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Single-cell technology depicts integrated tumor profiles including both tumor cells and tumor microenvironments, which theoretically enables more robust diagnosis than traditional diagnostic standards based on only pathology. However, the inherent challenges of single-cell RNA sequencing (scRNA-seq) data, such as high dimensionality, low signal-to-noise ratio (SNR), sparse and non-Euclidean nature, pose significant obstacles for traditional diagnostic approaches. The diagnostic value of single-cell technology has been largely unexplored despite the potential advantages. Here, we present a graph neural network-based framework tailored for molecular diagnosis of primary liver tumors using scRNA-seq data. Our approach capitalizes on the biological plausibility inherent in the intercellular communication networks within tumor samples. By integrating pathway activation features within cell clusters and modeling unidirectional inter-cellular communication, we achieve robust discrimination between malignant tumors (including hepatocellular carcinoma, HCC, and intrahepatic cholangiocarcinoma, iCCA) and benign tumors (focal nodular hyperplasia, FNH) by scRNA data of all tissue cells and immunocytes only. The efficacy to distinguish iCCA from HCC was further validated on public datasets. Through extending the application of high-throughput scRNA-seq data into diagnosis approaches focusing on integrated tumor microenvironment profiles rather than a few tumor markers, this framework also sheds light on minimal-invasive diagnostic methods based on migrating/circulating immunocytes., (© 2024. The Author(s).)

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB + and CD8 proliferating proportions and a low Macro CD5L + proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB + and CD8 proliferating proportions are increased, but the CD8 GZMK + proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB + to CD8 GZMK + facilitates good response to the therapy, while Macro CD5L + -CD8 GZMB + crosstalk impairs the response by increasing CTLA4 in CD8 GZMB + . Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8 + T-cell status conversion and exhaustion induced by Macro CD5L + in influencing the response, suggesting future avenues for cancer treatment optimization., (© 2024. The Author(s).)

Advanced intrahepatic cholangiocarcinoma (ICC) has a dismal prognosis. Here, we report the efficacy and safety of combining toripalimab, lenvatinib, and gemcitabine plus oxaliplatin (GEMOX) as first-line therapy for advanced ICC. Thirty patients with pathologically confirmed advanced ICC received intravenous gemcitabine (1 g/m 2 ) on Days 1 and 8 and oxaliplatin (85 mg/m 2 ) Q3W for six cycles along with intravenous toripalimab (240 mg) Q3W and oral lenvatinib (8 mg) once daily for one year. The expression of programmed death-ligand 1 (PD-L1) and genetic status was investigated in paraffin-embedded tissues using immunohistochemistry and whole-exome sequencing (WES) analysis. The primary endpoint was the objective response rate (ORR). Secondary outcomes included safety, overall survival (OS), progression-free survival (PFS), disease control rate (DCR) and duration of response (DoR). As of July 1, 2022, the median follow-up time was 23.5 months, and the ORR was 80%. Twenty-three patients achieved partial response, and one achieved complete response. Patients (21/30) with DNA damage response (DDR)-related gene mutations showed a higher ORR, while patients (14/30) with tumor area positivity ≥1 (PD-L1 staining) showed a trend of high ORR, but without significant difference. The median OS, PFS, and DoR were 22.5, 10.2, and 11.0 months, respectively. The DCR was 93.3%. Further, 56.7% of patients experienced manageable grade ≥3 adverse events (AEs), commonly neutropenia (40.0%) and leukocytopenia (23.3%). In conclusion, toripalimab plus lenvatinib and GEMOX are promising first-line regimens for the treatment of advanced ICC. A phase-III, multicenter, double-blinded, randomized study to validate our findings was approved by the National Medical Products Administration (NMPA, No. 2021LP01825).Trial registration Clinical trials: NCT03951597., (© 2023. The Author(s).)

Background: Perineural invasion (PNI) is associated with metastasis in malignancies, including intrahepatic cholangiocarcinoma (ICC), and is correlated with poor prognosis., Methods: The study included three large cohorts: ZS-ICC and TMA cohorts from our team, MSK cohort from a public database, and a small cohort named cohort 4. Prognostic implications of PNI were investigated in MSK cohort and TMA cohort. PNI-related genomic and transcriptomic profiles were analyzed in MSK and ZS-ICC cohorts. GO, KEGG, and ssGSEA analyses were performed. Immunohistochemistry was used to investigate the relationship between PNI and markers of neurons, hydrolases, and immune cells. The efficacy of adjuvant therapy in ICC patients with PNI was also assessed., Results: A total of 30.6% and 20.7% ICC patients had PNI in MSK and TMA cohorts respectively. Patients with PNI presented with malignant phenotypes such as high CA19-9, the large bile duct type, lymph node invasion, and shortened overall survival (OS) and relapse-free survival (RFS). Nerves involved in PNI positively express tyrosine hydroxylase (TH), a marker of sympathetic nerves. Patients with PNI showed high mutation frequency of KRAS and an immune suppressive metastasis prone niche of decreased NK cell, increased neutrophil, and elevated PD-L1, CD80, and CD86 expression. Patients with PNI had an extended OS after adjuvant therapy with TEGIO, GEMOX, or capecitabine., Conclusion: Our study deciphered the genomic features and the immune suppressive metastasis-prone niche in ICC with PNI. Patients with PNI showed a poor prognosis after surgery but a good response to adjuvant chemotherapy., (© 2022. The Author(s).)

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4 + lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues ( P <.001), and patients with a high density of CTLA-4 + tumor-infiltrating lymphocytes (TILs CTLA-4 High ) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILs CTLA-4 Low ( P <.001 and P = .024, respectively). Similarly, patients with high FOXP3 + TILs (TILs FOXP3 High ) had poorer prognoses than patients with low FOXP3 + TILs ( P  = .021, P = .034, respectively), and the density of CTLA-4 + TILs was positively correlated with FOXP3 + TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (Tumor PD-L1 High ) and/or TILs CTLA-4 High presented worse OS and a higher recurrence rate than patients with TILs CTLA-4 Low Tumor PD-L1 Low . Moreover, multiple tumors, lymph node metastasis, and high Tumor PD-L1 /TILs CTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. Tumor PD-L1 /TILs CTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guo, Lu, Zeng, Zhou, Sun, Yang, Pei, Meng, Shen, Zhang, Cai, Huang, Ke, Shi, Zhou, Fan, Chen, Yang, Shi and Huang.)

An automatic inspection system for biochip's print quality is presented in this paper. It consists of an automatic mechanical control, a CCD sensor for getting the image of PET boart, and the special computer software for image processing and recognition. Experimental results indicate that this system is capable of providing a precise and effective realtime inspection for biochips' print quality.