Scalable AI-driven tumor-stroma ratio quantification for prognostic stratification in stage II-III colorectal cancer

Introduction: The tumor-stroma ratio (TSR), defined as the proportion of stromal content within the primary tumor, has been proposed as a prognostic histopathologic marker for colorectal cancer (CRC). Higher stromal content is often linked to poorer prognosis in stage II-III disease. However, TSR assessment remains manual and is prone to interobserver variability, while existing AI algorithms - often trained and tested on single-institution datasets - lack independent validation for generalizability. In this study, we developed an AI-driven TSR quantification model trained on the public Cancer Genome Atlas (TCGA) pathologist-annotated regions and validated it using predictions on (i) TCGA whole-slide images (WSI) and (ii) two large independent CRC cohorts (NHS/HPFS tissue microarray (TMA)). Methods: We trained a SegFormer semantic segmentation model on 99,871 image tiles derived from pathologist-annotated regions (J.V.) from 469 TCGA H&E images spanning stage I-IV CRC. The model was trained to segment three major tissue categories: tumor, stroma, and others. For validation, we applied the model to 375 stage II-III CRC samples within the TCGA cohort (a subset of the 469 training images, evaluated beyond annotated regions) and to 537 NHS/HPFS TMA images. TSR was defined as the area of stroma divided by the sum of stromal and tumor areas. Patients were stratified into TSR-high and TSR-low groups using the cohort median as the cut-off. Prognostic associations were evaluated using Cox proportional hazard model. Results: Computed TSR values range from 18.17% to 100% with a median of 88.56% for the NHS/HPFS cohort; and range from 11.72% to 99.87% with a median of 70.30% for the TCGA cohort. In the NHS/HPFS cohort, AI-derived TSR-high group showed significantly worse outcomes for overall survival (HR = 1.38; 95% CI = 1.06-1.81; P = 0.017) and CRC-specific survival (HR = 1.49; 95% CI = 1.03-2.15; P = 0.035). However, in the TCGA cohort, TSR stratification showed no significant prognostic value for either pathologist-annotated or WSI-AI-derived TSR for overall survival (HR = 1.12, 95% CI = 0.69-1.82; P = 0.647, and HR = 0.97; 95% CI = 0.59-1.60; P = 0.909, respectively) and CRC-specific survival (HR = 0.79, 95% CI = 0.40-1.58; P = 0.513, and HR = 0.88; 95% CI = 0.43-1.77; P = 0.723, respectively). Discussion: While AI-derived TSR showed prognostic significance in the NHS/HPFS cohort, the lack of significance in TCGA suggests limited generalizability across cohorts. Notably, this is, to our knowledge, the first study to evaluate TSR using the well-established TCGA CRC dataset. These findings highlight the need to further dissect the stromal composition - through molecular staining or AI-driven profiling of immune populations, stromal subtypes, and tumor-immune spatial interactions - to capture the biological mechanisms underlying TSR and improve its robustness as a prognostic biomarker.