 of the dead patients had liver metastasis. There were 4 patients with disease-free survival and 5 patients with disease progression, including 3 cases of abdominal metastasis and 2 cases of liver metastasis. The 3-year survival rate of patients was 65.9 %, and the 3-year progression-free survival rate was 30.7 %. Gene LRP1B point mutation was associated with poor prognosis (P＜0.001). There was no significant improvement in the prognosis of patients treated with immunotherapy compared with those treated with chemotherapy alone (P=0.595), but the prognosis of patients treated with postoperative chemotherapy or postoperative chemotherapy plus immunotherapy was better than that of patients treated with surgery alone (P＜0.05). Conclusions: Elevated serum AFP with GAED is a highly invasive tumor with unique molecular characteristics, often accompanied by multiple molecular events. TP53 mutation is the most common type of gene mutation. In addition, some cases are accompanied by HER2 amplification and gene rearrangement.
目的： 探讨血清甲胎蛋白（AFP）升高伴肠母细胞分化胃腺癌（GAED）患者的免疫表型和分子生物学特征。 方法： 收集2018—2020年山西省肿瘤医院收治的13例血清AFP升高且伴有GAED患者的临床病理资料，应用免疫组织化学（IHC）及二代测序方法对13例血清AFP升高伴GAED患者的病理组织进行免疫标志物和分子生物学特征分析，生存分析采用Kaplan-Meier法和Log rank检验。 结果： 13例GAED患者中，男12例，女1例，年龄41～70岁，中位年龄64岁，病灶位置以胃窦部（5例）、胃体部（4例）为主。IHC显示可表达瘤胚胎蛋白（AFP、SALL4、GPC3）、肠上皮分化蛋白（CDX-2、CD10）和一些原始肠上皮表型标志物（OCT3/4、Claudin6），联合应用多种标志物诊断可降低漏诊率。13例患者中，12例存在至少1个基因突变（1个基因突变：1例，2～5个基因突变：3例，6～15个基因突变：8例），1例未检测到突变。突变频率最高的基因是TP53（10例），其他突变基因包括EPHB1（3例）、ATRX（2例）、EPHA5（2例）、GATA3（2例）、LRP1B（2例）和MAP2K4（2例）。13例患者中3例存在基因结构变异，分别为C14orf177-GNAS、AIM1-FGFR3、EPHA6-ROS1基因重排。13例患者均存在拷贝数变异，11例患者存在2个以上基因的拷贝数变异。常见的扩增基因为IRS2（5例）、PTEN（5例）、GNAS（4例）、CCNE1（3例）、CEBPA（3例）、PCK1（3例）、ERBB2（2例），常见的缺失基因为SOX2（5例）、MYC（5例）。13例患者中，死亡4例，死亡患者中有2例出现肝转移；无病生存患者4例，5例患者病情进展，其中3例出现腹腔转移，2例出现肝转移。血清AFP升高伴GAED患者3年生存率为65.9%，3年无进展生存率为30.7%。基因LRP1B点突变与预后不良有关（P＜0.001）。行免疫治疗较单纯化疗的患者预后无明显改善（P=0.595），但进行术后化疗或术后化疗加免疫治疗较只进行手术患者预后好（均P＜0.05）。 结论： 血清AFP升高伴GAED是一种具有高度侵袭性的肿瘤，有独特的分子特征，往往同时伴有多个分子事件，TP53突变是其最常见的基因突变类型，此外，部分患者伴有人表皮生长因子受体2扩增和基因重排。.

Biomarkers screening is a benefit approach for early diagnosis of major diseases. In this study, magnetic nanoparticles (MNPs) have been utilized as labels to establish a multi-line immunochromatography (MNP-MLIC) for simultaneous detection of carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 19-9), and alpha-fetoprotein (AFP) in a single serum sample. Under the optimal parameters, the three biomarkers can be rapidly and simultaneously qualitative screening within 15 min by naked eye. As for quantitative detection, the MNP-MLIC test strips were precisely positioned and captured by a smartphone, and signals on the test and control lines were extracted by ImageJ software. The signal ratio of test and control lines has been calculated and used to plot quantitative standard curves with the logarithmic concentration, of which the correlation coefficients are more than 0.99, and the limit of detection for CEA, CA 19-9, and AFP were 0.60 ng/mL, 1.21 U/mL, and 0.93 ng/mL, respectively. The recoveries of blank serum were 75.0 ~ 112.5% with the relative standard deviation ranging from 2.5 to 15.3%, and the specificity investigation demonstrated that the MNP-MLIC is highly specific to the three biomarkers. In conclusion, the developed MNP-MLIC offers a rapid, simple, accurate, and highly specific method for simultaneously detecting multiple biomarkers in serum samples, which provides an efficient and accurate approach for the early diagnosis of diseases.

BACKGROUND: Hepatic arterial infusionchemotherapy (HAIC) is a promising option for large unresectable hepatocellular carcinoma (HCC). Identifying patients who could benefit from continuous HAIC remains a challenge. We aimed to establish an objective model to guide the decision for retreatment with HAIC.
METHODS: Between 2015 and 2020, the data of patients with large unresectable HCC without macrovascular invasion or extrahepatic spread undergoing multiple HAIC cycles from 3 different centers were retrieved. We investigated the basic tumor parameters and the effect of HAIC on liver function and tumor response, and their impact on overall survival (OS). A point score (ARH, Assessment for Retreatment with HAIC) was built by using a stepwise Cox regression model in the training cohort (n = 112) and was validated in an independent validation cohort (n = 71).
RESULTS: The high α-fetoprotein before the second cycle of HAIC, an increase in Child-Pugh score, and undesirable radiologic tumor responses remained independent negative prognostic factors and were used to create the ARH score. The prognosis of HCC patients deteriorated significantly with the increase in ARH score. The median OS of patients with ARH score 0-2 points and ≥ 2.5 points were 19.37 months and 11.60 months (P < 0.001). All of these results had been confirmed in the external validation cohort and demonstrated significance across multiple subgroups.
CONCLUSIONS: The ARH score makes an excellent prediction of the prognosis of HCC patients who received retreatment of HAIC. Patients with an ARH score ≥ 2.5 prior to the second cycle of HAIC may not profit from further sessions.

The conventional electrochemical detection strategy for alpha-fetoprotein (AFP) is limited by the antigen-antibody (Ag-Ab) reactions and suffers from low sensitivity and poor reproducibility due to the inconsistency of Ab-modified electrodes. Herein, we designed and explored a sandwich-type electrochemical sensor for highly sensitive detection of AFP based on aptamer (Apt)-AFP-Ab interaction mode with silver@gold (Ag@Au) core-shell nanoparticles (NPs) as a signal amplifier. AuNPs were electrodeposited onto MXene (Ti3C2TX)-modified glassy carbon electrode (GCE) to get AuNPs/MXene/GCE and further used as the signal amplification substrate. The tetrahedral DNA-linked AFP aptamers were immobilized onto AuNPs/MXene/GCE surface via Au-S bonds and used as the sensing and recognition platform for AFP capturing. Ag@AuNPs with core-shell structures were synthesized, characterized, and bound with Ab as detection elements by catalyzing H2O2 reduction. In the presence of AFP, a stable Apt-AFP-Ab sandwich structure was formed owing to the high affinities of aptamer and Ab toward the target AFP. The catalytic current produced by H2O2 reduction increased linearly with the logarithm of AFP concentration from 5 × 10-4 ng/mL to 1 × 105 ng/mL, accompanied by a low detection limit (1.6 × 10-4 ng/mL). Moreover, the novel sandwich-type electrochemical sensor shows high sensitivity, outstanding selectivity, and promising performance in the analysis of actual samples, displaying a broad application prospect in bioanalysis.

BACKGROUND: At present, liver transplantation (LT) is one of the best treatments for hepatocellular carcinoma (HCC). Accurately predicting the survival status after LT can significantly improve the survival rate after LT, and ensure the best way to make rational use of liver organs.
OBJECTIVE: To develop a model for predicting prognosis after LT in patients with HCC.
METHODS: Clinical data and follow-up information of 160 patients with HCC who underwent LT were collected and evaluated. The expression levels of alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin, Golgi protein 73, cytokeratin-18 epitopes M30 and M65 were measured using a fully automated chemiluminescence analyzer. The best cutoff value of biomarkers was determined using the Youden index. Cox regression analysis was used to identify the independent risk factors. A forest model was constructed using the random forest method. We evaluated the accuracy of the nomogram using the area under the curve, using the calibration curve to assess consistency. A decision curve analysis (DCA) was used to evaluate the clinical utility of the nomograms.
RESULTS: The total tumor diameter (TTD), vascular invasion (VI), AFP, and cytokeratin-18 epitopes M30 (CK18-M30) were identified as important risk factors for outcome after LT. The nomogram had a higher predictive accuracy than the Milan, University of California, San Francisco, and Hangzhou criteria. The calibration curve analyses indicated a good fit. The survival and recurrence-free survival (RFS) of high-risk groups were significantly lower than those of low- and middle-risk groups (P < 0.001). The DCA shows that the model has better clinical practicability.
CONCLUSIONS: The study developed a predictive nomogram based on TTD, VI, AFP, and CK18-M30 that could accurately predict overall survival and RFS after LT. It can screen for patients with better postoperative prognosis, and improve long-term survival for LT patients.