Masanori Kotake

LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer

BackgroundLong interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed.MethodsFormalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples.ResultsLINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC.ConclusionsLINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.

Anticipation of umbistoma in laparoscopic anterior rectal resection: Ileostomy and ostomy closure

Objective: The use of laparoscopic surgery for rectal disease is expected to provide good cosmetic benefits for patients postoperatively. However, this expectation is significantly reduced when a diverting ileostomy is created. We present a new technique that reduces the size of the skin wound by constructing a diverting ileostomy in the umbilicus. This procedure, diverting umbilical ileostomy (umbistoma) does not require special tools for its construction and closure. Methods: Twenty-nine patients underwent treatment with umbilical diverting stoma, including five women and 24 men, with a mean age of 70 years (range: 40-88 years). At the time of ostomy closure, a new umbilicus was formed by subcutaneously suturing the wound to the fascia. In addition, we did not close the new umbilical upper and lower spaces, so as to allow open drainage of the healing wound. Results: All procedures were completed successfully without any perioperative complications. Conclusions: Our findings suggest that the umbilical diverting stoma could provide improved safety and cosmetic advantages in laparoscopic rectal resection.

Laparoscopic versus Open stoma creation: A retrospective analysis

Objectives: This study aimed to compare open stoma (OS) creation with laparoscopic stoma (LS) creation considering the operation time, blood loss, time of oral intake, and complications. We also compared multiport LS and single-incision laparoscopic stoma (SILS) creation. Methods: We reviewed the demographic data, diagnosis, indications, operation time, blood loss, time of oral intake, operative procedure, and complications of 50 patients who underwent stoma creation between April 2014 and April 2016. Results: The mean blood loss was significantly lower in the LS group (7.85±18.4 ml) than in the OS group (38.1±73.2 ml; P=0.02). There were no statistical differences between the groups in terms of the operation time (LS, 72.1±32.7 min; OS, 61.2±31.2 min; P=0.23) or time of oral intake (LS, 1.0±0 days; OS, 1.91±2.71 days; P=0.17). Peristomal skin problems occurred in 11 patients (47.8%) in the OS group and 5 patients (18.5%) in the LS group. There were no statistically significant differences between the SILS and multiport LS groups, considering the operation time, amount of bleeding, and time of oral intake. Conclusions: LS is comparable with OS in terms of operation time and time of oral intake and may cause lesser blood loss. Considering its advantages, LS is a useful approach for patients requiring biopsies or intra-abdominal inspection. SILS is a minimally invasive technique, suitable for patients in whom the stoma site is preoperatively decided.

Laparoscopic pelvic lymph node dissection for malignant foot melanoma

A 39 year‐old woman with malignant foot melanoma underwent wide excision of the primary tumor with a safety margin and sentinel lymph node biopsy (SLNB) for the right inguinal lymph node. SLNB was positive and a computed tomography (CT) scan revealed right iliac lymph node swelling. Positron emission tomography computed tomography (PET–CT) scan of the lymph nodes revealed abnormal uptake of fluorodeoxyglucose (FDG). We performed a laparoscopic pelvic lymph node obturator, iliac lymph node) dissection. During the operation, several black lymph nodes were observed in the iliac lymph node. Pathologically, the iliac lymph node consisted of metastasized atypical melanocytes. This surgical method for pelvic lymph node dissection is not a standard procedure among institutions. There have been no reported cases of malignant melanoma with pelvic lymph node metastasis treated by laparoscopic surgery. However, due to the minimally invasive technique, this method is worth considering to be used for pelvic lymph node dissection in malignant melanoma as well as other cancers in the field of urology or gynecology.

Abstract 1708: LINE-1 methylation level is a molecular marker with little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Epigenetic alterations including hypermethylation of gene promoter regions and global hypomethylation of DNA are common in colorectal cancer (CRC). We recently reported that long interspersed nuclear element 1 (LINE-1) methylation level in tumor, a surrogate of global methylaiton, predicts prognosis and response to oral fluoropyrimidines of CRC patients. This suggests that the LINE-1 methylation level is a useful marker for tailored therapy in CRC. However, the LINE-1 methylation level in tumor may be heterogeneous because the DNA methylaion is reversible, which is a problem when the LINE-1 methylation level in tumor is used for decision making in clinic. In this study we investigated LINE-1 methylation level in cancer cells isolated from multiple tumor sites to evaluate intra-patient heterogeneity of the LINE-1 methylation level in tumor tissues. Methods: Primary tumor, metastatic lymph nodes, and distant metastases in 41 CRC patients were used for measurement of LINE-1 methylation level. Cancer cells in primary tumors were collected from tumor center and invasion front by laser capture microdissection (LCMD). Cancer cells in metastatic lymph nodes and distant metastases were also collected by LCMD. The LINE-1 methylation level was measured by multicolor MethyLight assay following bisulfite modification of DNA and was correlated with clinicopathological parameters. Results: There was no significant difference in the LINE-1 methylation level between cancer cells from the center and those from invasion front of the primary tumor tissue. In the same patient, there was no significant difference in the LINE-1 methylation level between the samples from primary tumor, metastatic lymph nodes, and distant metastases. The LINE-1 methylation level was decreased as the tumor stage advances. Discussion: The results suggest that the LINE-1 methylation level is a molecular marker with little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer. Prediction of patients’ prognosis using LINE-1 methylation level may be possible by multiple tumor specimen such as biopsy specimen by colonoscopy and surgically resected tumor. Measuring LINE-1 methylation level in primary tumor may be useful for prediction of response to fluoropyrimidines in metastatic tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1708. doi:1538-7445.AM2012-1708