Clinical Significance of Multi-Cancer Genome Profiling: Data from a Single Hospital in Japan.

BACKGROUND/AIM: Multi-cancer genome profiling (multi-CGP) testing intends to predict the therapeutic efficacy of anticancer medication treatments for eligible patients as part of "precision cancer care." The number of cases in which a new treatment was applied based on multi-CGP testing has been reported to be between 10% and 20% for all patients in Japan. This study aimed to determine the significance of multi-CGP testing in Japan by analyzing clinical data from multi-CGP testing in various solid cancers at our Hospital. PATIENTS AND METHODS: A total of 230 patients examined by one of three tests for multi-CGP including NCC Oncopanel, FoundationOne CDx, and FoundationOne Liquid were retrospectively enrolled. Adequate treatment for each patient was discussed at the expert panel meeting according to the results from the genome profiling tests. RESULTS: The most frequent cancer types enrolled in this study were pancreas cancer, bowel cancer, and biliary cancer. Of the 230 cases, 106 (46%) were druggable cases, and 21 (9.1%) were administered medication. Partial response (PR) effect was found in 7 (33.3%) of the 21 cases, of which 3 were biliary cancer and 3 had a BRCA2 mutation. Of all the 21 cases, 7 (33.3%) had the maximum treatment benefit of PR. Three cases of biliary tumors were found in the 7 PR cases within the 21 cases. CONCLUSION: Of 230 patients, 21 were administered medication following multi-CGP testing data, especially frequent in biliary tumor patients. Multi-CGP testing might be particularly beneficial to patients with biliary tumors in Japan.

Establishment of a gastric cancer cell line with high microsatellite instability, OCUM-13, derived from Borrmann type-2 primary tumor.

Gastric cancer (GC) with microsatellite instability (MSI) has been reported to be sensitive to immunotherapy, however some of GC cases with MSI remain resistant to immunotherapy. Cancer cell lines showing MSI might be useful for the analysis of mechanisms of immunotherapy, while only a few GC cell lines with MSI are available so far. In this study, we established a unique GC cell line with MSI, OCUM-13, from a primary GC with abundant tumor-infiltrating lymphocytes. MSI assay indicated that OCUM-13 cells as well as the primary tumor showed a band shift in more than 3 of 5 microsatellite loci, suggesting that OCUM-13 did have high MSI. The subcutaneous inoculation of OCUM-13 cells into mice performed tumor formation. Insulin-like growth factor 1 receptor inhibitor decreased the growth of OCUM-13 cells. The newly established cell line with MSI, OCUM-13, might be useful for the analysis of cancer therapy for GC with MSI.

Hyaluronan driven by epithelial aPKC deficiency remodels the microenvironment and creates a vulnerability in mesenchymal colorectal cancer.

Mesenchymal colorectal cancer (mCRC) is microsatellite stable (MSS), highly desmoplastic, with CD8+ T cells excluded to the stromal periphery, resistant to immunotherapy, and driven by low levels of the atypical protein kinase Cs (aPKCs) in the intestinal epithelium. We show here that a salient feature of these tumors is the accumulation of hyaluronan (HA) which, along with reduced aPKC levels, predicts poor survival. HA promotes epithelial heterogeneity and the emergence of a tumor fetal metaplastic cell (TFMC) population endowed with invasive cancer features through a network of interactions with activated fibroblasts. TFMCs are sensitive to HA deposition, and their metaplastic markers have prognostic value. We demonstrate that in vivo HA degradation with a clinical dose of hyaluronidase impairs mCRC tumorigenesis and liver metastasis and enables immune checkpoint blockade therapy by promoting the recruitment of B and CD8+ T cells, including a proportion with resident memory features, and by blocking immunosuppression.

Lipocalin-2 negatively regulates epithelial-mesenchymal transition through matrix metalloprotease-2 downregulation in gastric cancer.

BACKGROUND: Although the role of Lipocalin-2 (LCN2) in cancer development has been focused on recent studies, the molecular mechanisms and clinical relevance of LCN2 in gastric cancer (GC) still remain unclear. METHODS: Transcriptome analysis of GC samples from public human data was performed according to Lauren's classification and molecular classification. In vitro, Western blotting, RT-PCR, wound healing assay and invasion assay were performed to reveal the function and mechanisms of LCN2 in cell proliferation, migration and invasion using LCN2 knockdown cells. Gene set enrichment analysis (GSEA) of GC samples from public human data was analyzed according to LCN2 expression. The clinical significance of LCN2 expression was investigated in GC patients from public data and our hospital. RESULTS: LCN2 was downregulated in diffuse-type GC, as well as in Epithelial-Mesenchymal Transition (EMT) type GC. LCN2 downregulation significantly promoted proliferation, invasion and migration of GC cells. The molecular mechanisms of LCN2 downregulation contribute to Matrix Metalloproteinases-2 (MMP2) stimulation which enhances EMT signaling in GC cells. GSEA revealed that LCN2 downregulation in human samples was involved in EMT signaling. Low LCN2 protein and mRNA levels were significantly associated with poor prognosis in patients with GC. LCN2 mRNA level was an independent prognostic factor for overall survival in GC patients. CONCLUSIONS: LCN2 has a critical role in EMT signaling via MMP2 activity during GC progression. Thus, LCN2 might be a promising therapeutic target to revert EMT signaling in GC patients with poor outcomes.

CXCR2 signaling might have a tumor-suppressive role in patients with cholangiocarcinoma.

BACKGROUND: We reported that chemokine C-X-C motif receptor 2 (CXCR2) signaling appears to play an important role in the pathogenic signaling of gastric cancer (GC), and although CXCR2 may have a role in other solid cancers, the significance of CXCR2 in cholangiocarcinoma (CCA) has not been evaluated. Herein, we determined the clinicopathologic significance of CXCL1-CXCR2 signaling in CCA. MATERIALS AND METHODS: Two human CCA cell lines, OCUG-1 and HuCCT1, were used. CXCR2 expression was examined by western blotting. We investigated the effects of CXCL1 on the proliferation (by MTT assay) and migration activity (by a wound-healing assay) of each cell line. Our immunohistochemical study of the cases of 178 CCA patients examined the expression levels of CXCR2 and CXCL1, and we analyzed the relationship between these expression levels and the patients' clinicopathologic features. RESULTS: CXCR2 was expressed on both CCA cell lines. CXCL1 significantly inhibited both the proliferative activity and migratory activity of both cell lines. CXCL1 and CXCR2 were immunohistochemically expressed in 73% and 18% of the CCA cases, respectively. The CXCL1-positive group was significantly associated with negative lymph node metastasis (p = 0.043). The CXCR2-positive group showed significantly better survival (p = 0.042, Kaplan-Meier). A multivariate logistic regression analysis revealed that CXCR2 expression (p = 0.031) and lymph node metastasis (p = 0.004) were significantly correlated with the CCA patients' overall survival. CONCLUSION: CXCR2 signaling might exert a tumor-suppressive effect on CCA cells. CXCR2 might be a useful independent prognostic marker for CCA patients after surgical resection.

Dipeptidyl Peptidase-4 from Cancer-associated Fibroblasts Stimulates the Proliferation of Scirrhous-type Gastric Cancer Cells.

BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs. MATERIALS AND METHODS: OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array. RESULTS: The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM. CONCLUSION: DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.

Gastric cancer stem cells survive in stress environments via their autophagy system.

Cancer stem cells (CSCs) play an important role in the progression of carcinoma and have a high potential for survival in stress environments. However, the mechanisms of survival potential of CSCs have been unclear. The aim of this study was to clarify the significance of autophagy systems of CSCs under stress environments. Four gastric cancer cell line were used. Side population (SP) cells were sorted from the parent cells, as CSC rich cells. The expression of stem cell markers was examined by RT-PCR. The viability of cancer cells under starvation and hypoxia was evaluated. The expression level of the autophagy molecule LC3B-II was examined by western blot. The numbers of autophagosomes and autolysosomes were counted by electron microscope. SP cells of OCUM-12 showed a higher expression of stem cell markers and higher viability in starvation and hypoxia. Western blot and electron microscope examinations indicated that the autophagy was more induced in SP cells than in parent cells. The autophagy inhibitor significantly decreased the viability under the stress environments. These findings suggested that Cancer stem cells of gastric cancer might maintain their viability via the autophagy system. Autophagy inhibitors might be a promising therapeutic agent for gastric cancer.

EMMPRIN in extracellular vesicles from peritoneal mesothelial cells stimulates the invasion activity of diffuse-type gastric cancer cells.

Peritoneal metastasis of gastric cancer (GC) results in extremely poor prognoses. The peritoneal cavity is covered by a monolayer of peritoneal mesothelial cells (PMCs). Interactions between GC cells and PMCs might play a pivotal role in peritoneal metastasis. Extracellular vesicles (EVs) correlate with intercellular communication. Although intercellular communication between cancer cells and PMCs might be associated with the peritoneal metastatic process, the role of EVs from PMCs remains unclear. We investigated the effects of EVs from PMCs on GC cells. Three GC cell lines (OCUM-12, NUGC-3, and MKN74) and four mesothelial cell lines were used. The effects of EVs derived from the PMCs on the invasion and migration of GC cells were evaluated by Matrigel invasion assay. Factors contained in the PMC EVs were analyzed; extra-cellular matrix metalloproteinase inducer (EMMPRIN) was detected in the EVs. The effects of an EMMPRIN inhibitor on the invasion-stimulating activity of EVs were examined. The EMMPRIN expressions of 110 GCs were evaluated by immunohistochemistry. PMC EVs significantly promoted the invasion of diffuse-type GC cells, i.e., OCUM-12 and NUGC-3 cells. EMMPRIN in the EVs stimulated the invasion of OCUM-12 and NUGC-3 cells. The invasion-stimulating activity of PMC EVs was inhibited by the EMMPRIN inhibitor. A high EMMPRIN expression in PMCs was significantly associated with worse cancer-specific survival and peritoneal-recurrence-free survival. EMMPRIN in EVs from PMCs might stimulate the malignant progression of diffuse-type GC. EMMPRIN might be a useful prognostic marker of recurrence in GC patients.

The clinicopathologic significance of Tks5 expression of peritoneal mesothelial cells in gastric cancer patients.

BACKGROUND: Gastric cancer (GC) patients frequently develop peritoneal metastasis. Recently, it has been reported that peritoneal mesothelial cells (PMCs) activated by GC cells acquire a migratory capacity and promote GC cell invasion. The invasiveness of PMCs reportedly depends on the activity of Tks5, an adaptor protein required for invadopodia formation. However, the relationship between clinicopathologic features and Tks5 expression in PMCs has been poorly documented. In this study, we evaluated the clinicopathologic significance of the Tks5 expression of PMCs in GC patients. MATERIALS AND METHODS: A total of 110 GC patients who underwent gastrectomy were enrolled in this study. Tks5 expressions in PMCs from the greater omentum, lesser omentum and retroperitoneum were evaluated by immunohistochemistry. We analyzed the correlation between Tks5 expressions in PMCs and the patients' clinicopathologic features. RESULTS: Tks5 expression was found in 71 (64.5%) of the 110 patients, while 39 (35.5%) were Tks5-negative. Tks5 positivity was significantly (p = 0.038) associated with a greater tumor depth (i.e., T3/4 compared with T1/T2). Peritoneal recurrence was found in 12 of 98 cases within 3 years of surgery. The 3-year peritoneal recurrence-free survival (PRFS) rate in Tks5-positive cases was significantly poorer than that in Tks5-negative cases (80.1% vs 97.4%, p = 0.024). Multivariate analysis revealed that Tks5 positivity and lymph node metastasis were independent factors for PRFS. CONCLUSION: Tks5 is frequently expressed in PMCs in advanced-stage gastric cancer. Tks5 might be a useful predictor for peritoneal recurrence in GC patients.

Clinicopathologic significance of TROP2 and phospho-TROP2 in gastric cancer.

Trophoblast cell-surface antigen 2 (TROP2) is a transmembrane glycoprotein expressed in epithelial cells. Increased TROP2 expression has been reported to be associated with malignant progression in most carcinomas; however, TROP2 has a tumor-suppressive function in certain types of cancer. Since the function of TROP2 is controversial, the present study subsequently aimed to clarify the clinicopathologic significance of TROP2 and pTROP2 expression in human gastric cancer (GC). The cases of 704 patients with GC who underwent gastrectomy were retrospectively analyzed. The expression levels of TROP2 and pTROP2 in each tumor were evaluated by immunohistochemistry. The association between the clinicopathologic features of patients with GC and the levels of TROP2 and pTROP2 in their tumors was analyzed. Increased TROP2 and pTROP2 expression was identified in 330 (46.9%) and 306 (43.5%) of the 704 patients with GC, respectively. Increased TROP2 expression was associated with the histological intestinal type, high tumor invasion depth (T3/T4), lymph node metastasis, lymphatic invasion and venous invasion. By contrast, increased pTROP2 expression was associated with intestinal type, low tumor invasion depth (T1/2), no lymph node metastasis and no lymphatic invasion. Increased TROP2 expression was associated with poorer overall survival (OS) (P<0.01; log rank test), whereas increased pTROP2 expression was significantly associated with improved OS (P<0.01; log rank test). In conclusion, increased expression levels of TROP2, but not pTROP2, may be associated with the metastatic ability of GC, resulting in poor prognosis of patients with GC.