In metastatic colorectal cancer showing microsatellite instability-high (MSI-H)/mismatch repair deficiency (dMMR), the accumulation of DNA replication errors is thought to increase the tumor mutational burden and neoantigen load, thereby facilitating T cell-mediated antitumor immune responses. Accordingly, immune checkpoint inhibitors (ICIs) such as pembrolizumab, nivolumab, and ipilimumab represent important therapeutic options for MSI-H/dMMR metastatic colorectal cancer (1-4).

We encountered a case of rectal adenocarcinoma with liver metastases that was treated with ICIs but did not show a clinical response. Using pretreatment rectal biopsy specimens, we performed an immunohistochemical analysis focusing on tumor morphology, mismatch repair (MMR) protein expression, human leukocyte antigen (HLA)-related molecules, and infiltration of T lymphocytes and macrophages.

A 53-year-old Japanese woman presented to our hospital with epigastric pain, hematochezia, and buttock pain. She had no significant medical history or comorbidities. Blood tests showed mild elevation of hepatobiliary enzymes, and tumor markers were markedly elevated, with carcinoembryonic antigen (CEA) of 5,459 ng/ml and carbohydrate antigen 19-9 (CA19-9) of 24,065 U/ml. Colonoscopy revealed a flat elevated lesion involving approximately two-thirds of the circumference of the lower rectum (Figure 1A). Endoscopically, the lesion appeared to be predominantly adenomatous. However, contrast-enhanced computed tomography (CT) showed rectal wall thickening and perirectal fat stranding, indicating advanced cancer (Figure 1B). Multiple hypovascular masses were also observed in the liver (Figure 1C). Clinically, the patient was diagnosed with unresectable advanced rectal cancer with multiple liver metastases, and the clinical stage was determined as cT4N2M1, stage IV (Union for International Cancer Control TNM Classification of Malignant Tumours).

Histologically, most of the rectal biopsy tissue consisted of tubulovillous adenoma, with a coexisting invasive adenocarcinoma component. The adenocarcinoma component included a low-grade (gland-forming or well-to-moderately differentiated) component and a high-grade (poorly differentiated) adenocarcinoma component with unclear luminal formation (Figure 2 and Figure 3). The carcinoma component was confined to a small portion of the biopsy tissue and estimated to account for approximately 5% of the biopsy specimens.

Prominent inflammatory cell infiltration composed mainly of lymphocytes was observed in the tumor stroma, and increased lymphocyte infiltration was identified especially in the high-grade adenocarcinoma component. This finding raised the possibility of impaired MMR function, so immunohistochemistry for MMR proteins was performed. Loss of MLH1 and MSH2 expression was observed in the high-grade adenocarcinoma component, whereas MLH1 and MSH2 expression was retained in the adenomatous and low-grade adenocarcinoma components. MSH6 and PMS2 were positive in all components. Tissue-based comprehensive genomic profiling was not performed because of the low tumor cell content. Liver metastatic lesions were not biopsied.

Based on the demonstration of a dMMR component and the presence of unresectable metastatic rectal cancer, pembrolizumab was started as first-line therapy. Contrast-enhanced CT after two courses of pembrolizumab showed enlargement of liver metastases, peritoneal metastases, ascites, and lung metastases, and the disease was assessed as progressive disease. FOLFOX plus bevacizumab was started, and the disease was assessed as stable disease. At the time of this report, the patient was alive and continuing systemic therapy (Figure 1D); approximately nine months had elapsed since her initial presentation to our hospital, and approximately 8 months since the initiation of pembrolizumab as first-line therapy. This study was approved by the ethics committee of Izumi General Medical Center. Written informed consent was obtained from the patient.

Additional histopathological examination. To investigate possible factors underlying the insufficient efficacy of ICI therapy, additional immunohistochemical analyses were performed using pretreatment rectal biopsy specimens (Figure 2 and Figure 3). The following primary antibodies were used: CD8 (clone C8/144B; Nichirei, Tokyo, Japan), CD204 (clone SRA-E5; Cosmo Bio, Tokyo, Japan), HLA-A/B/C (clone EMR8-5; Hokuto, Sapporo, Japan), HLA-DR (clone TAL 1B5; Santa Cruz Biotechnology, Dallas, TX, USA), HLA-E (clone MEM-E/02; Abcam, Cambridge, UK), CD3 (clone SP7; Nichirei), and HLA-G (clone E8N9C; Cell Signaling Technology, Danvers, MA, USA).

HLA-A/B/C was expressed in tumor cells of all components. HLA-DR was also expressed in tumor cells of all tumor components; however, expression was heterogeneous. HLA-E was expressed in tumor cells in all tumor components. HLA-G was detected in a part of the adenomatous and low-grade adenocarcinoma components (Figure 3).

Regarding immune cell infiltration, numerous CD3-positive T lymphocytes infiltrated the stroma and were also observed within the tumor epithelium, especially in the high-grade adenocarcinoma component. CD8-positive T lymphocytes were present, whereas CD3-positive CD8-negative T lymphocytes, presumed to be CD4-positive T lymphocytes, were more predominant. CD204-positive macrophages mainly infiltrated the stroma (Figure 2).

These findings indicated component-dependent differences in MMR protein expression and HLA-DR expression, demonstrating intratumoral heterogeneity both morphologically and immunohistochemically.

In MSI-H/dMMR metastatic colorectal cancer, the efficacy of pembrolizumab and nivolumab plus ipilimumab has been demonstrated in the KEYNOTE-177 and CheckMate 8HW trials, respectively (3, 4). The distinctive feature of the present case was the absence of a clinical response to these ICIs. In the pretreatment rectal biopsy specimens, low-grade adenocarcinoma and high-grade adenocarcinoma components coexisted in a background of tubulovillous adenoma. In addition, MLH1 and MSH2 expression was lost in the high-grade adenocarcinoma component, but retained in the adenomatous and low-grade components. MMR protein expression is usually homogeneous in colorectal cancer, but rare cases with heterogeneous expression have been reported. Berrino et al. (5) reported MMR protein expression heterogeneity in nine (4.5%) of 200 colorectal cancers. In the present case, only the high-grade adenocarcinoma component showed dMMR, suggesting that the immunological nature of the entire tumor cannot be inferred simply from the MMR status of a single component.

Tumor antigen-presentation mechanisms also contribute to the responsiveness to ICI therapy. The HLA class I antigen-presentation machinery is important for presenting intracellular antigens from the tumor to CD8-positive T cells (6, 7). HLA-DR is an MHC class II molecule that may be involved in antitumor immunity mediated by CD4-positive T cells (8, 9). In the present case, HLA class I expression was retained in all components. HLA-DR expression was restricted to a part of tumor cells in adenoma and low-grade components, whereas almost all tumor cells in high-grade adenocarcinoma components expressed HLA-DR. Because CD4-positive T cells were prominent among the infiltrating T cells in the present case, the HLA-DR-positive high-grade adenocarcinoma component was suggested to be targeted by CD4-positive T cells.

HLA-E is a nonclassical HLA class I molecule that can suppress the function of NK and CD8-positive T cells through the CD94/NKG2A pathway (10-12). HLA-G is another nonclassical HLA molecule that can exert immunosuppressive effects, and its association with prognosis in colorectal cancer has been reported (13-15). Because HLA-G expression was partial in the present case, it should not be overinterpreted; however, the presence of a small subset of cells expressing immunosuppressive molecules is noteworthy as one aspect of intratumoral heterogeneity.

Infiltration of CD204-positive macrophages is also important when considering immune escape. Tumor-associated macrophages can contribute to tumor progression through the production of immunosuppressive factors, angiogenesis, tumor cell activation, and the promotion of invasion and metastasis (16). Involvement of CD204-positive macrophages in the malignant transformation of colorectal adenoma has also been reported (17). In the present case, T-lymphocyte infiltration coexisted with infiltration of CD204-positive macrophages, suggesting a tumor immune microenvironment in which both antitumor immunity and immunosuppression were present. Lee et al. (18) reported that MSI in rectal cancer was associated with changes in systemic inflammatory markers during neoadjuvant chemoradiotherapy, supporting the concept that MSI status is linked to inflammatory or immunologic responses. Furthermore, Liu et al. (19) demonstrated that immune cell subpopulations in the colon cancer microenvironment, including dendritic cell and macrophage subsets, were associated with prognosis, highlighting the clinical relevance of immune-cell composition in colorectal cancer.

In the present case, the histological type and immunological characteristics of the liver metastatic lesions were important for understanding ICI responsiveness. In contrast to the present nonresponsive case, Hosokawa et al. (20) reported an MSI-H colorectal cancer case with liver metastasis that showed a pathological complete response to pembrolizumab, with strong HLA class I and HLA-DR expression and increased CD8-positive T-cell infiltration in the primary lesion. In colorectal cancer, the metastatic pattern is influenced by histological subtype, and liver metastasis has been reported to be more frequent in low-grade adenocarcinoma than in high-grade adenocarcinoma (21). Because liver metastatic lesions were not biopsied, no definitive conclusion can be drawn; however, it is possible that the liver metastases were predominantly composed of the low-grade adenocarcinoma component with retained MMR protein expression, which may have been one factor underlying the poor response to ICIs.

Study limitations. The histopathological evaluation was based only on pretreatment rectal biopsy specimens, and the entire rectal tumor could not be evaluated. Tissue-based comprehensive genomic profiling was not performed because of the low tumor cell content. In addition, histological evaluation of liver metastatic lesions and post-ICI treatment specimens was not performed.

Although this case was initially diagnosed as dMMR rectal adenocarcinoma, no clinical response to ICI therapy was observed. MMR protein loss was restricted to the high-grade adenocarcinoma component, whereas MMR protein expression was retained in the low-grade adenocarcinoma component. Because liver metastases of colorectal cancer have been reported to arise more frequently from low-grade adenocarcinoma than from high-grade adenocarcinoma, it is possible that the metastatic lesions in the present case were mainly derived from the low-grade component. If so, the MMR status of the low-grade component may have been more relevant for treatment selection than that of the minor high-grade dMMR component. Therefore, in colorectal cancer with marked intratumoral heterogeneity, MMR assessment should carefully consider the tumor component that is most likely to be responsible for metastatic disease.

The Authors declare no conflicts of interest in relation to this study.

Kohei Okura and Yoshihiro Komohara: Performed histological and immunohistochemical analyses, interpreted pathological findings, and drafted the manuscript. Haruka Takenaka, Hiroshi Fujita and Norihisa Hanada: Managed the patient, collected clinical data, and contributed to interpretation of the clinical course and treatment response. Yoshihiro Komohara: Supervised the study and critically reviewed the manuscript. All Authors read and approved the final manuscript.

The Authors thank K.I. Stainer Inc. (Kumamoto, Japan) for the technical assistance.

This research received no external funding.

No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.