Go Nakajima

Association of insulin-like growth factor binding protein-3 expression with melanoma progression

Previous studies from our laboratory have identified several endothelial cell–associated marker genes implicated in human melanoma metastasis via tumor vasculogenic mimicry. In this study, we used dual model systems composed of melanoma cell lines and clinical melanoma samples to validate the importance of insulin-like growth factor binding protein-3 (IGFBP-3) as a marker involved in disease progression. Gene expression analysis was done using a microarray approach for both primary and metastatic melanoma samples. The expression of IGFBP-3 was decreased using a small interfering RNA (siRNA) knockdown approach and quantified with real-time quantitative reverse transcription-PCR analysis. The expression of insulin-like growth factor binding protein 3 (IGFBP-3) was up-regulated by nearly 16-fold in WM266-4 compared with WM35 cells. A subsequent parallel analysis using freshly isolated primary and metastatic melanoma cell samples and melanoma tissue array confirmed the previous findings. The functional significance of IGFBP-3 in melanoma invasion was further investigated using a siRNA gene knockdown approach, with the expression of IGFBP-3 markedly reduced. Additionally, siRNA knockdown resulted in a significant reduction in cell motility, migration, and invasive capacity of WM266-4 cells in vitro. These results strongly suggest that IGFBP-3 expression may be a vital cell motility, migration, and proliferation factor necessary for melanoma metastasis and is an important biomarker in human melanoma. [Mol Cancer Ther 2006;5(12):3078–84]

Amphiregulin and Epiregulin mRNA expression in primary colorectal cancer and corresponding liver metastases

BackgroundAmphiregulin (AREG) and Epiregulin (EREG), ligands of EGFR, are reported to be predictive biomarkers of colorectal cancer patients treated with Cetuximab, an anti-EGFR antibody. The purpose of this study is to determine the correlation of AREG and EREG expression between primary colorectal cancer and corresponding liver metastases.MethodsOne hundred twenty colorectal cancer patients with liver metastases (100 with synchronous metastases, 20 with metachronous) were evaluated. No patients had ever received anti-EGFR antibody agents. AREG and EREG mRNA expression from both the primary tumor and liver metastases were measured using real-time RT-PCR. KRAS codon 12, 13 mutation status was analyzed by direct sequencing.ResultsModest, but significant, correlations were observed between primary tumor and corresponding liver metastases in both AREG mRNA expression (Rs = 0.54, p < 0.0001) and EREG mRNA expression (Rs = 0.58, p < 0.0001). AREG and EREG mRNA expression was strongly correlated in both the primary tumor (Rs = 0.81, p < 0.0001) and the liver metastases (Rs = 0.87, p < 0.0001). No significant survival difference was observed between low and high AREG or EREG patients when all 120 patients were analyzed. However, when divided by KRAS status, KRAS wild-type patients with low EREG mRNA levels in the primary site showed significantly better overall survival rates than those with high levels (p = 0.018). In multivariate analysis, low EREG expression was significantly associated with better overall survival (p = 0.006).ConclusionsAREG and EREG expression showed a modest correlation between primary tumor and liver metastases. As EREG mRNA expression was associated with decreased survival, it is appeared to be a useful prognostic marker in KRAS wild-type patients who never received anti-EGFR therapy.

Multi-level gene expression profiles affected by thymidylate synthase and 5-fluorouracil in colon cancer

BackgroundThymidylate synthase (TS) is a critical target for cancer chemotherapy and is one of the most extensively studied biomarkers for fluoropyrimidine-based chemotherapy. In addition to its critical role in enzyme catalysis, TS functions as an RNA binding protein to regulate the expression of its own mRNA translation and other cellular mRNAs, such as p53, at the translational level. In this study, a comprehensive gene expression analysis at the levels of both transcriptional and post-transcriptional regulation was conducted to identify response markers using human genome array with TS-depleted human colon cancer HCT-C18 (TS-) cells and HCT-C18 (TS+) cells stably transfected with the human TS cDNA expression plasmid.ResultsA total of 38 genes were found to be significantly affected by TS based on the expression profiles of steady state mRNA transcripts. However, based on the expression profiles of polysome associated mRNA transcripts, over 149 genes were affected by TS overexpression. This indicates that additional post-transcriptionally controlled genes can be captured with profiling polysome associated mRNA population. This unique approach provides a comprehensive overview of genes affected by TS. Additional novel post-transcriptionally regulated genes affected by 5-fluorouracil (5-FU) treatment were also discovered via similar approach.ConclusionTo our knowledge, this is the first time that a comprehensive gene expression profile regulated by TS and 5-FU was analyzed at the multiple steps of gene regulation. This study will provide candidate markers that can be potentially used for predicting therapeutic outcomes for fluoropyrimidine-based cancer chemotherapy.

Drug Monitoring During FOLFOX6 Therapy in a Rectal Cancer Patient on Chronic Hemodialysis

Long-term hemodialysis is considered to be a significant risk factor for cancer, but little is known about the use of oxaliplatin in patients on chronic hemodialysis. A 58-year-old man on chronic hemodialysis was treated for unresectable rectal cancer with synchronous hepatic metastasis by FOLFOX6 therapy with therapeutic drug monitoring. Plasma levels of total platinum, ultrafiltrate (free) platinum and 5-fluorouracil were monitored from the start of oxaliplatin administration to 120 h after the end of oxaliplatin infusion. Pharmacokinetic data of free platinum showed a bimodal pattern, decreased rapidly during the first dialysis and subsequently rose until 48 h after oxaliplatin infusion. The free platinum area under the curve was 15.7-18.9 microg h/ml when 40 mg/m(2) of oxaliplatin was administered, which was comparable to the area under the curve at 85 mg/m(2) in patient with normal renal function. The total platinum level reached a peak immediately before dialysis and gradually decreased. The 5-fluorouracil level decreased rapidly after the start of dialysis and remained constant during the continuous infusion of 5-fluorouracil. Tumor response was judged to be stable disease for >6 months, and no peripheral neuropathy or other toxicity was observed even after 11 courses. FOLFOX6 therapy with reduced dose of oxaliplatin had been safely performed for >6 months without any severe toxicity. The serum levels of free platinum showed bimodal pattern, and this second peak increased the area under the curve of free platinum. This pattern seems to be unique in patients on hemodialysis.

PTEN mRNA expression is less pronounced in left- than right-sided colon cancer: a retrospective observational study.

BackgroundSeveral recent studies have reported that patients with metastatic colorectal cancer (CRC) whose primary tumor is located in left side of the colon have more favorable responses to anti-epidermal growth factor receptor (EGFR) antibody therapy than those with right-sided tumors. However, the mechanism for this phenomenon is unknown.MethodsFifty-two cases of primary CRC with liver metastases were analyzed in this retrospective study. The mRNA levels of 19 signal transduction genes in both primary tumor and liver metastases were measured by real-time reverse transcription polymerase chain reaction. The purposes of this study were (1) to determine the correspondence between signal transduction gene expressions in primary tumors and corresponding liver metastases, and (2) to determine whether expression levels of these genes differ by primary tumor location.ResultsmRNA expression levels of 14 of 19 signal transduction genes, including PTEN, ERBB2, MET, HGF, AREG, and EREG, showed significant correlations between the primary tumor and corresponding liver metastases. When the mRNA levels of the primary tumors were compared by tumor location, only PTEN mRNA expression differed significantly between left and right-sided CRC (median PTEN expression: left 1.00 vs. right 1.68; p = 0.017). When rectal cancers were separated from left-sided colon cancers, PTEN mRNA levels increased progressively from rectum to right-sided colon (median; rectum 0.84, left colon 1.23, right colon 1.68, p = 0.013). PTEN mRNA expression in liver metastases also differed significantly according to primary tumor location (median; left 0.92 vs. right 1.27, p = 0.048). There was no difference in overall survival between patients with high versus low levels of PTEN mRNA (p = 0.59).ConclusionsOur data suggest that the PIK3/AKT/mTOR pathway is more active in left- than right-sided CRC, which provides a possible explanation for the fact that efficacy of anti-EGFR therapy differs by location of primary tumor.

Elevated levels of mRNAs encoding dihydropyrimidine dehydrogenase and thymidylate synthase are associated with improved survival of patients with hepatocellular carcinoma treated with S-1

Thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) are associated with the response of tumors to fluoropyrimidines. The aim of the present study was to investigate the association between the levels of TYMS and DPYD mRNAs and the efficacy of S-1 for treating patients with HCC. A total of 35 patients with HCC who received S-1 upon recurrence (S-1 group) and 20 patients who never received a fluoropyrimidine (control group) were studied. The levels of TYMS and DPYD mRNA in surgically resected specimens were determined using reverse transcription-polymerase chain reaction assays. Overall survival (OS) time of S-1 group patients with high levels of DPYD mRNA was significantly longer compared with that of patients with low levels (median 501 days vs. 225 days; P=0.016). Similarly, the OS time of those patients with high levels of TYMS mRNA was significantly longer compared with those with low levels (median 503 days vs. 239 days; P=0.0076). By contrast, there was no difference in OS time of the control group between patients with high and low levels of DPYD and TYMS mRNAs. The levels of TYMS and DPYD mRNAs may serve as predictive markers for patients with HCC who receive S-1 chemotherapy.

Abstract CT124: A phase II study of bevacizumab and irinotecan as second-line therapy for patients with metastatic colorectal cancer previously treated with fluoropyrimidine, oxaliplatin, and bevacizumab

Background: FOLFIRI plus bevacizumab (BV) is widely used as second-line chemotherapy for patients with metastatic colorectal cancer (mCRC) previously treated with FOLFOX/XELOX plus bevacizumab. However, there is insufficient data characterizing the effectiveness of administration of fluoropyrimidine beyond first disease progression. FOLFIRI requires using a CV catheter. Oral fluoropyrimidine may cause severe diarrhea when administered with irinotecan (CPT-11). CPT-11+BV may overcome those problems. Methods: Patients with mCRC previously treated with at least four courses of fluoropyrimidine, oxaliplatin, and bevacizumab were designated to receive 150 mg/m2 of CPT-11 and 10 mg/kg of BV, every 2 weeks as second-line therapy. The primary endpoint was progression-free survival (PFS), and secondary endpoints included response rate (RR), overall survival (OS), and adverse events. Results: Thirty patients from six institutes were enrolled from March 2011 to January 2014. Twenty-five (83.3%) patients were refractory to first-line therapy and eight patients (16.6%) were unable to tolerate first-line therapy. The median PFS was 5.7 months (95% CI; 4.2-7.3 months) and the RR was 6.7% (range, 0.8-22.1%. Grades 3-4 adverse events included leucopenia (36.7%), neutropenia (50%), thrombocytopenia (26.7%), anemia (30%), diarrhea (3.3%), anorexia (6.7%), and hypertension (3.3%). Relative dose intensities were 94.5% and 96.3% for CPT-11 and BV, respectively. Median OS was 11.8 months (6.3 months-not reached). Conclusions: Administration of CPT-11 plus BV to patients with mCRC achieved comparable efficacy with relatively lower toxicities compared with the results of previous studies using FOLFIRI plus BV as second-line therapy. The dose intensity of CPT-11 was judged satisfactory. Clinical trial information: UMIN000005228 Citation Format: Go Nakajima, Hidekazu Kuramochi, Masayuki Ando, Michio Itabashi, Kazuyuki Kawakami, Mie Hamano, Eiichi Hirai, Takayuki Iino, Hajime Yokomizo, Ryuji Okuyama, Tatsuo Araida, Kazuhiko Yoshimatsu, Shingo Kameoka, Kazuhiko Hayashi. A phase II study of bevacizumab and irinotecan as second-line therapy for patients with metastatic colorectal cancer previously treated with fluoropyrimidine, oxaliplatin, and bevacizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT124.

ERCC1 mRNA levels as a useful prognostic biomarker for extrahepatic cholangiocarcinoma with R0 resection.

239 Background: To date, no prognostic biomarker for biliary tract carcinoma has been identified. In previous studies of biliary tract carcinoma, no reliable data was found due to the varying composition of the cancer type (gallbladder, cholangiocarcinoma, and ampullary carcinoma), differences in tumor location, a mixture of curative and non-curative operations, and differences in operative methods. Methods: Fifty extrahepatic cholangiocarcinoma patients who underwent a pancreatoduodenectomy with R0 resection at the Tokyo Women’s Medical University Hospital were examined. All patients were pathologically diagnosed as having papillary or tubular adenocarcinoma. T-RNA was extracted from FFPE samples, and mRNA expression levels were measured by real-time RT-PCR. Results: In the preliminary analysis, 10 patients who have survived more than 5 years (LS group) and 10 patients who had a relapse within 2 years (SS group) were selected. EGFR, AREG, EREG, MMP-9, CDH-1, PARP1, and ERCC1 mRNA expression were examined...

Abstract 779: Osteopontin gene expression is associated with 5-fluorouracil drug resistance in colon cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Resistance to chemotherapeutic drugs complicates the treatment of cancer patients. The extracellular matrix protein osteopontin (OPN) plays multiple roles in the proliferation and metastasis of cancer cells. We therefore attempted to determine whether OPN expression correlated with drug resistance. Methods: OPN expression in the HCT 116 colon cancer cell line was inhibited by an OPN-short interfering RNA (siRNA). We determined the cytotoxic effect (IC50) of 5-fluorouracil (5FU) on these cells. Patients with recurrence or colorectal cancer or harbored residual tumor cells were treated with S-1-based chemotherapy. The levels of OPN mRNA expression in the tumors were determined and compared with the patients’ responses to chemotherapy. Results: The IC50 of 5FU for HCT 116 cells transfected with the OPN-siRNA was 32.9 μM. In contrast, the IC50 values for cells transfected with a negative control siRNA, mock-transfected cells, or untreated cells were 6.58 μM, 7.08 μM, and 6.76 μM, respectively (P<0.001). The level of OPN mRNA expression in the S-1 non-responder group was significantly lower than that of the responder group (P = 0.0387). Survival analysis revealed no significant difference between the responder and non-responder groups (P = 0.8737). Conclusions: The level of OPN expression plays a role in the resistance of colorectal tumor cells to 5FU. Citation Format: Go Nakajima, Hidekazu Kuramochi, Masakazu Yamamoto, Kazuhiko Hayashi. Osteopontin gene expression is associated with 5-fluorouracil drug resistance in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2014-779

Abstract 5038: Non-cording microRNA hsa-miR-199a expression in hepatocellular carcinoma correlates with patient survival

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL [Aim] MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at a post-transcriptional level. Previous study from our group identified forty nine miRNAs that are expressed differentially by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor tissue and non-tumor liver tissue using next-generation sequencer system. In this study, the expression of hsa-miR-199a and hsa-miR-199b, one of the extracted targets from our study, were investigated by real-time PCR using validation set HCC samples, and were examined the correlation between the miRNA expression and clinical data. [Patients and Methods] Forty-eight patients diagnosed as HCC had undergone surgical removal from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Womens Medical University, Japan. The formalin-fixed paraffin embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10μm sections, and the tumor samples were collected using Laser-captured microdissection and total RNA including miRNA fraction were isolated. After synthesized cDNA using miRNA specific primer, real-time PCR was performed using miRNA specific primer/probe. [Results] The expression level of hsa-miR-199a and hsa-miR-199b were down-regulated in tumor compared to non-tumor (P<0.0001, P=0.0002, respectively). The patients were divided into two groups, higher/lower expression of each miRNA, the group of the higher hsa-miR-199a expression was significantly longer overall survival time compared to the lower expression group (P=0.0303). However, hsa-miR-199b was not significant difference (P=0.3801). [Conclusion] The hsa-miR-199a may be potential prognostic factor for HCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5038. doi:1538-7445.AM2012-5038