Case Report Adenocarcinoma with Neuroendocrine Differentiation of the Colon Accompanying Osteoclast-Like Giant Cells

after surgery. Conclusion . This is the ﬁ rst report of colonic adenocarcinoma with neuroendocrine di ﬀ erentiation accompanying OGCs. Histopathologic factors of the poor prognosis in the present case included (a) the presence of more than 2% cells with neuroendocrine di ﬀ erentiation, (b) in ﬁ ltration of CD163/CD68-positive OGCs at the invasion front, (c) de ﬁ ciency of dMMR proteins, and (d) PD-L1 expression.


Introduction
Colorectal carcinoma is the third most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide, accounting for about 700,000 deaths/year [1,2]. About 30% of patients with colorectal carcinoma present with local lymph node spread but no distant metastasis at the time of diagnosis (Union Internationale Contre le Cancer, stage III). The five-year survival in stage III colorectal cancer ranges from 89% for stage IIIA to 36% for stage IIIC [3]. Neuroendocrine marker-positive cells are occasionally distributed in adenocarcinoma of the colon and rectum [4][5][6]. It has been clarified that neuroendocrine differentiation in colorectal cancer is associated with poorer grade of tumor differentiation, nodal and distant metastasis, and other unfavorable features, contributing to a worse clinical outcome [7].
Approximately 4% of metastatic colorectal cancers are associated with high microsatellite instability (MSI-H), indicating a deficient function of DNA mismatch repair (dMMR) proteins [8]. Expression of the immune checkpoint protein, programmed death-ligand 1 (PD-L1), on the tumor cells is well correlated with dysfunction of dMMR proteins [9,10].
Osteoclast-like giant cells (OGCs) of histiocytic lineage are morphologically and immunophenotypically distinguished from multinucleated stromal giant cells or neoplastic multinucleated cells [11]. OGCs may represent an immune cellular population involved in the tumor growth. Some colorectal carcinomas may accompany tumor-infiltrating lymphocytes (TILs) in the stroma, representing tumorspecific immune responses [12]. TILs have been proposed as a crucial prognostic indicator. The density of TILs has a strong power for predicting patients' survival, as comparable with the well-established tumor, node, and metastasis classification system [13].
The present report describes clinicopathological features of adenocarcinoma with neuroendocrine differentiation of the transverse colon, accompanying OGCs and TILs. We believe that the cooccurrence of neuroendocrine features and OGCs in colorectal cancer has not been reported so far.

Case Presentation
An 86-year-old Japanese woman complaining of lower abdominal pain was referred to Shimada Municipal Hospital, Shimada, Shizuoka, Japan. She had no particular familial or environmental history. Ultrasonography, abdominal computed tomography, and colonoscopy suggested double cancer of the transverse and sigmoid colon. Both tumors were simultaneously removed by right hemicolectomy and sigmoidectomy with lymphadenectomy. The sigmoid colon cancer was ulcer-forming, 4 × 3 cm sized, well-differentiated papillotubular adenocarcinoma of common type, reaching the subserosal layer. Lymphatic, venous, and perineural invasions were observed but without lymph node metastasis. The transverse colon cancer grossly showed a circumferential 7 × 6 cm mass with gray-to black-colored ulceration (Figure 1(a)). The cancer directly invaded the area of the right gastroepiploic artery and vein and spread to the regional lymph nodes. Histologically, the mildly pleomorphic tumor cells formed both tubuloglandular components and irregular-shaped medullary solid nests (Figure 1(b)). Lymphovascular invasion was positive.
The carcinoma cells with solid nest formation were focally labeled for synaptophysin (13% of the tumor cells) (Figure 1(e)). Chromogranin A and CD56 were negative. No synaptophysin-positive tumor cells were identified in the lymph node metastasis.
In order to confirm the neuroendocrine nature, the cancer cells with synaptophysin immunoreactivity were targeted at electron microscopic evaluation by digging a small piece from the formalin-fixed, paraffin-embedded block. With this approach, the fine morphologic preservation was fairly good even after paraffin embedding [14]. The tumor cells formed desmosomal junctions. Cellular process formation was not evident. Neuroendocrine-type cored round electron-dense granules were dispersed among fine cytoskeletal filaments in the cytoplasm. The size of the granules ranged from 140 to 380 nm with the mean 220 nm (Figure 1(f)).
The cancer cells were strongly immunoreactive for HLA-DR (Figure 1(h)). PD-L1 decorated with a monoclonal antibody clone 22C3 was expressed on 70-80% of the tumor cells (Figure 1(i)). In situ hybridization analysis for Epstein-Barr virus-encoded small nuclear RNA was negative. Ki-67 (clone: MIB-1) labeling index was more than 80%.
The histopathological features of the transverse colon tumor were consistent with poorly differentiated adenocarcinoma with neuroendocrine differentiation accompanying OGCs and TILs, pT4bN2M0 (p-stage IIIC). Ten months after surgery, the tumor relapsed in the abdominal lymph nodes and disseminated onto the peritoneal cavity. At the 14th postoperative month, the patient vomited vast amounts of blood due to cancer invasion to the gastric prepylorus, and the patient expired 16 months after surgery. No autopsy was performed.

Discussion
The transverse colon cancer in the present case showed medullary poorly differentiated growth with deficient dMMR status. The deficient MMR status in colon cancer revealed by immunohistochemistry is commonly associated with right-sided localization, female sex, high histological grade, and dense inflammatory infiltration in the tumor stroma [8]. These features were reproduced in the present case. Furthermore, the tumor in the present case was quite unique for association of neuroendocrine differentiation and OGC reaction.
In sporadic colorectal cancer, neuroendocrine cells are detected in 8-77.5% of cases, largely depending on the method employed for assessing neuroendocrine cell population [7]. Neuroendocrine features are also noted in half of hereditary nonpolyposis colorectal cancers with defective dMMR proteins [15]. Using a multivariate Cox regression model, the presence of more than 2% of cells with neuroendocrine differentiation indicates a poor prognosis in stages III and IV colorectal cancer [16].
TILs at the invasive front of colorectal carcinoma play an important role in modulating invasion and progression of the cancer cells [12]. The extent of stromal TILs was negatively correlated with morphologic features of tumor progression, including venous, lymphatic, and perineural dissemination and nodal and distant metastases [17]. It is known that the expression of HLA-DR on the cancer cells is intimately related with cancer immunity via CD4-positive T-lymphocytes [18]. Colorectal cancer expressing HLA-DR is often associated with TILs and shows a better prognosis [19].
In contrast, an elevated ratio of CD163/CD68 in macrophages at the tumor invasion front was closely associated 2 Case Reports in Pathology with an aggressive phenotype and poor prognosis in colorectal cancer [20]. To the best of our knowledge, this is the second case of colorectal carcinoma with OGCs: the first case was described by Eshun-Wilson in 1973 [21]. Neuroendocrine tumors rarely accompany OGCs [22]. Correlation between the occurrence of OGCs and PD-L1 expression was studied in pancreatic cancer: in undifferentiated pancreatic carcinoma with OGCs, PD-L1 immunoreactivity was detected in the neoplastic cells in 63% cases, and PD-L1 expression indicated a poor prognosis [23]. MSI-H, caused by loss of function of MLH1 protein by CpG island hypermethylation in the promoter of MLH1 gene [24], is seen in 10% to 13% of colorectal cancers. The MSI-H colorectal cancer is poorly differentiated with a medullary growth pattern and is often associated with TILs [9]. Immune checkpoint inhibitors, such as nivolumab and pembrolizumab targeting programmed death 1 (PD-1) on the inflammatory cells, are effective against metastatic dMMR-deficient and MSI-H colorectal cancer [9,10].

Conclusion
As far as we know, this is the first report of colonic adenocarcinoma with neuroendocrine differentiation accompanying OGCs. The patient died 16 months after surgery. The histopathologic factors indicating the poor prognosis in the 3 Case Reports in Pathology present case included (a) the presence of more than 2% cells with neuroendocrine differentiation, (b) infiltration of CD163/CD68-positive OGCs at the invasion front, (c) deficiency of dMMR proteins in the cancer cell nucleus, and (d) PD-L1 expression on the cancer cells.

Data Availability
All data generated or analyzed during this study are included in this published article.

Disclosure
This case was presented at the 276th meeting of Shizuoka Pathology Seminar at Shizuoka, Japan, 2019.

Conflicts of Interest
The authors declare that there is no conflict of interests regarding the publication of the present paper.

Authors' Contributions
Each author has sufficiently participated in the work to take public responsibility for appropriate portions of the content. MT performed histopathological diagnosis of the resected sample, analyzed the data, drafted the figure, and made a major contribution to writing the manuscript. TI performed clinical evaluations, surgical treatment, and clinical followup. YT analyzed histopathological features and brushed the manuscript up.