The anti-tumor effect of trifluridine via induction of aberrant mitosis is unaffected by mutations modulating p53 activity.

The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

Assessment of the Changes in Mitochondrial Gene Polymorphism in Ulcerative Colitis and the Etiology of Ulcerative Colitis-associated Colorectal Cancer.

BACKGROUND: Mitochondria are energy-producing organelles, and dysfunction in these organelles causes various types of disease. Although several studies have identified mutations in nuclear DNA that are associated with the etiology of ulcerative colitis (UC), information regarding mitochondrial DNA (mtDNA) in UC is limited. This study aimed to investigate the mitochondrial DNA polymorphism underlying the etiology of UC and UC-associated colorectal cancer. MATERIALS AND METHODS: Next-generation sequencing was performed to assess mitochondrial DNA mutations in 12 patients with UC-associated cancer. The mtDNA mutations in the non-neoplastic mucosa, tumor tissues, and healthy controls were compared. RESULTS: The incidence of mutations of nicotinamide adenine dinucleotide phosphate ubiquinone oxidase subunit, ATP synthetase, and tRNA was higher in non-neoplastic mucosa in those with UC compared with the healthy controls. However, no statistically significant differences were observed in mutations between the tumor tissues and non-neoplastic mucosa in UC. CONCLUSION: Significant mutations in mtDNA were observed in the non-neoplastic mucosa of patients with UC-associated cancer.

CD44/CD133-Positive Colorectal Cancer Stem Cells are Sensitive to Trifluridine Exposure.

Cancer stem cells (CSCs) are involved in metastatic colorectal cancer recurrence, but no effective therapy targeting these cells is currently available. Because trifluridine (FTD)/tipiracil therapy is used for refractory colorectal cancer, we sought to determine whether FTD is effective against CSC-like cells. CD44+CD133+ high-expressing and other populations of human DLD-1 colon cancer cells were separately isolated through fluorescence-activated cell sorting. The sphere-forming activity of each population and the anti-sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells were then measured. CD44+CD133+ DLD-1 cells formed substantially more spheres than other cells. Moreover, treating CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) inhibited sphere formation, and this was superior to the effect of subtoxic concentrations (1 µM) of 5-FU. The associated inhibition rates for FTD and 5-FU were 58.2% and 26.1%, respectively. Further, CD44+CD133+ DLD-1 cells expressed higher levels of thymidine kinase 1, which is responsible for FTD phosphorylation, than DLD-1 cells, and FTD was incorporated into the DNA of CD44+CD133+ DLD-1 cells. Thus, our data show that FTD treatment is effective against CSC-like cells and might be applied as CSC-targeting chemotherapy for tumor subtypes with high CD44 and CD133 expression.

ChIP-seq Analysis to Explore DNA Replication Profile in Trifluridine-treated Human Colorectal Cancer Cells In Vitro.

BACKGROUND/AIM: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil that has been approved for the treatment of metastatic colorectal cancer. In this study, a comprehensive analysis of DNA replication profile in FTD-treated colon cancer cells was performed. MATERIALS AND METHODS: HCT-116 cells were exposed to BrdU or FTD and subjected to DNA immunoprecipitation. Immunoprecipitated DNA was sequenced; the density of aligned reads along the genome was calculated. Peak finding, gene ontology, and motif analysis were performed using MACS, GREAT, and MEME, respectively. RESULTS: We identified 6,043 and 5,080 high-confidence FTD and BrdU peaks in HCT-116 cells, respectively. Of 6,043 FTD peaks, 2,911 peaks were uncommon to BrdU. We observed that FTD was preferentially incorporated into genomic regions containing simple repeats, CpG islands, and gene bodies. Conserved motifs in FTD peaks contained dinucleotide repeats such as (GT)n. CONCLUSION: Global FTD incorporation patterns delineated FTD, preferentially incorporating loci in cancer cells.

Thymidine Kinase 1 Loss Confers Trifluridine Resistance without Affecting 5-Fluorouracil Metabolism and Cytotoxicity.

Acquired resistance to therapeutic drugs is a serious problem for patients with cancer receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analogue-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1-TK1 -/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The current data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. Mol Cancer Res; 16(10); 1483-90. ©2018 AACR.

Trifluridine/tipiracil overcomes the resistance of human gastric 5-fluorouracil-refractory cells with high thymidylate synthase expression.

Trifluridine/tipiracil (FTD/TPI or TFTD, also known as TAS-102) is a combination of the antineoplastic thymidine analog, FTD, and thymidine phosphorylase inhibitor, TPI (molar ratio 1:0.5). FTD/TPI was approved in Japan, the United States, and the European Union for the treatment of unresectable advanced or recurrent colorectal cancer. We evaluated the in vitro and in vivo efficacy and mechanisms of action of FTD and FTD/TPI against 5-fluorouracil (5-FU)-resistant MKN45/5FU, MKN74/5FU, and KATOIII/5FU human gastric cancer cells overexpressing thymidylate synthase (TS) and their respective parent cell lines. MKN45/5FU and KATOIII/5FU cells were not cross-resistant to FTD, whereas MKN45/5FU cells were 3.7-fold more resistant than the parental cells in vitro. FTD was also incorporated into genomic DNA in a concentration-dependent manner in 5-FU-resistant and parental cells. Additionally, deoxyuridine monophosphate levels in MKN45/5FU cells after 24-h FTD treatment were 3.0-fold higher than those in parental cells, and FTD treatment for 72 h induced G2/M arrest in MKN45/5FU cells, unlike the S phase arrest in MKN45 cells. Thus, TS-overexpressing MKN45/5FU cells, but not MKN74/5FU and KATOIII/5FU cells, showed partial cross-resistance to FTD. However, FTD/TPI (administered orally twice a day) exhibited antitumor activity to the same extent in MKN45 and MKN45/5FU xenograft mouse models, overcoming in vitro cross-resistance to FTD. DNA incorporation rather than TS inhibition seems to be the main action of FTD under these in vivo conditions. Thus, FTD/TPI is a promising chemotherapeutic agent against gastric cancers recurring following 5-FU therapy.

Synergistic anticancer activity of a novel oral chemotherapeutic agent containing trifluridine and tipiracil in combination with anti-PD-1 blockade in microsatellite stable-type murine colorectal cancer cells.

Trifluridine/tipiracil (FTD/TPI) is a combination of FTD, an antineoplastic thymidine-based nucleoside analog, and TPI, which acts to enhance the bioavailability of FTD in vivo. It is used to treat patients with unresectable advanced or recurrent colorectal cancer that is refractory to standard therapies. We investigated the anticancer activity of FTD/TPI combined with anti-mouse programed cell death 1 (PD-1) monoclonal antibody (mAb) against CMT-93 cells, which are microsatellite stable (MSS)-type murine colorectal cancer cells. Tumor growth inhibition (TGI) after treatment with anti-mouse PD-1 mAb monotherapy (0.1 mg, i.p., days 1, 5, 9) and FTD/TPI monotherapy (150 mg/kg/day, p.o., days 1-14) were 86.7% and 52.7%, respectively, and that of the combination was 98.4%. The TGI of the combination therapy was significantly greater than that of each monotherapy (P<0.05). The combination therapy caused complete tumor regression in four out of five mice without body-weight reduction, but neither of the monotherapies resulted in complete tumor regression. Low dose FTD/TPI (75 and 100 mg/kg) combined with anti-mouse PD-1 mAb also showed significant antitumor activity against CMT-93 tumors. Flow cytometric analysis revealed that a higher CD8+ T cell ratio among total lymphocytes and a lower regulatory T cells (Tregs) ratio in CD4+ T cells in the combination group compared with that in the control group. These results suggested that the combination therapy induced a cytotoxic response from infiltrated cytotoxic CD8+ T cells and reduced immunosuppressive activity as indicated by decreased Tregs. In this study, the combination therapy was found to have synergistically greater antitumor activity against CMT-93 cells. These preclinical findings indicated that FTD/TPI and anti-mouse PD-1 mAb combination therapy may be a promising treatment option, even for MSS-type colorectal cancer.

Improved chemoradiation treatment using trifluridine in human colorectal cancer cells in vitro.

We aimed to assess the combined effect of trifluridine (FTD) and ionizing radiation (IR) on colorectal cancer cells in vitro. Colorectal cancer cells, HT-29, HCT-15, and HCT 116, showing low, medium, and high sensitivity to IR, respectively, were treated with the combinations of FTD and IR, and evaluated by the clonogenic survival assay. The radiation dose modification factors (DMFs) were calculated as the ratio of radiation doses producing equivalent surviving fractions following the FTD/IR treatment, or IR alone. DMFs of 4 µM FTD followed by 8 Gy of IR were 2.7, 1.5, and 1.2 for HT-29, HCT-15, and HCT 116, respectively, whereas those of 8 Gy of IR followed by FTD were 1.6, 1.4, and 1.0 for these cells, respectively. Intracellular DNA double-strand break levels after IR and FTD were significantly higher than those observed following the IR treatment alone, regardless of whether the IR was applied before or after FTD. RAD51 expression levels were shown to be increased in FTD and IR treated cells. Apoptotic proteins, such as cleaved PARP and cleaved caspase-3, were detected in cells treated with the combination of FTD and IR, while their expression was not significantly induced after IR or FTD treatment alone. These findings suggest that FTD enhances the efficacy of IR and provide a rationale for designing novel combination chemoradiotherapy regimens containing FTD for patients with rectal cancer that are insensitive to the radiation treatment.

Colitic Cancer Develops Through Mutational Alteration Distinct from that in Sporadic Colorectal Cancer: A Comparative Analysis of Mutational Rates at Each Step.

BACKGROUND: Patients with ulcerative colitis (UC) are at risk of UC-associated colorectal cancer (CRC); however, little is known about genetic alterations occurring during UC carcinogenesis. We examined mutational changes in patients with colitic cancer and the features that differed between the carcinogenesis of UC and sporadic CRC. MATERIAL AND METHODS: Specimens were obtained from the non-neoplastic mucosa and cancer cells of 12 patients with colitic cancer. The mutational rate of oncogenes in colitic cancer was analyzed and compared to that of oncogenes in sporadic CRC. RESULTS: We observed a lower mutation rate in adenomatous polyposis coli (APC) (16.7%(2/12) vs. 75.9%(161/212), respectively, p=0.0001) and KRAS (16.7%(2/12) vs. 42% (89/212), respectively, p=0.04) in colitic cancer than in sporadic CRC. With respect to cadherin 1 (CDH1) and fibroblast growth factor receptor 2 (FGFR2), the mutational rates for non-neoplastic colorectal mucosa were similar to those in sporadic CRC. CONCLUSION: We demonstrated that mutational rates for APC and KRAS differ between colitic cancer and sporadic CRC. Furthermore, we revealed that CDH1 and FGFR2 become mutated at an earlier stage in colitic carcinogenesis than in sporadic CRC.

MicroRNA profiles involved in trifluridine resistance.

Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which is approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. A microRNA analysis of three colorectal cell lines was conducted to investigate causes of FTD resistance. Drug resistant sublines of DLD-1, HCT-116, and RKO cells were developed by continuous administration of increasing doses of FTD for 5 months. The let-7d-5p gene, which maps to chromosome 9q22.32, was downregulated in the FTD-resistant DLD-1 sublines. DLD-1 cells became more resistant to FTD when let-7d-5p was knocked down and more sensitive when let-7d-5p was overexpressed. The FTD-resistant sublines were not cross-resistant to 5-fluorouracil (5-FU); 5-FU sensitivity was affected only slightly when let-7d-5p as overexpressed or knocked down. These data indicate that let-7d-5p increases sensitivity of FTD but not 5-FU and that let-7d-5p is a potential clinical marker of treatment sensitivity.

Increased Copy Number Variation of mtDNA in an Array-based Digital PCR Assay Predicts Ulcerative Colitis-associated Colorectal Cancer.

AIM: Mitochondrial dysfunction plays a central role in carcinogenesis in numerous cancer-related diseases. We examined the copy number variation of mitochondrial DNA (mtDNA) and the expression of energy-producing genes in relation to ulcerative colitis (UC)-associated carcinogenesis. MATERIALS AND METHODS: We studied 17 patients with UC-associated adenocarcinoma (UC-Ca) and 16 without UC-associated adenocarcinoma (UC-nonCa). The copy number of mtDNA in non-dysplastic mucosa in both groups was quantified by an array-based digital polymerase chain reaction (PCR) assay. Simultaneously, gene expression related to mitochondrial energy metabolism was determined by a PCR array. RESULTS: We observed a higher copy number of mtDNA in non-dysplastic mucosa in the UC-Ca group compared to the UC-nonCa group (484.2 vs. 747.7 copies/cell, p=0.022). The sensitivity, specificity, positive predictive value, and negative predictive value for the detection of UC-associated adenocarcinoma by mtDNA copy number were 43.8%, 100%, 100%, and 60.9%, respectively. We observed an increased expression of mitochondrial genes related to energy metabolism together with an increased copy number of mtDNA. CONCLUSION: Mitochondrial function and its metabolic process play essential roles in UC carcinogenesis and are possible risk markers for the development of colitic cancer.

Inhibition of PI3K suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil.

Drug-tolerant cancer cell subpopulations are responsible for relapse after chemotherapy. By continuously exposing the gastric cancer cell line MKN45 to 5-FU for >100 passages, we established a 5-fluorouracil (5-FU)-tolerant line, MKN45/5FU. Orthotopic xenografts of MKN45/5FU cells in the stomach of nude mice revealed that these cells had a high potential to metastasize to sites such as the liver. Levels of phosphorylated phosphatidylinositide 3-kinase (PI3K) increased both in 5-FU-tolerant subpopulations according to the 5-FU dose, and in gastric submucosal orthotopic xenografts of MKN45/5FU cells. Sequential administration of 5-FU and a PI3K inhibitor, GDC-0941, targeted the downstream ribosomal S6 kinase phosphorylation to significantly suppress 5-FU-tolerant subpopulations and tumor propagation of orthotopic MKN45/5FU xenografts. These results suggest that administration of 5-FU followed by GDC-0941 may suppress disease relapse after 5-FU-based gastric cancer chemotherapy.

Prognostic impact of KRAS mutant type and MET amplification in metastatic and recurrent gastric cancer patients treated with first-line S-1 plus cisplatin chemotherapy.

Receptor tyrosine kinase (RTK)-related genes, including HER2, EGFR, MET, FGFR2 and KRAS, are target molecules that are clinically beneficial in gastric cancer (GC). We investigated the correlation between RTK-related genes and the curative effect of first-line S-1 plus cisplatin (SP) combination chemotherapy in metastatic and recurrent GC. We enrolled 150 patients with histopathologically confirmed metastatic and recurrent GC treated with SP. KRAS mutation was detected using direct sequencing. DNA copy number was measured by real-time PCR. Formalin-fixed paraffin-embedded specimens were examined immunohistochemically for HER2, EGFR, FGFR2 and MET. Among 144 patients, KRAS mutation was detected in five (3.5%) at codon 12 and one (0.7%) at codon 13. FGFR2, EGFR, HER2, MET and KRAS gene amplification was suggested in 4.4%, 5.9%, 9%, 3.7% and 10.3% of patients, respectively. KRAS mutation, but not KRAS amplification, was associated with significantly shorter overall and progression-free survival. MET membranous overexpression was associated with a significantly higher tumor response. MET amplification was associated with significantly shorter overall survival. We show for the first time that KRAS mutation and MET amplification are promising predictive markers in metastatic and recurrent GC patients treated with SP. KRAS status may be a useful prognostic marker in patients treated with SP.

Genome-wide DNA Copy-number Analysis in ACTS-CC Trial of Adjuvant Chemotherapy for Stage III Colonic Cancer.

BACKGROUND: The adjuvant chemotherapy trial of TS-1 for colon cancer phase III trial was designed to validate the non-inferiority of the oral fluoropyrimidine S-1 to uracil and tegafur/leucovorin as adjuvant chemotherapy for stage III colonic cancer. As a prospective biomarker study of this trial, DNA copy number was studied using formalin-fixed, paraffin-embedded specimens. MATERIALS AND METHODS: FFPE blocks were obtained from 795 patients of the 1,535 patients enrolled in the study. The quality of extracted DNA was assessed using arbitrarily primed polymerase chain reaction and microfluidic analysis. Genomic copy-number alterations in cancer were analyzed by high-density single-nucleotide polymorphism arrays. Copy-number changes in Japanese patients with colonic cancer were compared with those in Western countries using data from a previously reported meta-analysis. We then compared genome-wide segment copy number and clinicopathological features of colorectal cancer. RESULTS: Genome-wide copy number was analyzed in 161 samples and DNA copy-number alteration profiles showed frequent DNA copy-number gains at chromosome 7, 8q and 13, and losses at 4, 5q, 8p, 17p and 18q. The weighted kappa statistic from comparing copy-number alteration status with data from Western countries was 0.828 (95% confidence interval=0.786 -0.871). DNA copy-number alterations of 8,684 segments were compared with clinicopathological features in 161 patients. Location of the tumor correlated with genomic segments of chromosome 4, 5, 7, 8, 13, 14, 18 and 20. Differentiation of the tumor correlated with segments in chromosome 4, 6, 8, 11, 13, 14,15, 16, 17 and 20. CONCLUSION: Somatic copy-number alteration profiles of stage III colonic cancer in the Japanese ACTS-CC trial closely agreed with the results of previous Western studies. Location and differentiation of the tumor correlated with DNA copy-number alterations. Our findings will facilitate understanding the characteristics of colonic cancer. Further investigation may contribute to the exploration of valid biomarkers.

Novel 5-fluorouracil-resistant human esophageal squamous cell carcinoma cells with dihydropyrimidine dehydrogenase overexpression.

5-Fluorouracil (5-FU) is a key drug for the treatment of esophageal squamous cell carcinoma (ESCC); however, resistance to it remains a critical limitation to its clinical use. To clarify the mechanisms of 5-FU resistance of ESCC, we originally established 5-FU-resistant ESCC cells, TE-5R, by step-wise treatment with continuously increasing concentrations of 5-FU. The half maximal inhibitory concentration of 5-FU showed that TE-5R cells were 15.6-fold more resistant to 5-FU in comparison with parental TE-5 cells. TE-5R cells showed regional copy number amplification of chromosome 1p including the DPYD gene, as well as high mRNA and protein expressions of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU degradation. 5-FU treatment resulted in a significant decrease of the intracellular 5-FU concentration and increase of the concentration of α-fluoro-ureidopropionic acid (FUPA), a metabolite of 5-FU, in TE-5R compared with TE-5 cells in vitro. Conversely, gimeracil, a DPD inhibitor, markedly increased the intracellular 5-FU concentration, decreased the intracellular FUPA concentration, and attenuated 5-FU resistance of TE-5R cells. These results indicate that 5-FU resistance of TE-5R cells is due to the rapid degradation of 5-FU by DPD overexpression. The investigation of 5-FU-resistant ESCC with DPYD gene copy number amplification and consequent DPD overexpression may generate novel biological evidence to explore strategies against ESCC with 5-FU resistance.

Prediction of response to preoperative chemoradiotherapy in rectal cancer by using reverse transcriptase polymerase chain reaction analysis of four genes.

BACKGROUND: Patients with rectal cancer exhibit a wide spectrum of responses to chemoradiotherapy. Several gene expression signatures have been reported to predict the response to chemoradiotherapy in rectal cancer, but the lack of practical assays has restricted the clinical use of this technique. OBJECTIVE: We aimed to identify a set of discriminating genes that can be used for the clinical prediction of response to chemoradiotherapy in rectal cancer. DESIGN AND SETTINGS: This study is a retrospective analysis of tumor samples in a single institute. PATIENTS: Sixty-two patients who underwent preoperative chemoradiotherapy were studied. MAIN OUTCOME MEASURES: Gene expression was initially studied in 46 training samples by microarray analysis, and the association between gene expression and response to chemoradiotherapy was evaluated. Quantitative reverse transcriptase polymerase chain reaction was performed to validate the microarray expression levels of the discriminating genes. We developed a gene expression model for the prediction of response to chemoradiotherapy based on the reverse transcriptase polymerase chain reaction findings and validated it by using 16 independent test samples. RESULTS: We identified 24 discriminating probes with expression levels that differed significantly between responders and nonresponders. Among 18 genes identified by Gene Symbol, real-time reverse transcriptase polymerase chain reaction showed significant differences in the expression of 16 genes between responders and nonresponders. We constructed a predictive model by using different sets of these 16 genes, and the highest accuracy rate (89.1%) was obtained by using LRRIQ3, FRMD3, SAMD5, and TMC7. The predictive accuracy rate of this 4-gene signature in the independent set of 16 patients was 81.3%. LIMITATIONS: Validation in a different and large cohort of patients is necessary. CONCLUSIONS: The 4-gene signature identified in this study is closely associated with response to chemoradiotherapy in rectal cancer.

Combination therapy using oral S-1 and targeted agents against human tumor xenografts in nude mice.

In this study, combination therapies using the oral fluoropyrimidine tegafur-gimeracil-oteracil (S-1) with several targeted agents or antibodies, were evaluated. First, the effects of tyrosine kinase inhibitors (erlotinib hydrochloride, sorafenib tosilate and sunitinib malate) against human non-small cell lung cancer (NSCLC), breast cancer and colorectal cancer were evaluated in vivo. The effects of the combination of S-1 and targeted antibodies (bevacizumab and cetuximab) against human colorectal cancers was also evaluated in vivo. S-1 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, showed a significant inhibition of growth in human NSCLC (Lu-99 and PC-9 cell lines). The antitumor activity of the combination of S-1 and erlotinib against Lu-99 and PC-9 cancer cell lines was significantly superior to either monotherapy (P<0.05). Combination therapy using the multi-tyrosine kinase inhibitors, sorafenib or sunitinib, with S-1 against breast cancer (MX-1 cell line) and NSCLC (NCI-H460 cell line) was significantly superior to either monotherapy (P<0.01). The combination of the anti-vascular endothelial growth factor antibody bevacizumab or the anti-EGFR antibody, cetuximab, with S-1 against human colorectal cancer [Col-1, KM20C (bevacizumab) and DLD-1 (cetuximab) cell lines] and a 5-fluorouracil (5-FU)-resistant cell line (KM12C/5-FU) was significantly superior to either monotherapy (p<0.01). In particular, the growth of the Col-1 cells was completely inhibited by the combination of S-1 and bevacizumab. No toxic mortalities and no significant difference in the body weight changes of the animals treated with S-1 combined with the targeted agents or with the mono-therapies were observed; therefore, the treatments appeared to be well-tolerated. Our preclinical findings indicate that the combination therapies of S-1 and targeted agents are promising treatment options.

Recent approaches to identifying biomarkers for high-risk stage II colon cancer.

The use of adjuvant chemotherapy for stage II colon cancer remains controversial. The accurate assessment of the risk factors associated with recurrence in patients with stage II disease is the key to identifying the patients that are most likely to benefit from adjuvant chemotherapy. Recent guidelines advocate that adjuvant chemotherapy for high-risk stage II colon cancer should take into account factors such as the T stage, number of lymph nodes examined, tumor differentiation, and tumor perforation. In addition to these clinicopathological factors, there has also been intense interest in the identification of new prognostic or predictive biomarkers that can improve outcomes through better patient classification and selection for adjuvant chemotherapy. Recent advances in the field of molecular genetics have led to the identification of specific biomarkers involved in colorectal cancer progression, whereas gene expression microarray technology has led to the identification of molecular profiles able to predict recurrence or benefit from adjuvant chemotherapy. However, none of these has yet been validated in large prospective clinical trials. In this article, we review the current status of prognostic and predictive biomarkers for stage II colon cancer and provide an update on the most recent developments.

Efficacy of combination chemotherapy using oral fluoropyrimidine S-1 with oxaliplatin (SOX) against colorectal cancer in vivo.

Oxaliplatin is effective when used with 5-fluorouracil (5-FU) and leucovorin, or with capecitabine (COX) for the treatment of colorectal cancer. In this experiment, we investigated the optimal combination schedule and antitumor activity of oral S-1 with oxaliplatin combination therapy (SOX) against human colorectal cancer xenografts in vivo. Using human colon cancer COL-1-bearing nude mice, oxaliplatin was administered at a total dose of 8.3 mg/kg on day 1 alone, on day 8 alone, or in divided doses administered on days 1 and 8 with S-1 (6.9 mg/kg, days 1-14). The antitumor activity of SOX, administered according to the divided schedule was significantly superior to both monotherapies (p<0.01), and the toxicity was tolerable. However, administration on day 8 alone failed to significantly increase the antitumor activity, when compared with that of monotherapy, while administration on day 1 alone was toxic in this model. Next, the efficacy of SOX was compared with that of COX (360 mg/kg, days 1-14). The antitumor effect of SOX was significantly superior to that of COX (p<0.01), with an equivalent toxicity; moreover SOX suppressed COL-1 tumor growth for a longer period of time (2.2 times) than did COX. The antitumor activity of SOX against the 5-FU-resistant colorectal cancer cell line KM12C/5-FU was equivalent to that of COX. The evaluation of intermittent SOX administration in a clinical trial might be of critical value.

Predicting the response to preoperative radiation or chemoradiation by a microarray analysis of the gene expression profiles in rectal cancer.

Preoperative radiotherapy or chemoradiotherapy (CRT) has become a standard treatment for patients with locally advanced rectal cancer. However, there is a wide spectrum of responses to preoperative CRT, ranging from none to complete. There has been intense interest in the identification of molecular biomarkers to predict the response to preoperative CRT, in order to spare potentially non-responsive patients from unnecessary treatment. However, no specific molecular biomarkers have yet been definitively proven to be predictive of the response to CRT. Instead of focusing on specific factors, microarray-based gene expression profiling technology enables the simultaneous analysis of large numbers of genes, and might therefore have immense potential for predicting the response to preoperative CRT. We herein review published studies using a microarray-based analysis to identify gene expression profiles associated with the response of rectal cancer to radiation or CRT. Although some studies have reported gene expression signatures capable of high predictive accuracy, the compositions of these signatures have differed considerably, with little gene overlap. However, considering the promising data regarding gene profiling in breast cancer, the microarray analysis could still have potential to improve the management of locally advanced rectal cancer. Increasing the number of patients analyzed for more accurate prediction and the extensive validation of predictive classifiers in prospective clinical trials are necessary before such profiling can be incorporated into future clinical practice.