Immunohistochemical analysis of the distribution of RANKL: a case of disseminated carcinomatosis of bone marrow as the first presentation of relapse in curatively resected colorectal cancer

Poorly differentiated adenocarcinoma of colorectal carcinoma (CRC) is a rare condition with poor prognosis. In this report, we describe a case of a 69-year-old man who underwent laparoscopic low anterior resection after being diagnosed with stage IIIB CRC. At 10 months post-operation, he developed fever and loss of appetite. Laboratory examination revealed > 120.0 μg/dL fibrin degradation products and > 60.0 μg/dL D-dimer. Bone marrow (BM) examination showed malignant epithelioid infiltrate with CK20 and CDX2 expression, leading to diagnosis of disseminated carcinomatosis of BM, which is rare in CRC and indicative of widespread disease throughout the body. Furthermore, immunohistochemistry revealed high expression of receptor activator of nuclear factor κB ligand (RANKL) in tumor cells, including budding cells of CRC and BM tissues. Thus, RANKL expression, which is known to indicate metastatic behavior of cancer cells, may play a critical role in promoting osteoclast formation, which has been associated with the pathogenesis of BM lesions.


Introduction
Colorectal carcinoma (CRC) is the third most common type of cancer in the world [1]. Based on histopathological differentiation, CRC tumors can be classified under the well/ moderate group, in which tumors are mainly located on the left side of the colorectum, i.e., the sigmoid colon, or Por1 (solid subtype) and Por2 (non-solid subtype) groups, in which tumors are located on the right side of the colorectum, i.e., in the ascending and transverse colon. Poorly differentiated adenocarcinoma of colorectal carcinoma (PDACRC) accounts only for 7% of all CRC cases, and its clinicopathological characteristics have not been studied in detail owing to its low incidence rate [2].
The prognosis and treatment of CRC largely depend on the stage of the disease, as classified by the tumor, node, and metastasis classification [3]. Morphologically, tumor budding (TB) is an important prognostic factor in patients with CRC. TB is defined as the presence of single tumor cells or small clusters of up to 5 cells in the tumor stroma, and it has been linked to epithelial-mesenchymal transition [4]. Disseminated carcinomatosis of the bone marrow (DCBM) is a condition in which diffusely invading bone marrow (BM) metastases are frequently accompanied by disseminated intravascular coagulation (DIC) [5]. DCBM of solid tumors is typically incurable and fatal. Although almost all types of malignancies can metastasize to BM, the most common non-hematological malignancies are those of the prostate, lung, breast, and stomach [5,6]. DCBM of CRC is relatively rare, with a frequency of 0-2% among solid tumors [5,6], and it is not necessarily accompanied by DIC [6].
Receptor activator of nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor family of cytokines. It is typically expressed on osteoblasts and bone stromal cells. RANKL and its receptor RANK, which is expressed on the surface of osteoclast precursors, are involved in osteoclast survival, differentiation, and activation [7]. Previous studies have reported that the RANKL/RANK pathway is related to tumor progression and migration (especially bone metastasis) in gastric cancer [8]. Additionally, RANK is expressed in solid tumors involved in CRC [9]. In this study, we reported a case of DCBM and TB due to PDACRC and described the distribution of RANKL based on immunohistochemistry (IHC) to aid the prognosis of DCBM during development of CRC and BM lesions.

Case report
A 69-year-old man with bloody stools was admitted to our hospital in June 20XX. He had previously visited another hospital for bloody stools, and colonoscopy revealed the presence of a type 2 rectal tumor in the upper rectum without stenosis (Suppl. Figure 1a). On admission to the hospital, laparoscopy revealed low anterior resection + D3 (Suppl. Figure 1b), while pathological staging showed 37 × 25 mm, Type 2, 28% circ, por > tub2 > sig, pT4a(SE), BD3, INFc, Ly1c(SM + SS), V1c (SS)(EVG), Pn1a, pPM0 (180 mm), pDM0 (46 mm), pRM1, pN2 (17/21), pStage IIIb < LN > #251-15/15 [ND3], #252-2/6, and #253-0/0 ( Fig. 1a, b) [10]. Based on these findings, the patient was diagnosed with stage IIIb CRC. The region with the highest TB was observed at 200 × magnification to analyze tumor growth and count the number of tumor buds; the tumor buds were classified as BD 3: high grade (Fig. 1a, b) [10] Thereafter, tegafur and uracil plus leucovorin were administered as postoperative chemotherapy in August 20XX, which was terminated in February 20XX + 1. Further, lower gastrointestinal endoscopy in March 20XX or chest and abdominal computed tomography after 6 months revealed no recurrence (Suppl. Figure 1c, d). However, after 10 months of operation, he developed fever and loss of appetite. Laboratory examinations showed the following: white blood cell count, 10,600/μL; platelet count, 13.2 × 10 4 /μL; lactate dehydrogenase, 1257 U/L; alkaline phosphatase, 2798 U/L; C-reactive protein, 18.90 mg/dL; fibrin degradation products, > 120.0 μg/dL; and D-dimer, > 60.0 μg/dL (Suppl. Table 1). A sagittal T1-weighted magnetic resonance image of the whole spine showed multiple low signal foci (Suppl. Figure 1e). Moreover, a metastatic bone tumor was detected using Ga-67 scintigraphy (Suppl. Figure 1f), and metastatic carcinoma, involving approximately 70% of BM space, was observed on BM biopsy (Fig. 2a). The malignant epithelioid infiltrate revealed expression of CK20 (Fig. 2b) and CDX2 (Fig. 2c) but not CK7 (Fig. 2d), which was consistent with the typical observation of metastatic CRC. Moreover, IHC for RANKL:RANKL Polyclonal Antibody (bs0747R, dilution × 200; Bios Inc, Boston, MA, USA) in a serial section of the same specimen showed positive staining in the cytoplasm and plasma membrane of PDACRC cells and TB in the primary lesion (Fig. 3a), capillary (Fig. 3b), and cytoplasm of BM (Fig. 3c). RANKL-positive staining was also observed in the plasma membrane of metastatic CRCs in the capillaries and poorly differentiated clusters (PDCs), similar to the BM lesion. Non-tumor rectal tissue was virtually devoid of RANKL, which was only observed in a few vessels in the reactive lymphocyte aggregates of lamina propria on the endothelial surface membrane (Fig. 3d). Prior to chemotherapy, blood examination revealed that the patient had DIC syndrome. The patient died 26 days after DIC onset.

Discussion
Solid tumors in patients may be the cause of the rare occurrence of DIC during clinical course [4,5]. A previous review revealed that DCBM is particularly rare in CRC, with only 11 reported cases [9]. The average age of the 11 patients was 61.0 years (men, 9 cases; women, 2 cases). The primary tumor was located in the rectum (n = 6, 54.5%); sigmoid colon (n = 2); or ascending (n = 1), transverse (n = 1), and right side (no detailed site was noted) of the colon (n = 1). The histological subtypes of adenocarcinoma were signet ring-like cell (n = 3), poorly differentiated (n = 3), mucinous (n = 2), moderately differentiated (n = 2), and well-differentiated (n = 1) adenocarcinoma. In the current case, the lesion indicated that PDACRC tended to grow and spread rapidly. Moreover, the presence of lymphovascular invasion  indicated a heightened probability of the cancer spreading outside the colon [11]. Based on histology, the cancer subtype of the lesion in the current case was determined as PDACRC. Previous studies have shown that the frequency of PDACRC is considerably low, accounting for only 7.1% of all CRCs [2]. Moreover, the frequency of metastasis to the lymph nodes, liver, lung, and peritoneum is higher for the Por2 group than that for the Por1 group, while it does not substantially differ between the Por1 and well/moderate groups [2].
Most cases of PDACRC are diagnosed in the advanced stage of the disease, and the number of cases with metastasis in the para-aortic lymph nodes, synchronous hepatic metastasis, and synchronous peritoneal metastasis is higher than that in cases with well-and moderately differentiated CRC. Apart from a published review [2], to the best of our knowledge, this is the first report on a case of DCBM as the first presentation of relapse in curatively resected CRC.
In the current case, histology showed tumor budding and PDCs in the primary tumor (Fig. 2a). In addition to PDCs in the primary CRC at both the invasive front and within the tumor stroma [12], PDCs of CRC were found in the liver. Moreover, vessels or venous capillaries showed positive vascular invasion (Fig. 2b). Lymphovascular invasion is considered a major predictor of poor outcome, and it is associated with more aggressive and/or more advanced CRC [13]. DCBM, as the first presentation of relapse in cases of curatively resected CRC, is rare [13][14][15][16].
In 2021, TB was finally considered in histological subtypes of CRC [10,17]. Using the International Tumor Budding Consensus Conference criteria, we found that peritumoral budding in histological subtypes affected survival, analogous to our thesis on intratumoral budding; however, no specific morphological criteria have been established [17]. Pan-cytokeratin IHC can be helpful to better visualize TB, although the final bud count should be performed using Hematoxylin and Eosin staining [4,17].
The role of budding in tumor progression and patient prognosis was not investigated for specific markers. Nevertheless, PDC-involved TB in the rectum and BM tissues significantly expressed RANKL, while TB was associated with the RANKL-expressed phenotype. It is important to immunohistochemically investigate RANKL expression in tissue specimens for at least suspected BM metastasis from clinical and laboratory data.
Seventy-five percent of CRC tissues have been reported to be RANK positive [9]. Furthermore, the role of RANKL in CRC has been related to the BM environment or osteoclast activity in human samples [18]. To the best of our knowledge, this is the first study demonstrating high RANKL expression in CRC cells, indicating extensive invasion of BM in CRC without any metastasis. This study suggests that CRC cells may directly act on osteoclast precursor cells via RANKL to promote osteoclast formation, indicating a potential mechanism underlying the development of bone lesions in DCBM. Considering the multifactorial roles of RANK-RANKL axis in the immune system, there has been considerable interest in the drug, denosumab, as an anti-RANKL inhibitor, to improve response to immune checkpoint inhibitors in the treatment of cancer [19].
Clinically, an increase in serum progesterone and RANKL levels is associated with an increase in breast cancer risk in postmenopausal women [20]. Considering the different roles of RANKL/RANK pathway in bone metabolism and immune system functions, therapy targeting this axis may not only control primary tumor development, such as in the case of breast cancer, but also reduce bone metastasis [20]. Future studies should investigate the mechanism of RANKL and RANKL/RANK pathway in CRC.
In summary, our case report suggests the potential role of RANKL, expressed in CRC cells, in development of BM lesions in DCBM with CRC. Our findings provide insights regarding the development of therapeutic strategies for CRC.