Koji Murono

Elevated Neutrophil to Lymphocyte Ratio Predicts Poor Prognosis in Advanced Colorectal Cancer Patients Receiving Oxaliplatin-Based Chemotherapy

Background: The aim of this study was to assess whether the neutrophil to lymphocyte ratio (NLR) and other laboratory markers may predict the prognosis of advanced colorectal cancer (CRC) patients receiving palliative chemotherapy. Methods: The study population included 50 patients with far advanced or recurrent unresectable CRC who received oxaliplatin-based combination chemotherapy as first-line treatment in our hospital between June 2005 and November 2010. Seven clinical variables and 7 laboratory indices before chemotherapy were evaluated retrospectively as the possible prognostic factors of overall and progression-free survival. Results: During the study period, 27 patients (54%) died of CRC. Elevated NLR (≥4.0) was observed in 15 patients (30%). By univariate analysis, elevated NLR, performance status and hypoalbuminemia were significantly associated with both poor overall and progression-free survivals. Multivariate analysis showed that elevated NLR (hazard ratio 4.39, 95% confidence interval 1.82–10.7; p = 0.0013) and thrombocytosis (hazard ratio 5.02, 95% confidence interval 1.69–13.4; p = 0.0066) were independently associated with overall survival. Conclusion: Elevated NLR is a powerful predictor of poor response to oxaliplatin-based chemotherapy in patients with unresectable CRC. The ratio is a simply accessible and inexpensive but useful biomarker in CRC patients receiving chemotherapy.

Resistance of colon cancer to 5-fluorouracil may be overcome by combination with chloroquine, an in vivo study.

Autophagy is a complex of adaptive cellular response that enhances cancer cell survival in the face of cellular stresses such as chemotherapy. Recently, chloroquine diphosphate (CQ), a widely used antimalarial drug, has been studied as a potential inhibitor of autophagy. Here, we aimed to investigate the role of CQ in potentiating the effect of 5-fluorouracil (5-FU), the chemotherapeutic agent of first choice for the treatment of colorectal cancer, in an animal model of colon cancer. The mouse colon cancer cell line colon26 was used. For the in-vivo study, colon26 cells were injected subcutaneously into BALB/c mice, which were treated with saline as a control, CQ (50 mg/kg/day), 5-FU (30 mg/kg/day), or the combination therapy (CQ plus 5-FU). The tumor volume ratio and body weight were monitored. After the sacrifice, tumor tissue protein extracts and tumor sections were prepared and subjected to immunoblotting for the analysis of autophagy-related and apoptosis-related proteins, and the terminal transferase uridyl end labeling assay. The combination of CQ resulted in the inhibition of 5-FU-induced autophagy and a significant enhancement in the 5-FU-induced inhibition of tumor growth. Furthermore, the combination treatment of CQ and 5-FU resulted in a significant increase in the ratio of apoptotic cells compared with other treatments. The expression levels of the proapoptotic proteins, namely Bad and Bax, were increased by the CQ treatment in the protein extracts from tumors. Our findings suggest that the combination therapy of CQ and 5-FU should be considered as an effective strategy for the treatment of colorectal cancer.

Hypoxia enhances colon cancer migration and invasion through promotion of epithelial-mesenchymal transition.

BACKGROUND A hypoxic environment exists in most solid tumors because in rapidly growing tumors, the development of angiogenic vasculature is heterogenous, usually not enough to overcome the necessary oxygen supply. In an ischemic condition, cancer cells develop escape mechanisms to survive and leave the unfavorable environment. That result in the acquisition of increased potential for local invasion and evasion to distant organs. However, the escape mechanisms of cancer cells from hypoxic stress have not been fully characterized. MATERIALS AND METHODS The human colon cancer cell line LoVo was cultured in hypoxia, and the adhesive and migratory properties were analyzed. The expression of cell surface and cytoplasmic molecules was also investigated. RESULTS Under hypoxic conditions, cells developed epithelial-mesenchymal transition. The expression levels of α2, α5, and β1 integrins were significantly upregulated and, as a consequence, the ability to adhere to and migrate on collagen and fibronectin was increased. On the other hand, the expression of 67-kDa laminin receptor and the abilities to adhere to and migrate on laminin were decreased. Additionally, the expression of CXCR4 was significantly increased on cells cultured in hypoxia, and the chemotactic activity to stromal cell-derived factor 1α was remarkably increased. CONCLUSIONS Hypoxic stress induced active epithelial-mesenchymal transition in colon cancer cells, with the typical morphologic and functional changes. These morphologic and functional changes of β1 integrins, the 67-kDa laminin receptor, and CXCR4 may be essential for the acquisition of the invasive and metastatic features in colorectal cancer.

Level of arterial ligation in sigmoid colon and rectal cancer surgery.

BackgroundCurative resection of sigmoid colon and rectal cancer includes “high tie” of the inferior mesenteric artery (IMA). However, IMA ligation compromises blood flow to the anastomosis, which may increase the leakage rate, and it is unclear whether this confers a survival advantage. Accordingly, the IMA may be ligated at a point just below the origin of the left colic artery (LCA) “low tie” combined with lymph node dissection (LND) around the origin of the IMA (low tie with LND). However, no study has investigated the detailed prognostic results between “high tie” and “low tie with LND.” The aim of this study was to assess the utility of “low tie with LND” on survival in patients with sigmoid colon or rectal cancer.MethodsA total of 189 sigmoid colon or rectal cancer patients who underwent curative operation from 1997 to 2007 were enrolled in this study. The patient’s medical records were reviewed to obtain clinicopathological information. Overall survival (OS) and relapse-free survival (RFS) rates were calculated using the Kaplan-Meier method, with differences assessed using log-rank test.ResultsForty-two and 147 patients were ligated at the origin of the IMA (high tie) and just below the origin of the LCA combined with LND around the origin of the IMA (low tie with LND), respectively. No significant differences were observed in the complication rate and OS and RFS rates in the two groups. Further, no significant difference was observed in the OS and RFS rates in the lymph node-positive cases in the two groups.Conclusions“Low tie with LND” is anatomically less invasive and is not inferior to “high tie” with prognostic point of view.

Anatomy of the inferior mesenteric artery evaluated using 3-dimensional CT angiography.

BACKGROUND: The branching of the inferior mesenteric artery and vein varies among individuals. Three-dimensional CT angiography is a less invasive modality than traditional angiographic examination to assess the artery and vein. OBJECTIVE: We aimed to demonstrate the clinical applicability of CT angiography by evaluating bifurcations of the inferior mesenteric artery and the positional relationship between the inferior mesenteric artery and vein. DESIGN: This was a prospective observational study of patients undergoing preoperative CT angiography. SETTINGS: This study was conducted at a single tertiary care institution in Japan. PATIENTS: A total of 471 consecutive patients who underwent preoperative CT angiography from April 2012 to December 2013 were prospectively enrolled. MAIN OUTCOME MEASURES: The branching pattern of the inferior mesenteric artery, the positional relationship between the inferior mesenteric artery and vein, and the associations between inferior mesenteric artery length and clinical features were evaluated. RESULTS: The length of the inferior mesenteric artery varied widely, from 10.1 to 82.2 mm. In 41.2% patients, the left colic artery arose independently from the sigmoid artery, and in 44.7% of the patients, the left colic artery and sigmoid artery had a common trunk, whereas the left colic artery did not exist in 5.1%. The left colic artery was located lateral to the inferior mesenteric vein at the level of the origin of the inferior mesenteric artery in 73.0% of the patients. The incidence of a short inferior mesenteric artery was significantly increased in men with high BMIs (75.0%). LIMITATIONS: Three-dimensional reconstruction was performed by the use of a single software, and angiographic examination was not performed. Therefore, accuracy and reliability of the 3-dimensional reconstruction could not be established for each modality. CONCLUSIONS: Using 3-dimensional CT angiography, preoperative understanding of the anatomic vascular variations can be easily obtained, which would help surgeons to safely perform laparoscopic surgery in the left-side colon and rectum.

LINE-1 hypomethylation status of circulating cell-free DNA in plasma as a biomarker for colorectal cancer

Colorectal cancer (CRC) is a serious public health problem and non-invasive biomarkers improving diagnosis or therapy are strongly required. Circulating cell-free DNA (cfDNA) has been a promising target for this purpose. In this study, we evaluated the potential of long interspersed nuclear element-1 (LINE-1) hypomethylation as a blood biomarker for CRC. LINE-1 hypomethylation level in plasma cfDNA in 114 CRC patients was retrospectively examined by absolute quantitative analysis of methylated alleles real-time PCR, and was expressed using LINE-1 hypomethylation index (LHI) [unmethylated copy number/ (methylated copy number + unmethylated copy number)]. Greater LHI values indicated enhanced hypomethylation. In our clinicopathological analysis, CRC patients with large tumors (≥6.0 cm), advanced N stage (≥2), and distant metastasis (M1) had statistically significantly higher cfDNA LHI than other CRC patients, suggesting cfDNA LHI as a disease progression biomarker for CRC. Furthermore, early stage I/II (n = 57) as well as advanced stage III/IV (n =57) CRC patients had significantly higher cfDNA LHI than healthy donors (n=53) [stage I/II: median 0.369 (95% confidence interval, 0.360–0.380) vs. 0.332 (0.325–0.339), P < 0.0001; stage III/IV: 0.372 (0.365–0.388) vs. 0.332 (0.325–0.339), P < 0.0001]. The receiver operating characteristic analysis showed that cfDNA LHI had the detection capacity of CRC with area under the curve(AUC) of 0.79 and 0.83 in stage I/II and stage III/IV CRC patients, respectively. The present study demonstrated for the first time the potential of plasma cfDNA LHI as a novel biomarker for CRC, particularly for early stage detection.

Temsirolimus and chloroquine cooperatively exhibit a potent antitumor effect against colorectal cancer cells.

PurposeTemsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored.MethodsWe evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo.ResultsIn vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio.ConclusionsBased on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

Barium enema and CT volumetry for predicting pathologic response to preoperative chemoradiotherapy in rectal cancer patients.

BACKGROUND: Preoperative chemoradiotherapy has been widely used for the prevention of local recurrence of locally advanced rectal cancer, and the effect of chemoradiotherapy is known to be associated with overall survival. OBJECTIVE: We aimed to evaluate the association of the pathologic response grade with tumor recurrence rate after chemoradiotherapy, using radiographic analysis and the Response Evaluation Criteria in Solid Tumors as the parameters. DESIGN: This study was conducted at a single tertiary care institution in Japan. SETTING: This was a retrospective cohort study of patients undergoing preoperative chemoradiotherapy. PATIENTS: A total of 101 low rectal cancer patients receiving preoperative chemoradiotherapy from July 2004 to August 2012 were enrolled. MAIN OUTCOME MEASURES: The tumor reduction rate was measured with the use of traditional Response Evaluation Criteria in Solid Tumors, barium enema, and CT volumetry, and the correlation between the reduction rate and the pathologic response grade was examined. RESULTS: The tumor reduction rate assessed according to Response Evaluation Criteria in Solid Tumors showed no association with the pathologic response grade (p =0.61). In contrast, the radiographic response rate by both barium enema and CT volumetry strongly correlated with the pathologic response grade (p < 0.0001 and p = 0.001). In terms of local tumor recurrence, those diagnosed as high responders by the pathologic response grade, Response Evaluation Criteria in Solid Tumors, barium enema, and CT volumetry had a lower recurrence rate (p =0.03, p =0.03, p =0.0002, and p =0.001). The difference between high responders and low responders was especially prominent by barium enema and CT volumetry. LIMITATIONS: The study is limited by its retrospective nature. CONCLUSIONS: Double-contrast barium enema and CT volumetry were superior to Response Evaluation Criteria in Solid Tumors in evaluating the effect of chemoradiotherapy and predicting the likelihood of tumor recurrence.

Changes in the plasma levels of cytokines/chemokines for predicting the response to chemoradiation therapy in rectal cancer patients.

In the present study, we aimed to characterize the predictive value of cytokines/chemokines in rectal cancer (RC) patients receiving chemoradiation therapy (CRT). Blood samples were obtained pre- and post-CRT from 35 patients with advanced RC, who received neoadjuvant CRT followed by surgery, and the correlation between plasma levels of cytokines/chemokines and the response to CRT was analyzed. The pre-CRT levels of soluble CD40-ligand (sCD40L) and the post-CRT levels of chemokine ligand-5 (CCL-5) were significantly associated with the depth of tumor invasion and with venous invasion. In addition, a significant decrease in sCD40L and CCL-5, as well as in platelet counts, was associated with a favorable response to CRT. A significant correlation between pre-CRT platelet counts and sCD40L was observed in patients with a favorable response. By contrast, higher post-CRT interleukin (IL)-6 was associated with a poor response. Platelets, immune system and cancer cells, cross-linked through various cytokines/chemokines, appear to play an important role in the response to CRT, and by understanding their roles, new approaches for the improvement of the therapy might be proposed.

Detection of carcinoembryonic antigen mRNA in peritoneal lavage by the transcription-reverse transcription concerted method indicates poor prognosis in patients with stage II and III colon cancer.

BACKGROUND Peritoneal dissemination and positive peritoneal lavage cytology are associated with poor prognosis in colorectal cancer. Carcinoembryonic antigen (CEA) messenger RNA (mRNA) is often used as a marker to detect micrometastases. We aimed to evaluate the prognostic significance of CEA mRNA in the peritoneal lavage of colon cancer patients. METHODS Colon cancer patients (n = 201) who underwent curative operative resection between August 2009 and February 2013 were enrolled. CEA mRNA in peritoneal lavage was measured using the transcription-reverse transcription concerted method, a quantitative RNA amplification method. The correlation between CEA mRNA and overall and peritoneal recurrence-free survival was evaluated. RESULTS Positive CEA mRNA in peritoneal lavage was an independent risk factor for overall recurrence-free survival in colon cancer (P < .0001). Positive CEA mRNA was a risk factor for poorer overall recurrence in stage II and III patients (P = .04 and P = .02, respectively). Moreover, among stage III patients with positive CEA mRNA, the postoperative chemotherapy group had significantly lower overall and peritoneal recurrence rates than the no postoperative chemotherapy group (P = .001). CONCLUSION Positive CEA mRNA in peritoneal lavage was associated with high overall recurrence rates in stage II and III colon cancer. Further study is necessary to determinate the efficacy of aggressive postoperative chemotherapy for stage II and III colon cancer patients with positive CEA mRNA.