Kiyoshi Eshima

Heterogeneity of KRAS status may explain the subset of discordant KRAS status between primary and metastatic colorectal cancer.

BACKGROUND: KRAS status is a useful predictive marker for anti-epidermal growth factor receptor antibody therapy. OBJECTIVE: This study aimed to examine the concordance rate of KRAS mutation status between corresponding primary and metastatic colorectal cancer lesions, and also among multiple metastatic tumors. Furthermore, we examined the heterogeneity of KRAS mutations with respect to discordant KRAS status between primary and metastatic tumors. DESIGN AND SETTINGS: This study was retrospective in design. PATIENTS: Forty-three patients with primary tumors and 113 metastatic tumors were studied. MAIN OUTCOME MEASURES: The KRAS mutational status was determined by the peptide nucleic acid clamp real-time polymerase chain reaction TaqMan assay. We also performed sequencing analysis to validate the KRAS mutational status. When KRAS status differed between primary and metastatic tumors, we examined the heterogeneity of KRAS status within individual primary tumors by microdissecting multiple samples in each patient. RESULTS: The frequency of KRAS mutations in primary tumors was 34.9%. A high concordance rate of KRAS (88.4–91.7%) mutations was observed between primary and metastatic tumors. All 5 cases (11.6%) with discordant KRAS status had heterogeneous KRAS status in primary tumors. However, in 10 concordant cases all microdissected areas showed an identical KRAS mutational status within each patient. The KRAS mutational statuses in all multiple liver and/or lung metastatic tumors were the same as those of the primary tumor. LIMITATIONS: We could not validate KRAS status in microdissected samples by the direct sequence method that was used in the present study, because the quantity of DNA was not sufficient to perform direct sequencing. CONCLUSION: KRAS status in a primary site may be used for selecting patients who would benefit from anti-epidermal growth factor receptor therapy. However, KRAS status can be heterogeneous within a primary tumor, and thus different parts of such tumors should be examined for KRAS status to correctly predict the KRAS status in metastatic lesions.

The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: a comparison between direct sequencing and real-time PCR.

PURPOSE Kirsten rat sarcoma (KRAS) gene mutations occur early in the progression of colorectal adenoma to carcinoma. The mutation status of the KRAS gene determines the benefits of molecular targeting drugs in patients with advanced colorectal cancer, although many methods are available to detect such mutations. The purpose of this study was to evaluate the influence of assay sensitivity on the detection frequency of mutated genes. METHODS Colorectal tumors in 224 colorectal cancer patients were characterized for KRAS mutations using PCR amplification following by direct sequencing as well as a peptide nucleic acid (PNA)-clamp real-time PCR-based assay. RESULTS KRAS mutations were observed in 32.1% (72/224) patients by direct sequencing, and 43.3% (97/224) by PNA-clamp PCR. The chi-square test revealed that the difference in the frequency of KRAS mutations determined by direct sequencing and PNA-clamped PCR (threshold for 1% detection) was statistically significant (p<0.015). CONCLUSIONS Our data suggest that assay method sensitivity clearly influences the detection frequency of mutated genes. As more sensitive assays detect more mutated genes in clinical samples, this must be taken into consideration when determining KRAS gene status in clinical practice.

Differential gene expression signatures between colorectal cancers with and without KRAS mutations: Crosstalk between the KRAS pathway and other signalling pathways

PURPOSE KRAS mutation is an important predictive marker in determining resistance to anti-Epidermal Growth Factor Receptor (EGFR) antibody therapies. In order to clarify whether not only KRAS related signalling pathways but also other signalling pathways are altered in patients with colorectal cancers (CRCs) with KRAS mutations, we examined the differences in the gene expression signatures between CRCs with and without KRAS mutation. PATIENTS AND METHODS One-hundred and thirteen patients who underwent a surgical resection of a primary CRC were examined. KRAS mutational status was determined using the Peptide Nucleic Acid (PNA)-clamp real-time polymerase chain reaction (PCR) TaqMan assay. Gene expression profiles were compared between CRCs with and without KRAS mutation using the Human Genome GeneChip array U133. RESULTS Among 113 CRCs, KRAS mutations were present in 35 tumours (31%). We identified 30 genes (probes) that were differentially expressed between CRCs with and without KRAS mutation (False Discovery Rate (FDR), p<0.01), by which we were able to predict the KRAS status with an accuracy of 90.3%. Thirty discriminating genes included TC21, paired-like homeodomain 1 (PITX1), Sprouty-2, dickkopf homologue 4 (DKK-4), SET and MYND domain containing 3 (SMYD3), mitogen-activated protein kinase kinase kinase 14 (MAP3K14) and c-mer Proto-oncogene tyrosine kinase (MerTK). These genes were related to not only KRAS related signalling pathway but also to other signalling pathways, such as the Wnt-signalling pathway, the NF-kappa B activation pathway and the TGF-beta signalling pathway. CONCLUSIONS KRAS mutant CRCs exhibited a distinct gene expression signature different from wild-type KRAS CRCs. Using human CRC samples, we were able to show that there is crosstalk between the KRAS-mediated pathway and other signalling pathways. These results are necessary to be taken into account in establishing chemotherapeutic strategies for patients with anti-EGFR-refractory KRAS mutant CRCs.

Prediction of liver metastasis after colorectal cancer using reverse transcription-polymerase chain reaction analysis of 10 genes

PURPOSE Liver metastasis is one of the major types of recurrence after surgery for colorectal cancer. Traditional methods of predicting liver metastasis are limited in their accuracy, suggesting the need to develop new predictors. We developed a 10-gene signature that is closely associated with the development of liver metastasis after colorectal cancer. PATIENTS AND METHODS We examined a total of 189 frozen specimens of primary colorectal cancers using both microarray and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Initially, we studied gene expression in colorectal cancer tissue from 160 randomly selected patients who had undergone surgical resection of colorectal cancer and evaluated the association between the level of gene expression and the occurrence of liver metastasis. We developed a gene-expression model for the prediction of liver metastasis based on the RT-PCR findings. We then used specimens from 29 other patients for validation. RESULTS The expression of 14 genes was correlated with liver metastasis according to both microarray and RT-PCR analysis. We constructed an accurate predictive model based on the results for 10 of these genes, which included epiregulin (EREG), amphiregulin (AREG), cyclooxygenase 2 (COX-2) and lymphocyte-specific protein tyrosine kinase (LCK). The 10-gene signature was an independent predictor of liver metastasis. The model was validated in the independent set of 29 patients. The predictive accuracy of the model in a test set of patients was 86.2%. CONCLUSION The 10-gene signature identified in this study is closely associated with the occurrence of liver metastasis in colorectal cancer patients.

Gene expression of mesenchyme forkhead 1 (FOXC2) significantly correlates with the degree of lymph node metastasis in colorectal cancer.

In stage III colorectal cancer, patients with N1 stage tumors show poorer outcome than patients with N2 stage tumors. Our objective was to identify genes that are predictive for the presence of lymph node metastasis, and to characterize the aggressiveness of lymph node metastases. Gene expression profiles of colorectal cancer were determined by microarray in training (n = 116) and test (n = 25) sets of patients. We identified 40 discriminating probes in patients with and without lymph node metastases. Using these probes, we could predict the presence of lymph node metastasis with an accuracy of 87.1% (training set) and 76.0% (test set). Among discriminating probes, FOXC2 expression was significantly correlated with the degree of lymph node metastasis. FOXC2 was expressed significantly and disparately in patients with N1 and N2 stage tumors as analyzed by real-time reverse transcriptase-polymerase chain reaction. FOXC2 appears to be involved in determining the aggressiveness of lymph node metastasis in colorectal cancer.

Gene expression signature and response to the use of leucovorin, fluorouracil and oxaliplatin in colorectal cancer patients.

PurposeFOLFOX (a combination of leucovorin, fluorouracil and oxaliplatin) has achieved substantial success in the treatment of colorectal cancer (CRC) patients. However, about half of all patients show resistance to this regimen and some develop adverse symptoms such as neurotoxicity. In order to select patients who would benefit most from this therapy, we aimed to build a predictor for the response to FOLFOX using microarray gene expression profiles of primary CRC samples.Patients and methodsForty patients who underwent surgery for primary lesions were examined. All patients had metastatic or recurrent CRC and received modified FOLFOX6. Responders and nonresponders were determined according to the best observed response at the end of the first-line treatment. Gene-expression profiles of primary CRC were determined using Human Genome GeneChip arrays U133. We identified discriminating genes whose expression differed significantly between responders and nonresponders and then carried out supervised class prediction using the k-nearest-neighbour method.ResultsWe identified 27 probes that were differentially expressed between responders and nonresponders at significant levels. Based on the expression of these genes, we constructed a FOLFOX response predictor with an overall accuracy of 92.5%. The sensitivity, specificity, positive and negative predictive values were 78.6%, 100%, 100% and 89.7%, respectively.ConclusionThe present model suggests the possibility of selecting patients who would benefit from FOLFOX therapy both in the metastatic and the adjuvant setting. To our knowledge, this is the first study to establish a prediction model for the response to FOLFOX chemotherapy based on gene expression by microarray analysis.

Predicting Ulcerative Colitis-Associated Colorectal Cancer Using Reverse-Transcription Polymerase Chain Reaction Analysis

BACKGROUND Widespread genetic alterations are present not only in ulcerative colitis (UC)-associated neoplastic lesions but also in the adjacent normal colonic mucosa. This suggests that genetic changes in nonneoplastic mucosa might be effective markers for predicting the development of UC-associated cancer (UC-Ca). This study aimed to build a predictive model for the development of UC-Ca based on gene expression levels measured by reverse-transcription polymerase chain reaction (RT-PCR) analysis in nonneoplastic rectal mucosa. PATIENTS AND METHODS Fifty-three UC patients were examined, of which 10 had UC-Ca and 43 did not (UC-NonCa). In addition to the 40 genes and transcripts previously shown to be predictive for developing UC-Ca in our microarray studies, 149 new genes, reported to be important in carcinogenesis, were selected for low density array (LDA) analysis. The expression of a total of 189 genes was examined by RT-PCR in nonneoplastic rectal mucosa. RESULTS We identified 20 genes showing differential expression in UC-Ca and UC-NonCa patients, including cancer-related genes such as CYP27B1, RUNX3, SAMSN1, EDIL3, NOL3, CXCL9, ITGB2, and LYN. Using these 20 genes, we were able to build a predictive model that distinguished patients with and without UC-Ca with a high accuracy rate of 83% and a negative predictive value of 100%. CONCLUSION This predictive model suggests that it is possible to identify UC patients at a high risk of developing cancer. These results have important implications for improving the efficacy of surveillance by colonoscopy and suggest directions for future research into the molecular mechanisms of UC-associated cancer.

Anticancer activity of the intraperitoneal-delivered DFP-10825, the cationic liposome-conjugated RNAi molecule targeting thymidylate synthase, on peritoneal disseminated ovarian cancer xenograft model

Introduction Peritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip) paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis. Methods We developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS) and a cationic liposome (DFP-10825) and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors. Results DFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA) levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect. Conclusion Collectively, the ip delivery of DFP-10825, TS shRNA conjugated with cationic liposome, shows a favorable antitumor activity without systemic adverse events via the stable localization of TS shRNA for a sufficient time and concentration in the peritoneal cavity of the peritoneally disseminated human ovarian cancer-bearing mice.

Development of new promising antimetabolite, DFP-11207 with self-controlled toxicity in rodents

To reduce 5-fluorouracil (5-FU)-induced serious toxicities without loss of antitumor activity, we have developed DFP-11207, a novel fluoropyrimidine, which consists of 1-ethoxymethyl-5-fluorouracil (EM-FU; a precursor form of 5-FU), 5-chloro-2,4-dihydroxypyridine (CDHP; an inhibitor of 5-FU degradation), and citrazinic acid (CTA; an inhibitor of 5-FU phosphorylation). In vitro studies of DFP-11207 indicated that it strongly inhibited the degradation of 5-FU by dihydropyrimidine dehydrogenase (DPD) in homogenates of the rat liver, and also inhibited the phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT) in tumor tissues in a similar magnitude of potency by CDHP and CTA, respectively. Especially, DFP-11207 inhibited the intracellular phosphorylation of 5-FU in tumor cells in a dose-dependent manner whereas CTA alone did not protect intracellular 5-FU phosphorylation. These results postulate that DFP-11207 rapidly entered into the cell and the free CTA produced from DFP-11207 inhibited the phosphorylation of 5-FU in the cell. Furthermore, following oral administration of DFP-11207, CTA was found to be highly retained in the gastrointestinal (GI) tract compared to other tissues in rats. Interestingly, EM-FU, the prodrug of 5-FU was found to specifically produce 5-FU by various species of liver microsomes. When DFP-11207 was administered to rats, the plasma level of 5-FU was persisted for a long-time with lower Cmax and longer half-life than that from other 5-FU prodrugs. The antitumor activity of DFP-11207 was evaluated in human tumor xenografts in nude rats and found that DFP-11207 showed an antitumor activity in a dose-dependent fashion and its efficacy is equivalent to reference 5-FU drugs. In striking contrast, DFP-11207 manifested no or less 5-FU-related toxicities, such as a decrease in body weights, GI injury, and myelosuppression, especially thrombocytopenia. Taken together, the preclinical evaluation of DFP-11207 strongly indicates that DFP-11207 be a potential new version of the oral fluoropyrimidine prodrug for further clinical development.

Abstract 2192: DFP-10825 IP delivery provides a new effective treatment option to peritoneal-disseminated cancers

Objective: Peritoneal disseminated ovarian and pancreatic cancers are the most difficult to be treated with conventional cytotoxic or molecular targeted drugs. The treatment option is very limited although an intraperitoneal (IP) paclitaxel has been available and shown to improve a prognosis in patients. Therefore, it is urgent to develop a new IP chemotherapeutic drug regulating the fast DNA synthesis in peritoneal disseminated tumors originated commonly from the ovary, pancreas and stomach. We have developed a unique RNAi molecule consisting of shRNA (55-mer) against TS and a cationic liposome (DFP-10825) and tested its anti-tumor activity and PK profile in peritoneal disseminated ascetic tumor models. Methods: We developed luciferase gene-transfected ovarian cancer (SKOV3-luc) and pancreatic cancer (PANC-1-luc) models in mice. After IP injection of 5x106 cells, DFP-10825 containing 20 µg TS shRNA (20 mg/mouse) was administered in an IP route (q3d x 4) to the tumor-bearing mice. In combination therapy, paclitaxel (10 mg/kg) was also IP administered to SKOV3-luc model to which the treatment was performed in the same schedule. The anti-tumor effect was assessed by measuring luciferase activity and tumor volume. Furthermore, the TS expression level in both ascetic tumor cells and solid tumors was measured by conventional RT-PCR. For PK study with DFP-10825 (especially TS shRNA), total RNAs were isolated at various time points from ascetic tumor cells and disseminated SKOV3-luc solid tumor models treated with DFP-10825 and TS shRNA levels were determined by Stem-loop RT-PCR. Results: IP DFP-10825 delivery significantly suppressed the growth of ascetic SKOV3-luc and PANC-1-luc tumor cells and extended the survival time of these tumor-bearing mice compared with that of control group. Combination with the IP paclitaxel augmented the efficacy of DFP-10825. After the IP administration, TS shRNA levels derived from DFP-10825 in ascetic fluid were maintained at nM range (0.7 - 4.3-nM) across 24 hours but not detected (below 5 pM) in the plasma, suggesting that TS shRNA be relatively stable in the peritoneal cavity to be able to exert its anti-tumor activity but not in blood. Also, TS expression (TS mRNA) in ascetic tumor cells was significantly suppressed, supporting the notion that the anti-tumor activity and host survival benefit by DFP-10825 in tumor-bearing mice are through MOA to knock down the TS level in tumors specifically. Conclusion: IP administration of newly developed DFP-10825, the TS shRNA conjugated with cationic liposome is localized stably in the peritoneal cavity and provides a new effective treatment option to the intractable peritoneal disseminated ovarian and pancreatic cancers without any systemic adverse events. Citation Format: Cheng Jin, Kenzo Iizuka, Kokoro Eshima, Masakazu Fukushima, Tatsuhiro Ishida, Cheng-Long Huang, Hiromi Wada, Kiyoshi Eshima. DFP-10825 IP delivery provides a new effective treatment option to peritoneal-disseminated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2017-2192