Colorectal cancer

Prognostic significance of the DICER1 mRNA expression in colorectal cancer

Author(s)：: Hisae Iinuma, Takuya Akahane, Junko Tamura, Atsushi Horiuchi, Keisuke Nakamura, Hideki Yamada, Tamuro Hayama, Takeshi Tsuchiya, Keijiro Nozawa, Syouichi Fujii, Keiji Matsuda, Yojiro Hashiguci
Affiliation(s)：: Dept. Surgery, Teikyo Univ. Sch. of Med.

Background: The cytoplasmic ribonuclease DICER1 is one of the key enzymes in the microRNA processing that regulates RNA-based gene silencing for production of mature microRNA. Recently, the influence of DICER1 expression in tumor tissues on the prognosis of patients with several cancers has been examined with controversial results among different cancer types. In particular, the clinical significance of DICER1 expression in colorectal cancer (CRC) patients still remains unclear. In this study, we investigated the correlation between the DICER1 mRNA levels of primary tumor tissues and the clinicopathological characteristics, and prognostic significance in CRC patients.
Patients and methods: We examined primary tumor tissues and normal adjacent tumor tissues from 260 patients with CRC (Dukes' stage A: 40 cases, Dukes' B: 68 cases, Dukes' C: 88 cases, Dukes' D: 64 cases). All samples were derived from patients who did not receive any chemotherapy or radiotherapy prior to surgery.
Immediately following surgical resection, tissues were frozen at -80℃until laser-capture micro-dissection (LMD). Total RNA of LMD sample was extracted by miRNeasy Mini Kit and cDNA was synthesized by SuperScript II reverse transcriptase system. The relative expression levels of DICER1 and GAPDH mRNA were determined by quantitative real time RT-PCR using a Light Cycler 480. The PCR conditions of these genes are 95℃ for 5 min, followed by 40 cycles 95℃ for 1 sec, 60℃ for 20 sec. All the samples were performed in triplicates. The expression levels of DICER1 mRNA were normalized to GAPDH mRNA expression. The study protocol conformed to the guidelines of the ethics committee. It was approved by the review board of Teikyo University and written informed consent was obtained from all patients.
Results: The expression levels of DICER1 mRNA decreased significantly in CRC tissues as compared to normal tissues (P=0.039). To evaluate the correlationship between the DICER1 mRNA levels and the clinicopathological characteristics, patients were divided into two groups (high and low) by the cut-off level based on the analysis of ROC curve. A statistically significant association was observed between DICER 1 mRNA expression and tumor size, depth of invasion, lymph node metastasis, lymphatic invasion and Dukes' stage. The prognostic significances of DICER1 mRNA levels were evaluated for the overall survival rates (OS) in all the patients (n=260), and disease-free survival rates (DFS) in the 196 patients who underwent curative surgery. The average follow-up time for OS was 39.4±25.8 months and that of DFS was 38.8±21.5 months. In Kaplan-Meier survival curve analysis, OS of patients with low DICER1 mRNA expression were significantly worse than those of patients with high DICER1 mRNA expression (P<0.001). DFS of patients with low DICER1 mRNA expression were also significantly worse than those of patients with high DICER1 mRNA expression (P<0.001). In the univariate Cox proportional hazard regression analysis for OS, tumor size, depth of invasion, lymph node metastasis, lymphatic invasion, venous invasion, histological type, liver metastasis, peritoneal dissemination, serum CEA, serum CA19-9, Dukes' stage and DICER1 mRNA level showed significance. In the multivariate analysis, Dukes' stage and DICER1 mRNA showed significance for OS. In the univariate analysis for DFS, depth of invasion, venous invasion, serum CEA, Dukes' stage and DICER1 mRNA showed significance. In the multivariate analysis, venous invasion, Dukes' stage and DICER1 mRNA showed significance for DFS. These results suggest that DICER1 mRNA expression in CRC tissues was an independent prognostic factor for OS (hazard ratio [HR], 0.30; 95% CI, 0.13 to 0.64; P=0.001) and DFS (HR, 0.23; 95% CI, 0.10 to 0.48; P=0.001).
Conclusion: This study clearly revealed that reduced DICER 1 mRNA of tumor tissues shows a prognostic significance in CRC patients. Our finding of DICER1 mRNA expression being an independent marker capable of predicting high-risk patients is potentially useful in the individualized management and monitoring of CRC patients. In the future, these molecules may serve as a novel target with beneficial therapeutic applications.

Strategy for cStage4 colorectal cancer: Should treat in ether of Chemotherapy or
resection of primary tumor ?

Author(s)：: Shigeyoshi Iwamoto, Katsuji Tokuhara, Kazuhiko Yoshioka, Satoshi Okazaki, A-ho Kwon
Affiliation(s)：: Department of Surgery, Kansai Medical University

Introduction: It is controversial that the primary tumor must be resect prior chemotherapy in cStage4 colorectal cancer, therefore some cases prognostic factor were metastatic sites. We report cases of cStage4 colorectal cancer which were underwent intensive chemotherapy prior the primary tumor resection.
Background: 176 cases of metastatic colorectal cancer were treated by L-OHP based chemotherapy plus bevacizumab/ cetuximab/ panitumumab in Sep.2007 to Dec. 2012. 49 cases were treated by intensive chemotherapy prior primary tumor resection, and 40 cases were underwent surgical resection of primary tumor after evaluation of chemotherapy response.
Results: 34 / 8 / 7 cases were treated by bevacizumab / cetuximab /panitumumab with L-OHP combined therapy as intensive treatment. 26 patient with obstructed primary lesion were underwent stoma surgery (53.1%). Evaluable lesions except primary lesion were liver (49.0%), lung (12.2%), LNs (30.6%). Response rate of chemotherapy were 67.3% (PR/NC/PD :33/12/4), and 40 cases were performed resection of primary lesion in PR and SD cases. Pathological G2 and G3 response in resected primary lesion were 18.4% (G1a/G1b/G2/G3:15/16/7/2). A GI perforation and ileus in chemotherapy and 3 cases of anastomotic leakage and 6 cases of SSI in perioperative period were observed respectively.
Discussion: It is seemed to contribute improvement of QOL and local control that start with systemic chemotherapy prior primary lesion resection, therefore some cStage4 cases were difficult to R0 resection also primary lesion and systemic chemotherapy could be reduced symptom with metastatic sites. Only 18.4% of the resected primary tumor were G2/3 pathological response by systemic chemotherapy, on the other hand preoperative chemo-radiation in rectal caner accomplished 67.3% of G2/3 pathological response.
Conclusion: The end-point of therapy in Stage4 colorectal cancer patient are QOL and prolong survival, and should be selected treatments depending on the patient condition.

A questionnaire on QOL and toxicity in colorectal cancer survivors received adjuvant chemotherapy.

Author(s)：: Hiroya Taniguchi¹, Yukiya Narita1, Koji Komori², Kenya Kimura², Takashi Kinoshita², Azusa Komori¹, Shiori Uegaki¹, Motoo Nomura¹, Souhei Nitta¹, Kazuhisa Yamaguchi¹, Shigenori Kadowaki¹, Daisuke Takahari¹, Takashi Ura¹, Masashi Andoh¹, Kei Muro^1,3
Affiliation(s)：: 1 Department of Clinical Oncology, Aichi Cancer Center Hospital
2 Department of Gastroenterological Surgery, Aichi Cancer Hospital
3 Aichi Cancer Network

Background: The relative risk of cancer recurrence with FOLFOX/CapeOX therapy (Ox) used as postoperative adjuvant chemotherapy for stage III colorectal cancer is relatively reduced by approximately 20% compared with that with fluorouracil + leucovorin (LV). The Japanese guidelines recommend tegafur–uracil + LV (UL) and capecitabine (C) as adjuvant chemotherapy, and a comparative trial of UL-C and S-1, an oral fluoropyrimidine, is underway. Purpose: The present study aimed to evaluate the quality of life (QOL) and extent of side effects in patients (survivors) who received adjuvant chemotherapy and to discern the differences in awareness of the side effects between patients and medical staff to examine the balance of Ox therapy risks and benefits. Subjects and methods: An anonymous self-administered questionnaire was distributed to patients with colorectal cancer, who received adjuvant chemotherapy without experiencing relapse, and doctors and nurses involved in chemotherapy administration. Evaluation items for the patients included were as follows: (1) current QOL using the EQ-5D™, a standardized instrument to measure health outcomes; (2) current sensory peripheral neuropathy [Common Terminology Criteria for Adverse Events (CTCAE), Functional Assessment of Cancer Therapy/Gynecologic Oncology Group Oxaliplatin-Specific Neurotoxicity questionnaire (NTX-12)]; and (3) side effects at the time of receiving adjuvant chemotherapy. Each side effect and the overall side effects were evaluated using a three-point scale. The medical staff members were asked to evaluate whether the side effects of Ox, S-1, and C were severe, moderate, or mild. Results: Responses were obtained from 144 patients (median age, 61 years; 6, 127, and 11 patients were in stages II, III, and IV, respectively) who underwent adjuvant chemotherapy at our hospital (recovery rate, 86%), 54 doctors from four institutions, and 84 nurses from four institutions. The median postoperative follow-up period was 2.8 years, and the adjuvant chemotherapy regimen was UL, C, S-1, Ox, and other in 25, 39, 31, 26, and 23 patients, respectively. Analysis of the patient responses showed a mean EQ-5D score of 0.877, 0.963, 0.968, and 0.941 in the Ox, UL, C, and S-1 groups, respectively. While the score of the Ox group was lower than that of the C and UL groups, the overall scores were good. The CTCAE grade 1:2 ratio was 65%, 4%,13%, and 19% in the Ox, UL, C, and S-1 groups, respectively, while the mean NTX-12 scores were 8.2, 1.3, 1.5,and 2.1 in the Ox, UL, C, and S-1 groups, respectively, indicating that the Ox scores were poor. Among patients who responded that the side effects were severe or moderate, hand–foot syndrome and numbness were most common in the C (56%) and Ox (88%) groups, respectively; loss of appetite/nausea tended to be more prevalent in the S-1 group (52%/48%) than in the UL (21% and 17%) and C (33% and 28%) groups. The proportion of patients who responded that the side effects were severe, moderate, or mild were 8%, 81%, and 12% in the Ox group; 4%, 25%, and 71% in the UL group; 3%, 46%, and 50% in the C group; and 23%, 57%, and 20% in the S-1 group, respectively, with the strength of side effects approximately in the order of OX/S-1, C, and U. (4) In contrast, evaluations submitted by the medical staff showed that 39% and 60% of doctors and 43% and 52% of nurses rated the side effects of Ox as severe and moderate, respectively. The side effects of C and S-1 were evaluated as moderate by 74% of doctors and 84% of nurses, which differed from those reported by the patients. Conclusion: Of the patients who received Ox, 81% responded that the side effects were moderate and the QOL was good, although there was a high rate of residual sensory peripheral neuropathy. Considering that Ox adjuvant chemotherapy may reduce the risk of recurrence by approximately 20%, the risk/benefit balance is acceptable.

Relationship between prognosis and nutrition in patients with unresectable CRC

Author(s)：: Yasuo Sakamoto, Ryuichi Karashima, Yu Imamura, Takatsugu Ishimoto, Shiro Iwagami, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Toru Beppu, Hideo Baba.
Affiliation(s)：: Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University

Purpose: The prognosis of the advanced or recurrent unresectable colorectal cancer (CRC) has improved using the new anticancer drugs and molecular target drugs. However, the prognosis is not enough yet. Moreover, such patients with unresectable CRC are often unable to take sufficient oral intake and the nutrition status of such patients is poor. The aim of this study is to evaluate the relationship between the prognosis and the nutrition before the chemotherapy in patients with unresectable colorectal cancer.
Method: From April 2005 to December 2012, 98 patients with advanced or recurrent unresectable CRC were enrolled in this study. They started receiving first-line chemotherapy in our hospital Patients performed surgical intervention, such as hepatectomy, lobectomy and the resection of local recurrence, were excluded. The nutrition status was evaluated using Onodera's Prognostic Nutrition Index (PNI). The statistical analysis used Chi-square test, Log-rank test, Kaplan-Meier method, and Pearson product-moment correlation coefficient. All patients agreed to our use of their data for this study, and provided their written informed consent.
Result: Median age of the patients was 68 years old (range: 34-82). Sixty men and 38 women were enrolled in this study. The primary site was colon in 68 cases and rectum in 30 cases. Most frequent metastatic site was liver, followed by lung. There were synchronous metastases in 74 patients, and metachronous metastases in 19 patients. Five patients with unresectable local recurrence were included in this study. Ninety-one patients were received oxaliplatin-based chemotherapy as first -line chemotherapy, and other 7 patients were received irinotecan-based chemotherapy. Forty-eight patients received the administration of molecular target drugs with anti-cancer drugs. Of 48 patients, bevacizumab, cetuximab, and panitumumab were used in 36, 4, and 8 patients, respectively. Median PNI in 98 patients was 43.9. Based on whether PNI is more or less than 44, the patients were divided in PNI high group or PNI low group.
Between both groups in the patients' characteristics, there was no statistical difference in age, gender, the primary tumor site, the timing of metastasis, and presence of liver metastasis or not. Similarly, there was no statistical difference whether use of oxaliplatin firstly or not, use of molecular target drug or not between both groups. Median survival time was 17months and 22month in PNI low group and high group, respectively, and the prognosis of PNI low group was poorer than PNI high group; (HR 1.58: 1.00-2.63, p=0.049). Moreover, the relationship was showed between overall survival and PNI by Pearson product-moment correlation coefficient (r=0.29, p=0.004).
Conclusion: We suggest that the prognosis of patients with low PNI was poorer than with high PNI, although there was limitation that this study was retrospective one.

Cohort study of 1st line chemotherapy for metastatic colorectal cancer (EMERaLD)

Author(s)：: Hiroshi Matsuoka¹, Toraji Amano², Yasuo Ohashi³
Affiliation(s)：: 1. Surgery, Fujita Healty University
2: Department of Medical Oncology, Hokkaido University
3: Department of Biostatistics, School of Public Health, The Tokyo Univercity

Background
An observational cohort study plays a crucial role to establish a database to assess clinical status of patients (pts). There is no database which shows the current status including treatments of mCRC pts in Japan.

Methods
We planned and conducted a large cross-sectional cohort study which investigates 1st line CT in mCRC to establish a database to improve future clinical practice in Japan.
Data from the following pts are eligible to the study: treated with 1st line CT including oxaliplatin and bevacizumab (BV) for mCRC; started treatment in or after January 2010. The primary objectives are to evaluate overall survival (OS), liver resection rate, R0 liver resection rate; the secondary objectives are to evaluate response rate (RR), progression-free survival (PFS). We performed a preplanned interim analysis of 6-month efficacy and safety data after the 1,000th registration.

Results
A total of 1,353 pts were recruited from 132 centers in Japan between October 2010 and September 2011, and we analyzed data on 1,005 pts of them. The background of 1,005 pts were the following; male/female, 614/391; median age of 65 (range, 27-89); ECOG PS 0/1/2/3, 854/139/10/2; site of primary tumor colon/rectum/others, 549/451/5; and site of metastatic disease liver/lung/others, 627/308/421; FOLFOX/ CapeOx/ others with BV, 437/540/28, KRAS-status wild/mutant/unknown, 225/168/612. Six-month efficacy data were the following; liver resection rate 10.5%, R0 liver resection rate 8.9%, RR 51.6%, PFS 83.9%. Six-month grade 3/4 (CTC-AEv3.0) adverse events related to BV were hypertension 2.4%, proteinuria 0.2%, thromboembolism 1.1%, bleeding 0.6%, and gastrointestinal perforation 1.5%. We also performed a sub-group analysis by regimens, KRAS-status, and so on.

Conclusions
We performed a preplanned interim analysis on 1,005 pts and this could be a large database to investigate 1st line CT in clinical practice of mCRC pts in Japan. We will further investigate and analyze the 2-year data including survival on all 1,353 pts.
This study is sponsored by the Public Health Research Center Foundation CSPOR in Japan.

Comparison of the efficacy of IRIS and IRIS/Bev: analysis of two phase II trials

Author(s)：: Kazuteru Hatanaka¹⁾, Satoshi Yuki²⁾, Hiraku Fukushima²⁾, Kazuaki Harada²⁾, Michio Nakamura³⁾, Mineo Kudo⁴⁾, Miki Tateyama⁵⁾, Takuto Miyagishima⁶⁾, Takashi Kato⁷⁾, Ichiro Iwanaga⁸⁾, Nobuaki Akakura⁹⁾, Tomofumi Takagi⁹⁾, Kencho Miyashita⁹⁾, Yuh Sakata¹⁰⁾, Yoshito Komatsu²⁾
Affiliation(s)：: 1：Department of Gastroenterology, Hakodate Municipal Hospital
2：Department of Gastroenterology and Hepatology/Cancer Center, Hokkaido University Hospital
3：Department of Gastroenterology, Sapporo City General Hospital
4：Department of Gastroenterology, Sapporo Hokuyu Hospital
5：Department of Internal Medicine, Tomakomai Nisshou Hospital
6：Department of Gastroenterology, Kushiro Rosai Hospital
7：Department of Internal Medicine, Hokkaido Gastroenterology Hospital
8：Department of Gastroenterology, Japanese Red Cross Kitami Hospital
9：Hokkaido Gastrointestinal Cancer Study Group(HGCSG)
10：CEO, Misawa City Hospital

Background: The safety and efficacy of first-line IRIS (S-1 in combination with irinotecan; Komatsu Y, et al. Oncology, 2011) and IRIS/Bev (IRIS in combination with bevacizumab (Bev); Komatsu Y, et al. Acta Oncol, 2012/Yuki S, et al. 2013 ASCO-GI) have been evaluated in patients with metastatic colorectal cancer (mCRC). To date, no randomized studies comparing these regimens have been performed. This retrospective analysis compared efficacy data for the two regimens from separate phase II studies performed at Hokkaido Gastrointestinal Cancer Study Group (HGCSG).

Methods: Patients with histologically confirmed unresectable metastatic or recurrent CRC and received no prior chemotherapy were enrolled. In the first trial, patients received irinotecan 100 mg/m2 on day 1,15 and oral S-1 40 mg/m2 twice daily on days 1-14 every 4 weeks (IRIS study: HGCSG0302). In the second trial, patients received the same regimen plus Bev 5 mg/kg on day 1,15 (IRIS/Bev study).

Results: A total of 40 and 52 patients were enrolled the IRIS and IRIS/Bev studies, respectively. Patient characteristics were generally similar in both groups, whereas there were more cases of good performance status and less number of metastatic organ in IRIS/Bev group. The median overall survival was 39.6 months in IRIS/Bev, as compared with 23.4 months in IRIS, corresponding to a hazard ratio for death of 0.418 (p<0.001). The median progression-free survival was 17.0 months in IRIS/Bev, as compared with 8.6 months in IRIS (hazard ratio for disease progression, 0.402; p<0.001); the corresponding response rate were 63.5 percent and 52.5 percent (p=0.393). In a multivariate analysis of PFS and OS, IRIS/Bev was significantly associated with longer PFS and OS compared with IRIS alone.

Conclusions: In this retrospective comparison of two studies, the addition of Bev to IRIS appeared to improve outcome compared with IRIS alone in the first-line treatment of patients with mCRC.

Adjuvant therapy in elderly colon cancer patients: subgroup analysis of ACTS-CC trial

Author(s)：: Megumi Ishiguro¹, Eiji Nakatani², Toshiaki Ishikawa³, Hiroyuki Uetake¹,
Kenichi Sugihara³
Affiliation(s)：: #1: Tokyo Medical and Dental University, Graduate School, Department of Translational Oncology
#2: Foundation for Biomedical Research and Innovation, Translational Research Informatics Center
#3: Tokyo Medical and Dental University, Graduate School, Department of Surgical Oncology

Background: The ACTS-CC trial (ClinicalTrials.gov:NCT00660894) is a randomized phase III trial designed to validate non-inferiority of S-1 to UFT/LV which is a standard adjuvant therapy for stage III colon cancer in Japan. Considering increase of elderly patients, 80 years was selected as upper limit of inclusion criteria. Adverse events (AEs) and disease free survival (DFS) at 3 years (primary endpoint) were previously reported. To evaluate the efficacy and safety in elderly patients (pts), a subgroup analysis was performed.
Patients and Methods: 20-80 aged pts with stage III colon cancer were randomly assigned to receive UFT/LV (UFT: 300 to 600 mg/day and, LV: 75 mg/day on days 1-28, followed by 7 days rest, 5 courses) or S-1 (80 to 120 mg/day on days 1-28, followed by 14 days rest, 4 courses). We compared AEs and DFS between group A (age < 70) and group B (age 71-80).
Results: A total of 1518 pts (760 in S-1 arm, 758 in UFT/LV arm) were analyzed. The proportion of pts of group A and B were 69% (1043 pts) and 31% (475 pts), respectively, and there was no difference in stage distribution between the age groups. Pts with PS1 were more in group B than in group A (2.6% in group A vs. 8.7% in group B).
In S-1 treatment, incidences (any grades) of anemia (29% in group A vs. 40% in group B), decreased platelet count (11% vs.16%), anorexia (29% vs. 38%) and fatigue (25% vs. 33%) were higher in group B than in group A. In > grade 3 AEs, anorexia (3% vs. 9%), nausea (1% vs. 3%) and fatigue (2% vs. 4%) were more frequent in group B.
In UFT/LV treatment, incidence (any grades) of anorexia (22% vs. 32%) and anemia (24% vs. 33%) were higher in group B, while that of elevation of AST (23% vs. 14%) and ALT (25% vs. 14%) were higher in group A. In > grade 3 AEs, the difference between the age groups was observed only in anorexia (3% vs. 6%).
Both in S-1 and UFT/LV treatment, there was no difference in the treatment completion rate between group A and B (78% and 74% in S-1, 75% and 71% in UFT/LV).
There was no difference in DFS between group A and B both in pts receiving S-1 (log-rank test: p=0.535, 3-year DFS rate: 75% and 78%) and in pts receiving UFT/LV (p=0.315, 73% and 69%).
Conclusions: Both S-1 and UFT/LV as adjuvant therapy for stage III colon cancer maintained their efficacy and feasibility in elderly pts (age 71-80).

Effectiveness of preoperative chemoradiotherapy for rectal cancer according to stem cell marker expression

Author(s)：: Eiji Shinto1, Hideki Ueno¹, Yoshihisa Naito¹, Atsushi Shikina¹, Koichi Okamoto¹, Toru Kubo¹, Satomi Fukazawa¹, Akinori Sekizawa¹, Hitoshi Tsuda², Osamu Matsubara², Wataru Ichikawa³, Katsumi Hayashi⁴, Yojiro Hashiguchi⁵, Junji Yamamoto¹, Kazuo Hase¹
Affiliation(s)：: Department of Surgery¹, Pathology², Clinical Oncology³ and Radiology⁴ National Defense Medical College, Saitama, Japan
Department of Surgery⁵, Teikyo university, Tokyo, Japan

BACKGROUND: Preoperative chemoradiotherapy (CRT) can cause tumor regression characterized by size reduction, downstaging, and even complete disappearance. Tumor regression has been associated with an increased probability of tumor resectability, high rate of sphincter-saving procedures, and increased postoperative local control. However, CRT is associated with severe toxicities such as anastomotic leakage, perineal wound infection, and it adversely affects the anal sphincter function after sphincter-preserving surgery. Thus, the prediction of tumor response is essential for tailoring treatments to individual patients. In this study, we investigated if the overexpression of cancer stem cell markers can be used as predictive markers of local control after preoperative CRT in patients with rectal cancer, because there is increasing evidence that cancer stem cells are more resistant to chemotherapy and radiotherapy than other types of cancer cells.
PATIENTS: We recruited 78 patients who underwent preoperative short-term CRT (20 Gy in 5 fractions with 400 mg/day Tegafur/Uracil over 1 week) and curative resection (CRT cases), and 61patients who underwent curative resection without CRT (surgery alone cases).
METHODS: We obtained pretreatment biopsy specimens and immunostained these specimens with antibodies for CD133, Sox2, Oct4, c-Myc. Immunoreactivity was evaluated semiquantitatively and the results were classified as immunopositive or immunonegative. The following cut-off values were used in this study: immunopositive for CD133 if ≥20% tumor cells exhibited positive apical/endoluminal surface staining; for Sox2, Oct4, c-Myc if ≥5%, ≥7%, ≥20%, respectively, of the tumor cells showed positive nuclear staining. Cancers with 2 or more marker expressions were judged to be high-grade positivity. The others were considered low-grade. In order to examine the prognostic significance, comparisons of local recurrence free survival were performed.
RESULTS: The incidences of positive CD133, Sox2, Oct4, c-Myc expressions were 19%, 19%, 35% and 34%, respectively. Expressions of these markers were not mutually correlated. Analyses of local recurrence free survival revealed that preoperative CRT (5-year survival of 92% in CRT cases versus 79% in surgery alone cases, P=0.021) and N status (5-year survival of 92% in node negative cases versus 80% in node positive cases, P=0.047) showed significant prognostic values. Multivaliate analysis disclosed that CRT (HR 3.3, P=0.029) was an independent prognostic indicator. Among patients with cancers judged to be high grade positive for stem cell marker, analyses of local recurrence free survival revealed that N status (93% versus 54%, P=0.008) and age (85% in cases of 65 years or under versus 56% in cases of 66 years or over, P=0.035) showed significant prognostic values, while preoperative CRT (75% versus 74%) was not associated with local control. Among patients with cancers judged to be low grade positive, analyses revealed that preoperative CRT (98% versus 81%, P=0.007) showed significant prognostic values, while N status (92% versus 92%) was not associated with local control.
CONCLUSIONS: Rectal cancer patients with cancers judged to be low grade positive for stem cell marker showed an apparent survival benefit from CRT, but not those with high grade positive cancers. The phenotypes according to the stem cell marker immunoreactivities are promising in identifying cancers with different responses to CRT.

Methylated miR-124, -137 and -34b/c as predictive biomarkers for ulcerative colitis-associated colorectal neoplasia

Author(s)：: Yuji Toiyama¹, Keun Hur², Masato Okigami¹, Mikio Kawamura¹, Aya Kawamoto¹, Yoshinaga Okugawa¹, Jyunichiro Hiro¹, Toshimitsu Araki¹, Koji Tanaka1, Yasuhiro Inoue¹, Keiichi Uchida¹, Yasuhiko Mohri¹, C. Richard Boland² and Ajay Goel², Masato Kusunoki¹
Affiliation(s)：: ¹ Department of Gastrointestinal and Pediatric Surgery, Division of Reparative
Medicine, Institute of Life Sciences, Graduate School of Medicine, Mie University,Mie 514-8507, Japan.
² Gastrointestinal Cancer Research Laboratory, Division of Gastroenterology,
Department of Internal Medicine, Charles A. Sammons Cancer Center and Baylor
Research Institute, Baylor University Medical Center, Dallas, Texas 75246-2017,USA.

Background:
Carcinogenesis in UC occurs in a histologically stepwise manner, sometimes called an "inflammation dysplasia carcinoma sequence". Carcinogenetic progression involves accumulation of genetic and epigenetic alterations, that can occur in both non-neoplastic and neoplastic epithelium of patients with UC-associated neoplasia; this is referred to as a "field defect.
Accumulating evidence suggests that microRNAs (miRNAs) can be targets of methylation-induced silencing in colorectal cancer, which can occur in an age-dependent manner, and constitutes an epigenetic "field defect". Recently, methylation of miR-124, -137, and -34b/c was reported to be common in CRCs with "field defects," and to increase with age in non-neoplastic colorectal epithelium. We hypothesized whether methylation of miR-124, -137 and -34b/c can predict colorectal neoplasia in patients with ulcerative colitis (UC).
Materials and Methods:
We examined 185 samples from 70 UC patients (45 patients without neoplasia, 12 with dysplasia and 13 with cancers) for the methylation status of miR-124, -137 and -34b/c by quantitative bisulfite pyrosequencing analysis. In addition, expression levels of these 3 miRNAs in dysplastic (n=12), cancerous (n=13) and non-neoplastic UC mucosa (n=20) were quantified by TaqMan qRT-PCR.
Results:
We evaluated associations between miR-124, -137, or 34b/c methylation levels and clinical findings in non-neoplastic mucosa from UC. MiR-124 and -137 methylation levels were significantly higher in rectal mucosa than in proximal mucosa, and were significantly associated with age and disease duration in rectal mucosa.
Next, we investigated miR-124, -137, and -34b/c methylation levels in non-neoplastic UC mucosa, dysplasia and cancer. Methylation levels of all 3 miRNAs were significantly higher in dysplasia and cancer compared to non-neoplastic UC mucosa. Receiver operating characteristic (ROC) analysis revealed that miR 124, -137, and miR-34b/c methylation levels were robust in discriminating cancer from non-neoplasia, with AUC values of 0.980, 0.974, and 0.970, respectively. Even more important from a diagnostic perspective, methylation levels of the three miRNAs could reliably differentiate dysplasia from non-neoplastic status, as evidenced by AUC values of 0.777, 0.907, and 0.863, respectively.
Furthermore, to assess the potential usefulness of methylation levels of miR-124, -137, and -34b/c to serve as biomarkers for early diagnosis of UC-associated neoplasia, we first investigated these miRNAs in non-neoplastic samples of rectal tissues from patients with neoplasia compared to those without. All three miRNA methylation levels were significantly higher in rectal mucosa from patients with cancer than in those without, indicating "field defect" were recognized in UC mucosa. Next, ROC curves were generated to assess the possibility of using miR-124, -137, and -34b/c methylation in non-neoplastic rectal tissues as biomarkers for patients with UC-associated neoplasia. Methylation levels of all 3 miRNAs in non-neoplastic rectal tissues could discriminate patients with cancer from those without neoplasia (miR-124 AUC value = 0.74; miR-137 AUC = 0.79; miR-34b/c AUC=0.72). Furthermore, methylated miR-137 in non-neoplastic rectal tissues allowed discrimination of UC patients with neoplasia from those without (AUC = 0.76), and independently predicted neoplasia (OR= 5.55; 95% CI: 1.40–22.05; P = 0.0148).
Expression levels of all 3 miRNAs were significantly down-regulated in dysplasia or cancer compared to non-neoplastic UC mucosa, and the expression and methylation were negatively correlated.
Conclusion:
In conclusion, we have shown for the first time that methylation of miR-124, -137, and -34b/c in rectal tissues can serve as robust biomarkers for early detection of UC-associated cancer; miR-137 methylation levels are particularly indicative of UC-associated neoplasia. Analysis of miR-137 methylation in rectal biopsy specimens may permit identification of UC patients at high risk for neoplasia elsewhere in the colorectum, or for developing it in the future. However, as the number of patients, especially those with UC-associated neoplasia, was limited in the present study, larger prospective trials will be needed to confirm the validity of their predictive value. In addition, restoration of these microRNAs by inhibition of DNA methylation could be the basis of new strategy to slow or reverse age-related UC-associated neoplasia.

Systemic siRNA delivery by 10nm carrier performed tumor-specific accumulation

Author(s)：: Xin Wu, Hirofumi Yamamoto, Mamoru Uemura, Taishi Hata, Junichi Nishimura, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki, and Masaki Mori
Affiliation(s)：: Department of Surgery, Gastroenterological Surgery, Graduate School of Medicine, Osaka University

Background: RNA interference technology is currently being applied in clinical trials for diseases of the eyes, skin, liver, and kidney. However, systemic delivery of small interfering RNA (siRNA) to solid tumors still faces hurdles. Particle size becomes a crucial factor that should be taken into account when engineering an in vivo systemic carrier. Recent nanoparticle siRNA delivery systems often result in distribution to normal organs, possibly due to the relatively large size of the nanoparticles (approximately 100 nm). At present, the tissue distribution and tumor accumulation of nanoparticles ranging from 5 to 30 nm remain to be uncovered. Here, we introduce a super apatite (sApa) delivery system that is expected to surmount the limitations of the current carriers by employing 10-nm ultra-nanoparticles. sApa ultra-nanoparticels consisting of inorganic ions (e.g., CO32-, Ca2+, and PO43-) are highly stable at the physiological pH 7.4, and quickly degradable at pH 5.5 in the endosomal compartments.

Methods: In order to investigate the in vitro transfection efficiency of siRNA in colon cancer cells using sApa, we performed intracellular siRNA measurement assay, western blot analysis, and cell proliferation assay, compared with Lipofectamine 2000. Furthermore, we prepared colon cancer solid tumor model to perform in vivo biodistribution study and antitumor activity study of sApa. Human colon cancer HCT116 cells (5.0×106 cells) were inoculated subcutaneously in both the left and right flanks of the mice to prepare the solid tumor model. A biodistribution study was performed when the tumors reached 10 mm in diameter. An antitumour activity study was performed when the HCT116 tumors were 5-6 mm in diameter.

Results and Discussion: We confirmed significantly higher in vitro transfection efficiency of siRNA by sApa, comparing with Lipofectamine 2000 (Lp). Quantitative flow cytometry analysis and fluorescence images showed that sApa successfully carried fluorescently labeled control siRNA into the human colon cancer cell lines (HCT116, HT29, KM12SM). At both 4 and 12 h, the mean fluorescence intensity (MFI) for the cells treated with sApa-siRNA was significantly higher than that with Lp-siRNA in HCT116, KM12SM (P = 0.0304 for each). To examine the knockdown efficiency of certain protein products by a specific siRNA, we chose survivin, a member of the inhibitor of apoptosis family. Survivin performs multiple functions, including cell cycle regulation and inhibition of cell death. Western blot analysis showed that survivin protein in HCT116 decreased more obviously with sApa transfection compared to Lp. The rapid and strong down-regulation of survivin protein expression by sApa treatment could drastically reduce (by 50%) the cell viability. As for in vivo delivery efficiency, the sApa exhibited significantly lower siRNA accumulation in liver and kidney, but higher siRNA distribution to tumors (2.7±0.26-fold) when compared with naked-siRNA treated tumors (0.95±0.084-fold, p<0.01). Accordingly we injected i.v. sApa incorporating survivin siRNA, and the tumor volume was significantly smaller in mice treated with sApa-survivin-siRNA than that treated with sApa incorporating control siRNA or saline (p<0.001). The total volume of siRNA delivered by the sApa system in our antitumor activity assay was up to 135 μg, which was a 6- to 11-fold lower dose compared with other reported systems delivered intravenously, indicating that sApa worked efficiently in an in vivo system.

Conclusion: The sApa ultra-nanoparticles drastically improved siRNA transfection in vitro and achieved tumor-specific accumulation and strong antitumor effects in mice when sApa ultra-nanoparticles containing survivin siRNA were injected into the tail vein. Our data suggest that the sApa delivery system is of vital use to experimental approaches both in vitro and in vivo, and 10-nm nanoparticles may constitute a new avenue in nanoparticle-based medicine.

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