Microsatellite instability (MSI) is the most validated molecular marker associated with longer disease-free survival and lower expected benefit from chemotherapy in localized colon cancer (CC) patients [1], [2]. The management of localized MSI-CC therefore differs significantly from that of microsatellite stable (MSS) cases in which assessment of high-risk histopathological factors is particularly important. Lymph node status is the main predictor of recurrence and the prime indicator of adjuvant treatment in CC at later stages [3]. However, it has been demonstrated that a combination of other high-risk histopathological features can be used to identify a subset of stage II CC cases showing worse survival than those with lymph node metastasis, highlighting the importance of protocolized and meticulous histological analysis [4], [5].

High Tumor budding (TB) grade and high tumor-associated stroma (TS) content are two morphological features linked to mesenchymal differentiation and to worse prognosis in localized stages [6], [7], [8]. TS is composed of extracellular matrix and a mixture of non-neoplastic cells including cancer-associated fibroblasts (CAFs), immune cells and endothelial cells [9]. CAF activation causes the secretion of several growth factors and cytokines such as transforming growth factor beta (TGFβ), inducing pro-tumorigenic effects and resulting in a mesenchymal phenotype [10], [11]. The formation of buds (isolated or small clusters of up to four tumor cells that detach from the main tumor mass) is considered the histological hallmark of epithelial-to-mesenchymal transition (EMT) [12], [13], [14]. Moreover, TS can induce EMT in cancer cells via cell-to-cell contact, and budding cancer cells interact with CAFs and immune cells to promote immune suppression, invasion, and metastasis [15], [16]. TS proportion and TB grade have been found to be significantly correlated in localized CC series [17] and both high TS content and high TB grade are more frequently encountered in tumors belonging to the mesenchymal CMS4 consensus molecular subtype, the most aggressive molecular subtype of CC [17], [18], [19].

The composition of the tumor microenvironment has a significant impact on the progression of colorectal cancer (CC) at early stages, as previously validated by the Immunoscore—a risk stratification algorithm based on the evaluation of CD3 + and CD8 + T-cells in the tumor core and invasive margin [20]. Beyond the T-cell immune response, B-cell aggregates, also known as tertiary lymphoid structures (TLSs), play a critical role in lymphocyte recruitment and maintaining an immunoresponsive microenvironment. A high TLS content is a feature commonly associated with microsatellite instability (MSI) in CC [21]. However, TLS density has been demonstrated as an independent indicator of better prognosis and chemosensitivity in various solid tumors, including colorectal carcinoma, regardless of mismatch repair (MMR) status [21], [22], [23]

In this study, we evaluate tumor budding (TB), tumor-associated stroma (TS), and the composition of the tumor microenvironment in patients with localized MMR-proficient CC. Our goal is to develop a comprehensive combined score that predicts the risk of recurrence and investigates its association with the tumor microenvironment composition in this specific clinical context.