Matthew Chau Hsien Ng

Figure S1: Quality-control parameters and sensitivity analyses of CLDN18.2 microenvironmental and pathway changes Figure S2: Additional sensitivity survival analyses of the YCC cohort and CheckMate 649 trial Figure S3: Features of cellular neighbourhoods and correlation with progression-free survival Figure S4: Supplementary analyses to digital spatial profiling Figure S5: B/plasma cell enrichment scores across the tumor-stroma interface

Supplementary Tables 1-4 from Spatial Organization of Tumor-Infiltrating B Cells Informs Immune Checkpoint Inhibitor Response in Claudin 18.2–Expressing Gastric Cancer

Purpose: Claudin 18.2 (CLDN18.2), a tight junction protein normally expressed in the gastric epithelium and rendered accessible during malignant transformation, has emerged as a key therapeutic target. Here, we seek to characterize the immune microenvironment of CLDN18.2-expressing gastric cancer and identify immune features associated with CLDN18.2 expression status. Experimental Design: Tumor-immune features were profiled in 103 patients with HER2-neg/low advanced gastric cancer treated with first-line immune checkpoint inhibitor (ICI) using multiplex immunohistochemistry [7,423,483 cells across 2,315 regions of interest (ROI)]. The findings were validated in five published whole-transcriptome sequencing/microarray cohorts (1,521 samples), including the landmark CheckMate 649 phase III trial. Spatial immune architecture was assessed by cellular neighborhood analysis. Digital spatial profiling (DSP) was performed on 480 ROIs from a CLDN18.2-annotated tissue microarray (75 patients). Results: CLDN18.2high tumors showed reproducible enrichment of humoral pathways, with higher CD20+ B-cell density and enrichment of B-cell/plasma-cell signatures. CLDN18.2 status alone was not associated with response or survival on first-line ICI therapy; in CLDN18.2low tumors, B-cell enrichment was associated with ICI benefit, unlike CLDN18.2high tumors. Spatial analyses indicated that CLDN18.2high tumors preferentially harbored tumor-infiltrating B cells, rather than tertiary lymphoid structure/lymphoid aggregate–like neighborhoods, and this tumor-compartment localization was associated with diminished ICI benefit. DSP confirmed that CD20+ B-cell enrichment was most pronounced within tumor regions, alongside compartment-specific activation of humoral and extrafollicular response programs and interferon-associated and remodeling signatures. Conclusions: CLDN18.2high gastric cancer exhibits a humoral-enriched microenvironment with increased tumor-infiltrating B cells and spatial immune organization associated with reduced ICI benefit. Spatial and humoral-state biomarkers may inform ongoing CLDN18.2-directed combination trials.

Background: Perioperative FLOT (5-fluorouracil, oxaliplatin and docetaxel) is a standard of care for patients with locally advanced gastro-oesophageal adenocarcinoma (GEA) in Western guidelines, but its use is limited in Asian patients. We report outcomes from a single Asian centre of perioperative FLOT with concomitant granulocyte colony-stimulating factor (GCSF) prophylaxis. Methods: A retrospective analysis of all 56 stage II to III GEA patients treated with perioperative FLOT at the National Cancer Centre Singapore between June 2017 and February 2024 was performed. All patients were discussed at a multidisciplinary tumour board, underwent preoperative laparoscopic staging, and received prophylactic GCSF with perioperative FLOT. Surgery was performed across four partner institutions. The primary endpoints were the tolerability of FLOT and pathological complete response (pCR). A univariate analysis of factors associated with survival and adverse events was also performed. Results: Overall, 33 patients (58.9%) completed eight cycles of pre- and postoperative FLOT, and 92.9% underwent resection. The commonest grade 3 to 4 adverse events (AEs) were diarrhoea (10.7%) and neutropenia (5.6%). The 30- and 90-day postoperative mortality rates were 0% and 1.9%, respectively. In resected tumours, the pCR was 15.4%. The median DFS was 27.5 months, but the median OS was not reached. The values for 1-, 2-, and 3-year DFS were 74.6%, 61.0%, and 46.5%, respectively. The values for 1-, 2-, and 3-year OS were 85.0%, 67.4%, and 61.0%, respectively. In the univariate analysis of patients who underwent resection, an ECOG status of 0 was associated with better DFS, while ypN0, R0 resection, and pathological stages 0-II were associated with better DFS and OS. Patients ≥ 65 years benefited from FLOT similarly to those p = 0.940) and OS (HR 1.08; p = 0.869), with similar rates of grade 3 to 4 AEs. Patients with a higher housing index (HI) were less likely to experience ≥grade 3 AEs compared to those with a lower HI (OR 0.16, p = 0.029). Conclusions: This study presents a unique real-world Asian experience of perioperative FLOT with prophylactic GCSF use, with low rates of G3 to 4 neutropenia. The tolerability of FLOT was similar to that reported in Western populations. Furthermore, similar survival and rates of grade 3 to 4 AEs were observed in elderly patients. Patients of lower socioeconomic status were more likely to experience severe AEs, highlighting the need to proactively support vulnerable groups during treatment.

Supplementary Table S7. Progression-free survival according to central review (intention-to-treat population).

Supplementary Table S6. Response according to central review (modified intention-to-treat population).

Supplementary Table S5. Response and disease control according to local assessment (intention-to-treat population).

Supplementary Table S9. Summary of adverse events (safety population).

Supplementary Table S11. Treatment-emergent adverse events occurring at grade ≥3 in ≥2 patients (safety population).

Supplementary Table S12. Grade ≥3 treatment-emergent adverse events considered by the investigator to be related to the study drug (safety population).