Hideki Ushiku

Immunohistochemical analysis of RTKs expression identified HER3 as a prognostic indicator of gastric cancer

Standard treatment in Japan for the 13th Japanese Gastric Cancer Association stage II/III advanced gastric cancer is postoperative adjuvant S‐1 administration after curative surgery. High expression of receptor type tyrosine kinases (RTKs) has repeatedly represented poor prognosis for cancers. However it has not been demonstrated whether RTKs have prognostic relevance for stage II/III gastric cancer with standard treatment. Tumor tissues were obtained from 167 stage II/III advanced gastric cancer patients who underwent curative surgery and received postoperative S‐1 chemotherapy from 2000 to 2010. Expression of the RTKs including EGFR, HER2, HER3, IGF‐1R, and EphA2 was analyzed using immunohistochemistry (IHC). Analysis using a multivariate proportional hazard model identified the most significant RTKs that represented independent prognostic relevance. When tumor HER3 expression was classified into IHC 1+/2+ (n = 98) and IHC 0 (n = 69), the cumulative 5‐year Relapse Free Survival (5y‐RFS) was 56.5 and 82.9%, respectively (P = 0.0034). Significant prognostic relevance was similarly confirmed for IGF‐1R (P = 0.014), and EGFR (P = 0.030), but not for EphA2 or HER2 expression. Intriguingly, HER3 expression was closely correlated with IGF‐1R (P < 0.0001, R = 0.41), and EphA2 (P < 0.0001, R = 0.34) expression. Multivariate proportional hazard model analysis identified HER3 (IHC 1+/2+) (HR; 1.53, 95% CI, 1.11–2.16, P = 0.0078) as the sole RTK that was a poor prognostic factor independent of stage. Of the 53 patients who recurred, 40 patients (75.5%) were HER3‐positive. Thus, of the RTKs studied, HER3 was the only RTK identified as an independent prognostic indicator of stage II/III advanced gastric cancer with standard treatment.

Reduced preoperative serum albumin and absence of peritoneal dissemination may be predictive factors for long-term survival with advanced gastric cancer with positive cytology test.

BACKGROUND Peritoneal lavage cytology cancer-positive (CY1) is a critical prognostic factor and is taken as representing stage IV in gastric cancer. There is no consensus treatment strategy for CY1-gastric cancer, and the detailed clinicopathological features remain obscure. PATIENTS AND METHODS Among 790 gastric cancer patients between 2005 and 2009, 52 cases of CY1 were identified (6.6%). A multivariate prognostic model was applied to the univariate prognostic factors to identify independent prognostic factors and factors associated with long-term survival in CY1-gastric cancer. RESULTS (1) Five-year overall survival (OS) was 17.6% in CY1-gastric cancer as compared with 93.9% in CYX and 77.7% in CY0 (77.7%), where tumors with pT2 or beyond were included in 11% of CYX, 73% of CY0, and 98% of CY1 cases. (2) On univariate analysis, factors associated with a negative prognosis were the presence of peritoneal dissemination (p = 0.029) and high preoperative serum albumin (p = 0.011) in CY1-gastric cancer. The multivariate Cox proportional hazards and logistic regression model using propensity score identified preoperative albumin as a critical independent prognostic indicator. (3) Long-term survivors were identified and, were often characterized by long-term postoperative adjuvant treatment. CONCLUSION Reduced preoperative serum albumin and absence of peritoneal dissemination may be predictive factors for long-term survival in patients with advanced gastric cancer with positive cytology test. Long-term postoperative adjuvant therapy might improve survival of patients with CY1.

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1) Gene in Primary Breast Cancer.

Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1) gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC) with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP) in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004). Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007). Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

Homeobox-Only Protein Expression Is a Critical Prognostic Indicator of Pancreatic Neuroendocrine Tumor and Is Regulated by Promoter DNA Hypermethylation.

Objectives We have identified homeobox-only protein (HOPX) as a tumor suppressor gene in various human cancer, and its expression was reduced by promoter DNA hypermethylation. Homeobox-only protein is strongly expressed on pancreatic islet cells; however, clinical relevance of HOPX expression has remained elusive in pancreatic neuroendocrine tumor (pNET). Methods We investigated 36 patients with pNET who undertook surgical resection between 1988 and 2012 for HOPX expression and DNA methylation to reveal its clinical significance. Results (1) Homeobox-only protein is strongly expressed on pancreatic islet cells by immunohistochemistry (IHC). Homeobox-only protein expression was recognized on pNET tumor cells for 1+ in 15, for 2+ in 16, and for 3+ in 5. (2) Homeobox-only protein IHC expression was significantly associated with prognosis (P = 0.03), and survival rate was 37.5%, 70.3%, and 100% in HOPX 1+, 2+, and 3+, respectively. (3) Promoter DNA methylation was quantitatively assessed, and HOPX hypermethylation is found in 6.3%, 11.8%, and 66.7% of G1/G2/G3 pNET, respectively (P = 0.02). (4) Multivariate Cox proportional hazards model identified HOPX IHC expression and HOPX promoter DNA hypermethylation as independent prognostic factors in pNET. Conclusions Homeobox-only protein expression is a critical prognostic indicator of pNET, and its regulation may be made through promoter DNA methylation.

The H19-PEG10/IGF2BP3 axis promotes gastric cancer progression in patients with high lymph node ratios

We previously demonstrated that the lymph node ratio (LNR) is a prognostic factor associated with EGFR expression, among first priority genes amplified or overexpressed in cancer. Here, we investigated the associations between high LNR and second, third, and fourth priority genes. We performed mRNA expression microarray analysis of tumor tissue from patients with stage III gastric cancer and high or low LNRs. Candidate high LNR-associated genes were further evaluated in 39 patients with stage III gastric cancer. The functional relevance of these genes was evaluated in gastric cancer cell lines. We focused on five genes: H19,PEG10, IGF2BP3, CD177, and PGA3. H19 and PEG10 were confirmed as high LNR-associated genes. H19, PEG10, and IGF2BP3 were found to promote each other’s expression. Knocking down H19 or PEG10 using RNAi decreased cell proliferation, invasion, anchorage-independent growth, and chemoresistance. These genes had a mutual relationship in MKN7 cells. H19 knockdown decreased expression of epithelial-mesenchymal transition-associated genes in MKN74 cells to suppress transformation. Thus, H19 promotes epithelial-mesenchymal transition in gastric cancer and is a potential therapeutic target.

The clinical significance of cysteine dioxygenase type 1 methylation in Barrett esophagus adenocarcinoma

Methylation of cysteine dioxygenase type 1 (CDO1) gene, a tumor suppressor gene, has been studied in various cancers; however, there is no information regarding Barrett esophagus cancer. In this study, the clinical significance of CDO1 methylation in Barrett esophagus adenocarcinoma (BEA) was clarified. CDO1 gene promoter methylation was analyzed for DNA from the patients specimens using quantitative methylation-specific polymerase chain reaction. Thirty-eight BEA patients who underwent resection were identified between 2000 and 2014. Hypermethylation of CDO1 gene was demonstrated to be frequently recognized even at early stage in BEA by quantitative methylation-specific polymerase chain reaction. In BEA, there is a robust prognostic difference between stage I and stage II/III/IV with regard to 5-year relapse-free survival (P = 0.0016) and 5-year overall survival (P = 0.0024), and the tumor size separated by 7 cm was also a prognostic factor. There was significant difference in CDO1 gene methylation according to the tumor size (P = 0.036). BEA patients with CDO1 gene methylation were shown marginally significantly poorer prognosis (P = 0.054) than otherwise patients. In conclusion, higher CDO1 gene methylation was seen in BEA at earlier stage than in squamous cell carcinoma, and it may account for aggressive phenotype of BEA.

Discrepancies between the K-ras mutational status of primary colorectal cancers and corresponding liver metastases are found in codon 13

K-ras mutation status has remained elusive in the metastatic liver tumors of colorectal cancer (CRC) in contrast to the primary CRC tumors. In this study, K-ras mutational status of the primary and corresponding liver metastatic tumors was investigated in the 43 CRC patients. Codons 12 and 13 of K-ras were directly sequenced, and a K-ras mutation was evident in 17 cases (39.5%). In 6 cases, the K-ras mutation was evident only in the liver metastasis, but not in the primary CRC, where the mutation was found in codon 13. This discrepancy between primary and metastatic lesions with regard to codon 13 of the K-ras gene may explain the clinical discrepancy of EGFR antibody therapy. In conclusion, the current data may lead to the development of personalized medicine for recurrent CRC, although further validation study is still required.

Epigenetic regulation of ZEB1-RAB25/ESRP1 axis plays a critical role in phenylbutyrate treatment-resistant breast cancer

Phenylbutyrate (PB) is a histone deacetylase antagonist that also exhibits antitumor activity. In this study, we used 7 breast cancer cell lines to identify biomarker candidates that predict PB sensitivity in breast cancer. Comprehensive gene expression profiles were compared using microarrays, and the importance of the identified genes to PB sensitivity was confirmed in gene transfection experiments. CRL and MDAMB453 cells were identified as PB-sensitive, while MDAMB231 cells were PB-resistant.RAB25 and ESRP1 were identified as key regulators of PB sensitivity, while ANKD1, ETS1, PTRF, IFI16 and KIAA1199 acted as PB resistance-related genes. Expression of these genes was dramatically altered by DNA demethylation treatments. RAB25 expression inhibited IFI16 and PTRF, while ESRP1 expression suppressed ANKRD1, ETS1, and KIAA1199. Both RAB25 and ESRP1 were suppressed by ZEB1, which was in turn regulated via epigenetic mechanisms. Thus, PB sensitivity is influenced by epigenetic expression alteration of ZEB1. The genes associated with PB sensitivity are downstream targets of ZEB1. Epigenetic regulation of ZEB1 may prove valuable as a critical biomarker for predicting resistance to breast cancer therapies.

Prognostic relevance of FGFR2 expression in stage II/III gastric cancer with curative resection and S‑1 chemotherapy

Curative gastrectomy and adjuvant chemotherapy using S-1 is a standard treatment for stage II/III gastric cancer in Japan. The purpose of the present study was to evaluate the prognostic relevance of fibroblast growth factor receptor (FGFR)2 expression in patients with stage II/III gastric cancer that underwent postoperative adjuvant chemotherapy with S-1. Formalin-fixed paraffin-embedded surgical specimens were retrospectively examined in 167 patients with stage II/III gastric cancer that underwent curative gastrectomy followed by adjuvant S1 chemotherapy. FGFR2 expression was measured using immunohistochemistry (IHC) staining. The IHC results for FGFR2 were as follows: Grade 1+, 32; grade 2+, 80; grade 3+, 55 patients. The FGFR2 expression level was not significantly associated with relapse-free or overall survival rates. However, in the diffuse type, the FGFR2 expression level tended to be negatively correlated with relapse-free survival. In particular, the proportion of patients who recurred >5 years following surgery was significantly larger in the FGFR2 grade 3+ group than in the grade 1+, 2+ group (4/22 vs. 1/35; P=0.047). The recurrent sites of long-term failure were mostly peritoneum among the diffuse type. To the best of our knowledge, the present study indicated for the first time that FGFR2 could predict long-term failure of adjuvant S-1 chemotherapy in curative advanced gastric cancer. There was no interaction between FGFR2 expression and patient survival outcomes in stage II/III gastric cancer. Patients with FGFR2 3+ in stage II/III gastric cancer should carefully be followed-up for >5 years after surgery.

Expression of KK-LC-1, a cancer/testis antigen, at non-tumour sites of the stomach carrying a tumour

Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a cancer/testis antigen (CTA) and predominant target for cancer immunotherapy. Our previous study indicated that KK-LC-1 was expressed in 82% of gastric cancers, and also in 79% of early stage of gastric cancers, with a correlation to Helicobacter pylori (H. pylori) infection. In addition, we found that KK-LC-1 was occasionally expressed at non-tumour sites of stomachs carrying tumours. Here, we investigated the characteristics of KK-LC-1 expression at non-tumour sites and the clinical utility of these phenomena. The gene expression of KK-LC-1 was detected at the non-tumour sites including pyloric glands. The most detectable corpus/gland subset had a KK-LC-1 expression rate of 77% in the pyloric gland of the lower corpus where H. pylori preferentially exists. KK-LC-1 expression rates were 67% or 32% with or without intestinal metaplasia, which also induced by H. pylori, respectively. Consequently, KK-LC-1 would be detected at the pre-cancerous condition of the stomach, and may be a useful marker to predict gastric cancer.