O-(N-acetyl-alpha-D-galactosaminyl)-L-threonine

2.1. Tn Antigen Expression in CRC

We conducted immunohistochemistry for Tn antigen using surgically resected whole tissue specimens, including 20 adenomas and 507 primary CRCs, in which 460 adjacent non-tumor mucosa were also available for evaluation. Immunoreactivity for Tn antigen staining in the cytoplasm and cell membrane were respectively evaluated and then combined to obtain the Tn score, as described in . In tumor adjacent mucosa, non-neoplastic epithelial cells often displayed weak to moderate granular staining predominantly in the supranuclear cytoplasm, but membranous staining was undetectable ( and A–F). The staining patterns of Tn antigen in adenomas were similar to those of non-tumor mucosa. We observed 35.2% of non-tumor mucosa, and 45.0% of adenomas were positive for Tn antigen expression (G). By contrast, in CRC tissues, the cytoplasmic and membranous expression of Tn antigen was observed in cancer cells with considerably varying degrees of staining intensity and its extent, occasionally accompanied by staining in the extracellular mucin deposit ( and A–F). Of the 507 CRC tissues, 189 (37.3%) were defined as Tn-negative (Tn score 0–3) and 318 (62.7%) were defined as Tn-positive (Tn score 4–24), and the latter was further classified into 277 Tn-weak (Tn score 4–15) (54.6%) and 41 Tn-strong (Tn score 16–24) (8.1%) tumors (G). In Tn-strong tumors, intense cytoplasmic and membranous staining was diffusely distributed throughout the tumor ( and A–C).

3. Discussion

The expression of Tn antigen in CRC has been studied since more than three decades ago. Earlier studies analyzed a relatively small number of specimens (less than 30 CRC tissues), and revealed that 72–82% of CRCs were found to be positive for Tn antigen expression [,,]. Oshikiri et al. reported that 68 of 146 CRCs (46.6%) were Tn positive []. More recently, Jiang et al. examined 186 CRC specimens, and 161 (86.6%) were determined to be positive for Tn antigen []. Although the Tn antigen positive rate varied among studies likely due to the detection approaches, including different monoclonal antibodies or lectins used, the expression pattern and the localization of Tn staining in CRC was highly consistent. In the present study, in a large cohort of CRC (n = 507), using a monoclonal antibody MLS128 for immunohistochemistry, we again confirmed that the majority (62.7%) of CRCs were positive for Tn antigen. Nevertheless, unlike previous studies described above, we herein identified a small subset of CRCs (8.1%) displaying strikingly intense and diffuse distributions of Tn antigen expression. Although none of the previous studies addressed the correlation between Tn antigen and MMR status, we found that the Tn-strong tumors were highly enriched within dMMR CRCs, where 16 of 40 (40.0%) of dMMR tumors were determined to be Tn-strong, which was in clear contrast to only 5.4% of pMMR tumors showing Tn-strong. More noteworthy is the fact that the unique subset of Tn-strong dMMR CRCs specifically lacked common immunological characteristics of dMMR CRC, such as dense CD8+ T cell infiltrate in the TME and PD-L1 expression on tumor cells. High levels of TILs have been considered not only an indicator of host immune response to the tumor, but also a favorable prognostic marker in CRC, independent of MMR status []. Particularly, in early stage patients with dMMR CRC, the pronounced anti-tumor immune response characterized by an increased density of intratumoral T cells seems to explain their generally good prognosis. A recent report demonstrated that a high number of TILs, along with high TMB, was correlated with clinical responses and survival benefit in patients with dMMR CRCs who were treated with ICIs []. By contrast, unresponsiveness to ICI treatment in pMMR CRCs is likely associated with low TMB and the lack of immune infiltration due to low tumor neoantigens. Our finding therefore suggests that Tn antigen overexpression is one of the underlying mechanisms of T cell exclusion in dMMR CRC. Although this study lacks direct assessment of the predictive role of Tn-strong for the efficacy of ICIs in patients with dMMR CRC, the Tn-strong dMMR subset may represent an immune cold subgroup of patients who do not respond well to the current immunotherapeutic strategies. Future studies would be required to address the prognostic as well as predictive roles of Tn-strong expression, particularly in patients with metastatic dMMR CRC who are treated with ICIs.

4.1. Patient Samples

We enrolled 20 patients with colon adenoma and 507 patients with stage 0 to IV primary CRC who underwent surgical resection at Fukushima Medical University Hospital between 2002 and 2013 without preoperative chemotherapy or radiotherapy. Their available formalin-fixed paraffin-embedded (FFPE) whole tissue sections were used for immunohistochemistry. Clinical information was retrospectively obtained from medical records. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Fukushima Medical University.