Circulating tumor DNA (ctDNA) provides a noninvasive approach to elucidate a patient’s genomic landscape and actionable information. Here, we design a ctDNA-based study of over 10,000 pan-cancer Chinese patients. Using parallel sequencing between plasma and white blood cells, 14% of plasma cell-free DNA samples contain clonal hematopoiesis (CH) variants, for which detectability increases with age. After eliminating CH variants, ctDNA is detected in 73.5% of plasma samples, with small cell lung cancer (91.1%) and prostate cancer (87.9%) showing the highest detectability. The landscape of putative driver genes revealed by ctDNA profiling is similar to that in a tissue-based database (R² = 0.87, p < 0.001) but also shows some discrepancies, such as higher EGFR (44.8% versus 25.2%) and lower KRAS (6.8% versus 27.2%) frequencies in non-small cell lung cancer, and a higher TP53 frequency in hepatocellular carcinoma (53.1% versus 28.6%). Up to 41.2% of plasma samples harbor drug-sensitive alterations. These findings may be helpful for identifying therapeutic targets and combined treatment strategies.

Background: The combination of immune checkpoint blockade and chemotherapy has revolutionized the treatment of advanced gastric cancer (GC). It is crucial to unravel chemotherapy-induced tumor microenvironment (TME) modulation and identify which immunotherapy would improve antitumor effect. Methods: In this study, tumor-associated immune cells (TAICs) infiltration in residual tumor after neoadjuvant chemotherapy (NAC) together with 1075 cases of treatment-naïve GC patients was analyzed first. Then we performed multiplex fluorescence staining of a panel of immune markers (CD3, CD4, CD8, FOXP3 and PDL1) and T cell receptor β-chain sequencing to phenotype and enumerate T cell subpopulations and clonal expansion in paired GC samples (prechemotherapy and postchemotherapy) from another cohort of 30 cases of stage II/III GC patients. Results: Infiltration of CD68⁺ macrophages in residual tumors after NAC was significantly decreased compared with treatment-naïve GC patients, while no significant difference observed with respect to other immune markers. In residual tumors, post-NAC CD8 +T cells and CD68+ macrophages levels were significantly associated with chemotherapy response. Post-NAC CD8+ T cell levels remained as an independent predictor for favorable prognosis. Furthermore, when comparing the paired samples before and after NAC from 30 cases of stage II/III GC patients, we found FOXP3+ regulatory T cells proportion significantly decreased after chemotherapy. Pre-NAC FOXP3+ T reg cells level was much richer in the response group and decreased more significantly in the stromal compartment. CD8+ cytotoxic T lymphocytes levels were elevated after chemotherapy, which was more significant in the group treated with XELOX regimen and in patients with better response, consistent with the TCR diversity elevation. Conclusions: These findings have deepened our understanding of the immune modulating effect of chemotherapy and suggest that the immune profile of specimens after standard chemotherapy should be considered for the personalized immunotherapy to ultimately improve clinical outcome in patients with GC.

Background: Insulin gene enhancer protein 1, (ISL1), a LIM‐homeodomain transcription factor, is involved in multiple tumors and is associated with insulin secretion and metabolic phenotypes. However, the role of ISL1 in stimulating glycolysis to promote tumorigenesis in gastric cancer (GC) is unclear. In this study, we aimed to characterize the expression pattern of ISL1 in GC patients and explore its molecular biological mechanism in glycolysis and tumorigenesis. Methods: We analyzed the expression and clinical significance of ISL1 in GC using immunohistochemistry and real‐time polymerase chain reaction (PCR). Flow cytometry and IncuCyte assays were used to measure cell proliferation after ISL1 knockdown. RNA‐sequencing was performed to identify differentially expressed genes, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) to reveal key signaling pathways likely regulated by ISL1 in GC. Alteration of the glycolytic ability of GC cells with ISL1 knockdown was validated by measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) and by detecting glucose consumption and lactate production. The expression of glucose transporter 4 (GLUT4) and ISL1 was assessed by Western blotting, immunohistochemistry, and immunofluorescent microscopy. The luciferase reporter activity and chromatin immunoprecipitation assays were performed to determine the transcriptional regulation of ISL1 on GLUT4. Results: High levels of ISL1 and GLUT4 expression was associated with short survival of GC patients. ISL1 knockdown inhibited cell proliferation both in vitro and in vivo . KEGG analysis and GSEA for RNA‐sequencing data indicated impairment of the glycolysis pathway in GC cells with ISL1 knockdown, which was validated by reduced glucose uptake and lactate production, decreased ECAR, and increased OCR. Mechanistic investigation indicated that ISL1 transcriptionally regulated GLUT4 through binding to its promoter. Conclusion: ISL1 facilitates glycolysis and tumorigenesis in GC via the transcriptional regulation of GLUT4.

Background: Epstein–Barr virus-associated gastric cancer(EBVaGC)has a unique tumor immune microenvironment. We performed a comprehensive analysis of the tumor-infiltrating immune cells in a cohort of EBVaGC in a Chinese population.Methods: Epstein–Barr encoding region (EBER) in situ hybridization was performed in 1,328 consecutive cases of surgically resected GC. Densities of immune cells, including T cells, B cells, natural killer cells, and macrophages from the patients were calculated after immunohistochemical staining with CD3, CD20, CD57, and CD68 antibodies in tissue microarrays, respectively.Results: EBVaGC patients accounted for 4.1% (55 of 1,328) cases in the overall population. The average age of patients with EBVaGC was lower than that of non-EBVaGC patients. Histologically, EBVaGC patients exhibited poorly differentiated adenocarcinoma (P = 0.004) and lower frequency of vascular invasion (P = 0.034). The density of CD3⁺ T lymphocytes (CD3, 23.84 ± 14.49 vs. 12.76 ± 8.93, P < 0.001) and CD68⁺ macrophages (CD68, 9.73 ± 5.25 vs. 5.44 ± 4.18, P < 0.001) was significantly higher in EBVaGC patients. CD3⁺ T cell density predicted better 5-year overall survival of EBVaGC patients (P = 0.022).Conclusions: EBVaGC patients were younger with low-differentiated adenocarcinoma and less vascular invasion. Increased infiltration of multiple immune cells affected the prognosis of patients, especially EBVaGC patients with more CD3⁺ T lymphocytes, who survived longer.

A good response to neoadjuvant chemotherapy (NACT) is strongly associated with a higher curative resection rate and favorable outcomes for patients with gastric cancer (GC). We examined the utility of serial circulating tumor DNA (ctDNA) testing for monitoring NACT response and prognosis in stage II–III GC. Seventy‐nine patients were enrolled to receive two cycles of NACT following gastrectomy with D2‐lymphadenectomy. Plasma at baseline, post‐NACT, and after surgery, and tissue at pretreatment and surgery were collected. We used a 425‐gene panel to detect genomic alterations (GAs). Results show that the mean cell‐free DNA concentration of patients with clinical stage III was significantly higher than patients with stage II (15.43 ng·mL⁻¹ vs 14.40 ng·mL⁻¹). After receiving NACT and surgery, the overall detection rate of ctDNA gradually reduced (59.5%, 50.8%, and 47.4% for baseline, post‐NACT, and postsurgery). The maximum variant allele frequency (max‐VAF) and the number of GAs decreased from 0.50% to 0.08% and from 2.9 to 1.7 after NACT. For patients with a partial response after NACT, the max‐VAF and the number of GAs declined significantly, but they increased for patients with progressive disease. Patients with detectable ctDNA at baseline, after NACT, or after surgery have a worse overall survival (OS) than patients with undetectable ctDNA. The estimated 3‐year OS was 73% for the post‐NACT ctDNA‐negative patients and 34% for ctDNA‐positive. Patients with perpetual negative ctDNA before and after NACT have the best prognosis. In conclusion, ctDNA was proposed as a potential biomarker to predict prognosis and monitor the NACT response for stage II–III GC patients.