Shinichiro Tahara

Immunohistochemical analysis of the novel marginal zone B-cell marker IRTA1 in malignant lymphoma

Marginal zone lymphoma (MZL) is a low-grade B-cell lymphoma derived from marginal zone B cells. Because of a lack of specific immunohistochemical markers, MZL is mainly diagnosed based on the cytological appearance and growth pattern of the tumor. Marginal zone B cells were recently shown to selectively express immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), but the antibody used in that study is not commercially available. We therefore investigated the IRTA1 expression in nonneoplastic lymphoid tissues and 261 malignant lymphomas, examining the ability of a commercially available antibody to accurately diagnose MZL. Among 37 MZLs, 23 of 25 extranodal MZLs of mucosa-associated lymphoid tissue (MALT lymphomas), 3 of 6 splenic MZLs and 3 of 6 nodal MZLs were positive for IRTA1. Among the 98 diffuse large B-cell lymphomas, 33 were positive for IRTA1, including 1 of 38 follicular lymphomas, and all precursor B-lymphoblastic (2/2) and T-lymphoblastic (7/7) leukemia/lymphomas. Other mature B-cell and T-cell lymphomas, and Hodgkin lymphoma were negative for IRTA1. In MALT lymphoma, positive cells were detected mainly in intraepithelial and subepithelial marginal zone B cells. In 1 case of grade 3 follicular lymphoma, IRTA1 was also expressed in the area of large cell transformation. When tumors were classified as germinal center B cell-like (GCB) or non-GCB using the algorithm of Hans, positive expression of IRTA1 was correlated significantly with non-GCB diffuse large B-cell lymphomas (P < .05). These results demonstrated the ability of the commercially available IRTA1 antibody to distinguish MALT lymphoma from other low-grade B-cell lymphomas.

CUBIC pathology: three-dimensional imaging for pathological diagnosis

The examination of hematoxylin and eosin (H&E)-stained tissues on glass slides by conventional light microscopy is the foundation for histopathological diagnosis. However, this conventional method has some limitations in x-y axes due to its relatively narrow range of observation area and in z-axis due to its two-dimensionality. In this study, we applied a CUBIC pipeline, which is the most powerful tissue-clearing and three-dimensional (3D)-imaging technique, to clinical pathology. CUBIC was applicable to 3D imaging of both normal and abnormal patient-derived, human lung and lymph node tissues. Notably, the combination of deparaffinization and CUBIC enabled 3D imaging of specimens derived from paraffin-embedded tissue blocks, allowing quantitative evaluation of nuclear and structural atypia of an archival malignant lymphoma tissue. Furthermore, to examine whether CUBIC can be applied to practical use in pathological diagnosis, we performed a histopathological screening of a lymph node metastasis based on CUBIC, which successfully improved the sensitivity in detecting minor metastatic carcinoma nodules in lymph nodes. Collectively, our results indicate that CUBIC significantly contributes to retrospective and prospective clinicopathological diagnosis, which might lead to the establishment of a novel field of medical science based on 3D histopathology.

S100A4 accelerates the proliferation and invasion of endometrioid carcinoma and is associated with the “MELF” pattern

Endometrioid carcinoma (EC) is one of the most common malignancies of the female genital system. Although the behavior of EC ranges from an excellent prognosis to aggressive disease with a poor outcome, the factors that determine its diversity have not been determined. Here, we show that S100A4, a calcium‐binding protein of the EF‐hand type, is correlated with the proliferation and invasion ability of EC. We demonstrated previously that EC cells with high aldehyde dehydrogenase (ALDH) activity were more tumorigenic than ALDH‐lo cells. Screening by shotgun proteomics demonstrated that the expression level of S100A4 in ALDH‐hi EC cells was significantly higher than that in ALDH‐lo cells. S100A4‐knockout cells generated by the CRISPR/Cas9 system showed reduced proliferation and invasion. These cells showed impaired AKT phosphorylation and matrix metalloproteinase‐2 activation, accounting for their impaired proliferation and invasion, respectively. Furthermore, in clinical EC samples, elevated expression of S100A4 was highly related to myometrial and lymphatic invasion in well to moderately differentiated EC. Notably, strong and diffuse expression of S100A4 was observed in tumor tissues with a microcystic, elongated and fragmented (“MELF”) pattern, which is associated with a highly invasive EC phenotype. Collectively, our results demonstrate not only that high expression of S100A4 contributes to an aggressive phenotype of EC, but also that its elevated expression is closely related to the MELF histopathological pattern.

Argininosuccinate Synthase 1-Deficiency Enhances the Cell Sensitivity to Arginine through Decreased DEPTOR Expression in Endometrial Cancer

Argininosuccinate synthetase 1 (ASS1) is a rate-limiting enzyme in arginine biosynthesis. Although ASS1 expression levels are often reduced in several tumors and low ASS1 expression can be a poor prognostic factor, the underlying mechanism has not been elucidated. In this study, we reveal a novel association between ASS1 and migration/invasion of endometrial tumors via regulation of mechanistic target of rapamycin complex (mTORC) 1 signaling. ASS1-knockout cells showed enhanced migration and invasion in response to arginine following arginine starvation. In ASS1-knockout cells, DEPTOR, an inhibitor of mTORC1 signal, was downregulated and mTORC1 signaling was more activated in response to arginine. ASS1 epigenetically enhanced DEPTOR expression by altering the histone methylation. Consistent with these findings, tumor cells at the invasive front of endometrioid carcinoma cases showed lower ASS1 and DEPTOR expression. Our findings suggest that ASS1 levels in each tumor cell are associated with invasion capability in response to arginine within the tumor microenvironment through mTORC1 signal regulation.

Requirement of CXCL12-CXCR7 signaling for CD20(-) CD138(-) double-negative population in lymphoplasmacytic lymphoma.

Cancer cells with tumorigenic potential are limited to a small subpopulation known as cancer-initiating cells (CICs). Recently we investigated a candidate of CICs of lymphoplasmacytic lymphoma (LPL), which is positive for both B-cell marker CD20 and plasma-cell marker CD138. We reported that the subpopulation of CD20− CD138− phenotype, in which both markers were negative was a candidate of CICs in LPL using LPL cell line, MWCL-1. CICs are known to be plastic under stressed condition, in which non-CICs are changed to CICs. In the present study, we investigated the plasticity of CICs of LPL, and found that hypoxia induced the conversion of CD20+ CD138− to CD20− CD138− phenotype. We then searched for markers preferentially expressed in CD20− CD138− subpopulation, and the chemokine receptor CXCR7 was isolated. When cultured with CXCL12, a ligand of CXCR7, the number of CD20− CD138− cells increased in a time- and dose-dependent manner. In addition, hypoxia enhanced the expression level of CXCL12 in MWCL-1. In clinical samples of LPL, a few tumor cells expressed CXCR7, in which CD20 expression was not detected. These results indicated that hypoxia and CXCL12-CXCR7 axis appeared to be advantageous microenvironments to CD20− CD138− cells.

Effect of glutamine on lymphoplasmacytic lymphoma, especially on the viewpoint of the differentiation into vulnerable subpopulation

Glutamine (Gln) is important not only for cell proliferation but also for differentiation. Although Gln is essential for plasmacytic differentiation of lymphocytes, no study has been done on the effect of Gln on differentiation of tumor cells, such as lymphoma. Here we examined the effect of Gln on plasmacytic differentiation of lymphoplasmacytic lymphoma (LPL) with its cell lines, MWCL-1 and RPCI-WM1. Gln promoted plasmacytic differentiation of LPL, and p38 MAPK signaling pathway mediated such differentiation. We previously reported that the subpopulation with plasmacytic differentiation was vulnerable to apoptosis in LPL. Although it is difficult to lead these findings to the radical therapy, they might help the treatment of LPL, in which stimulation of p38 MAPK by Gln induced differentiation of LPL into vulnerable subpopulation.

Usefulness of cytological analysis in the diagnosis of pancreatic undifferentiated rhabdoid carcinoma: Letter to the Editor

To the Editor: Pancreatic undifferentiated rhabdoid carcinoma is rare, and has not been described in the World Health Organization (WHO) Classification of Tumours. Agaimy et al. reported 14 cases of pancreatic undifferentiated rhabdoid carcinoma, and classified them into two subtypes; loss of SMARCB1/INI1 and alteration of KRAS. The morphology of tumor cells is different between the two subtypes; the former is monomorphic, whereas the latter is pleomorphic. To date, no cytological feature on pancreatic undifferentiated rhabdoid carcinoma has been reported. Here, we report a case of pancreatic undifferentiated rhabdoid carcinoma with loss of SMARCB1/INI1, in which metastases were found in tongue, muscle, liver and lung. Cytological specimens were obtained from the metastatic lesion, in which monomorphic tumor cells with eccentric nuclei and prominent nucleoli were discohesively arranged and occasionally had cytoplasmic inclusion bodies. This is the first report on the cytological features of pancreatic undifferentiated rhabdoid carcinoma. Since the molecular target therapy against loss of SMARCB1/INI1 is under development, the discrimination of pancreatic undifferentiated rhabdoid carcinoma subtypes would be necessary in future. Cytological analysis is less invasive, and useful for such discrimination even when the general condition of patients is poor. The patient was a 67-year-old woman, who was introduced to our hospital to examine a mass in the pancreas. She had a history of colon cancer resection 6 years prior and the follow-up computed tomography (CT) revealed a mass in the pancreas. The contrast-enhanced CT scan in our hospital showed a hypovascular mass measuring 1.9 cm in the body of the pancreas with surrounding swollen lymph nodes and a ring-enhanced lesion measuring 1.9 cm in the liver (Fig. S1). An endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) was carried out for the pancreatic mass and a biopsy was taken for the liver mass. She was diagnosed with pancreatic cancer and received chemotherapy. In the next half year, tumors occurred in multiple organs, such as lung, bone, submucosa of the tongue, and muscle. Fine needle aspiration cytology (FNAC) was conducted for the submucosal nodule of the tongue to confirm metastasis. After that, surgical resection was performed for the metastatic tumor of the tongue. Surgical resection was also performed for the intramuscular metastatic tumor in the right thigh in order to control the hemorrhage. Her general condition gradually deteriorated and she died 2 weeks after the resection of the intramuscular tumor. The tumor cells of the pancreatic mass (EUS-FNA), liver mass (biopsy), submucosal nodule of the tongue (surgical resection) and intramuscular mass (surgical resection) were morphologically similar. Monomorphic tumor cells with enlarged nuclei and acidophilic cytoplasm spread diffusely. Tumor cells were discohesive and loosely arranged without prominent desmoplastic reaction. No glandular formation was found. Tumor cells occasionally had eosinophilic cytoplasmic inclusion bodies (Fig. 1a, b). Immunohistochemically, tumor cells were diffusely positive for pan-cytokeratin (AE1/AE3), negative for leukocyte common antigen (LCA), S-100, and desmin. Moreover, cytoplasmic inclusion bodies were positive for AE1/AE3 and vimentin (Fig. 1c). Tumor cells were negative for SMARCB1/INI1 (Fig. 1d). These characteristics were different from the past colon cancer, the histological finding of which was moderately differentiated tubular adenocarcinoma. By the clinical course and image findings, we concluded that the tumor originated in the pancreas and diagnosed it as pancreatic undifferentiated rhabdoid carcinoma. We performed mutational analysis of KRAS exons 2 (codons 12, 13, 19) and 3 (codon 61), and found no mutation. In the FNAC specimen, monomorphic tumor cells with eccentric nuclei and prominent nucleoli were discohesively arranged. Tumor cells occasionally had cytoplasmic inclusion bodies. The characteristics of tumor cells in cytological specimen were similar to those in histological specimen (Fig. 1e, f). In this report, we describe the case of pancreatic undifferentiated rhabdoid carcinoma of an elderly woman. Although pancreatic undifferentiated rhabdoid carcinoma has not been described in the WHO Classification of Tumours in pancreatic cancer due to its rarity, several cases have recently been reported. Agaimy et al. reported 14 cases of pancreatic undifferentiated rhabdoid carcinoma, and classified them into two subtypes; loss of SMARCB1/ INI1 and alteration of KRAS. Four of 14 cases belonged to the former, in which tumor cells showed monomorphic anaplastic histology. The remaining 10 cases belonged to the latter, in which tumor cells showed pleomorphic giant cell histology. In fact, monomorphic anaplastic histology was characteristic in our case, in which SMARCB1/INI1 expression was lost and KRAS wasn’t mutated. We describe the histogenesis of pancreatic undifferentiated rhabdoid carcinoma. KRAS is activated by point mutations in >90% of ordinary ductal adenocarcinoma of the pancreas. This finding suggests that in two subtypes of pancreatic undifferentiated rhabdoid carcinoma, the KRAS

Adenylosuccinate lyase enhances aggressiveness of endometrial cancer by increasing killer cell lectin-like receptor C3 expression by fumarate

Adenylosuccinate lyase (ADSL) is an enzyme that plays important roles in de novo purine synthesis. Although ADSL was reported to be upregulated in various malignancies, such as colorectal, breast, and prostate cancer, as well as gliomas, the mechanism by which elevated ADSL expression contributes to cancer has not been elucidated. We previously performed a shotgun proteomics analysis to characterize specific proteins associated with the properties of the aldehyde dehydrogenase (ALDH)-high cell population, which was reported to be involved in tumorigenic potential, and showed that ADSL expression is upregulated in the ALDH-high population of endometrial cancer. Here, we showed that ADSL is involved in endometrial cancer aggressiveness by regulating expression of killer cell lectin-like receptor C3 (KLRC3), which is a receptor expressed on natural killer cells. Immunohistochemical analysis indicated that ADSL expression increased as endometrioid carcinoma specimens became more poorly differentiated and higher degree of primary tumor progression. Knockdown of ADSL in endometrial cancer cells decreased cell proliferation, migration, and invasive capability, and caused the cells to adopt a more rounded shape. DNA microarray analysis and quantitative real-time PCR showed that KLRC3 expression was decreased in ADSL knockdown cells. Knockdown of KLRC3 in endometrial cancer cells resulted in the same phenotype as knockdown of ADSL. Moreover, fumarate, which could be produced by ADSL and was recently shown to be an oncometabolite, recovered KLRC3 expression in ADSL knockdown cells, suggesting that fumarate produced by ADSL could regulate KLRC3 expression. Our findings indicate that ADSL enhances cell proliferation, migration, and invasive capability through regulation of KLRC3 expression by fumarate.

Origin of cancer-associated fibroblasts and tumor-associated macrophages in humans after sex-mismatched bone marrow transplantation

Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in tumor stroma play a key role in disease progression. Recent studies using mice models suggest that CAFs are partly derived from bone marrow and TAMs primarily originate from bone marrow-derived inflammatory monocytes. However, the origin of these cells in humans remains unclear. Hence, we investigated their human origin, using specimens from human secondary tumors that developed after sex-mismatched bone marrow transplantation, by modified immunofluorescent in situ hybridization analysis and triple immunostaining. We observed that most of the α-smooth muscle actin (αSMA)-positive CAFs in the mammary gland, liver, and oral mucosa specimens obtained 3–19 years after bone marrow transplantation are recipient-derived cells. In contrast, the majority of the peritumoral αSMA-negative fibroblast-like cells are actually bone marrow-derived HLA-DR-positive myeloid cells, such as macrophages and dendritic cells. Furthermore, almost all CD163-positive TAMs and macrophages present in the non-tumor areas are derived from bone marrow.Masako Kurashige et al. investigate the origin of cancer-associated fibroblasts and tumor-associated macrophages in humans. They find that these are derived from bone marrow following sex-mismatched bone marrow transplants.