The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination.

The histone methyltransferase enhancer of zeste 2 (EZH2) is known to be a polycomb protein homologous to Drosophila enhancer of zeste and catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27). We previously reported that EZH2 was overexpressed in various types of cancer and plays a crucial role in the cell cycle regulation of cancer cells. In the present study, we demonstrated that EZH2 has the function to monomethylate lysine 120 on histone H2B (H2BK120). EZH2-dependent H2BK120 methylation in cancer cells was confirmed with an H2BK120 methylation-specific antibody. Overexpression of EZH2 significantly attenuated the ubiquitination of H2BK120, a key posttranslational modification of histones for transcriptional regulation. Concordantly, knockdown of EZH2 increased the ubiquitination level of H2BK120, suggesting that the methylation of H2BK120 by EZH2 may competitively inhibit the ubiquitination of H2BK120. Subsequent chromatin immunoprecipitation-Seq and microarray analyses identified downstream candidate genes regulated by EZH2 through the methylation of H2BK120. This is the first report to describe a novel substrate of EZH2, H2BK120, unveiling a new aspect of EZH2 functions in human carcinogenesis.

Management for pancreatolithiasis: a Japanese multicenter study.

OBJECTIVES: A Japanese multicenter study of pancreatolithiasis was performed to investigate its clinical features and determine treatment strategies for pancreatolithiasis. METHODS: A retrospective study was performed on 916 patients managed in 34 institutions for a period of more than 5 years. RESULTS: The treatment methods were extracorporeal shock wave lithotripsy (ESWL) in 479 patients, surgery in 133, and endoscopy alone in 68. Fragmentation of stones after ESWL was achieved in 92.4% of the patients. However, complete stone clearance was achieved in 49.4% of the patients after ESWL alone. The complete stone clearance rate was lower after ESWL than after endoscopy (87.9%). The incidence of early complications was significantly higher after surgery (13.3%) than after ESWL (6.1%). The frequencies of total stone recurrence after ESWL (22.5%) and endoscopy (12.0%) were significantly higher than that after surgery (1.5%). After ESWL (17.6%), abdominal pain recurred significantly more frequently than after surgery (2.2%). Stones and abdominal pain most often recurred within 3 years after ESWL and endoscopy. CONCLUSIONS: First-line treatment of pancreatic stones should be ESWL alone or with endoscopy because of its minimal invasiveness and low incidence of early complications. Surgery should be performed on patients in whom ESWL and endoscopy failed.

Deregulation of the histone demethylase JMJD2A is involved in human carcinogenesis through regulation of the G(1)/S transition.

Although a number of JmjC-containing histone demethylases have been identified and biochemically characterized, pathological roles of their dysfunction in human disease such as cancer have not been well elucidated. Here, we report the Jumonji domain containing 2A (JMJD2A) is integral to proliferation of cancer cells. Quantitative real-time PCR analysis revealed higher expression of JMJD2A in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P<0.0001). Immunohistochemical analysis also showed positive staining for JMJD2A in 288 out of 403 lung cancer cases, whereas no staining was observed in lung normal tissues. Suppression of JMJD2A expression in lung and bladder cancer cells overexpressing this gene, using specific siRNAs, inhibited incorporation of BrdU and resulted in significant suppression of cell growth. Furthermore, JMJD2A appears to directly transactivate the expression of some tumor associated proteins including ADAM12 through the regulation of histone H3K9 methylation. As expression levels of JMJD2A are low in normal tissues, it may be feasible to develop specific inhibitors targeting the enzyme as anti-tumor agents which should have a minimal risk of adverse reaction.

Histone lysine methyltransferase SETD8 promotes carcinogenesis by deregulating PCNA expression.

Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.

Predictive factors for cholangiocarcinoma associated with hepatolithiasis determined on the basis of Japanese Multicenter study.

AIM: The aim of this study was to delineate predictive factors for cholangiocarcinoma in patients with hepatolithiasis, and to establish optimal management for hepatolithiasis from the viewpoint of carcinogenesis on the basis of a Japanese nationwide survey for hepatolithiasis. METHODS: The Hepatolithiasis Research Group was organized in 2006 by the Ministry of Health, Labour and Welfare of Japan, and conducted a nationwide survey. The research group collected data on 336 cases of hepatolithiasis in 2006, in a cross-sectional survey involving 2592 institutions in Japan. Predictive factors for cholangiocarcinoma associated with hepatolithiasis were analyzed by univariate and multivariate analyses of clinicopathological and therapeutic factors. RESULTS: Twenty-three patients had cholangiocarcinoma. Histories of choledocoenterostomy and liver atrophy were found to be significantly predictive factors by multivariate analysis. In 87.5% of cases of cholangiocarcinoma with liver atrophy, cholangiocarcinoma was located in the atrophic lobes. The method of reconstruction did not affect the incidence of cholangiocarcinoma (choledochojejunostomy vs. choledochoduodenostomy; side-to-end vs. side-to-side anastomosis). CONCLUSIONS: Choledocoenterostomy and liver atrophy may increase the risk of developing cholangiocarcinoma. Choledocoenterostomy is thus contraindicated in patients with hepatolithiasis. An aggressive resection strategy is recommended for an atrophic segment.

Which is the preference of choice either life with a stoma or evacuatory disorder following rectal cancer surgery?

BACKGROUND/AIMS: Low anastomosis with covering stoma is the standard operation for low rectal carcinoma. Some patients experience severe anorectal disorder, which makes us consider whether stoma closure should be performed or not. There was no comparative study between life with a stoma and life with evacuatory disorder. METHODOLOGY: Covering stoma was closed at 4 to 6 months after ultra-low anterior resection. Forty-five patients were evaluated by questionnaire in terms of their bowel evacuation and anorectal manometry before ultra-low anterior resection and 6 months after stoma closure. They were also questioned about their subjective preference regarding the life before and after stoma closure. RESULTS: After stoma closure, frequency of daily bowel movement was significantly increased up to 5 times (range 2-15). Incontinence score was also significantly worsened from 0 to 8, postoperatively. All patients complained of any influence in their social life. Ninety-three percent (42/45) of the cases were dissatisfied with evacuation, postoperatively. Eighty-nine percent (40/45) of the cases had postoperative evacuatory disorder defined by the present study. Under these backgrounds, all patients replied that evacuation from the anus was superior to life with a stoma even during postoperative evacuatory disorder status. CONCLUSION: Even when patients had evacuatory disorder, they preferred life without a stoma according to their subjective opinion.

Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker.

The emphasis in anticancer drug discovery has always been on finding a drug with great antitumor potential but few side-effects. This can be achieved if the drug is specific for a molecular site found only in tumor cells. Here, we find the enhancer of zeste homolog 2 (EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real-time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non-small-cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (P = 0.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD-18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor-specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow-up in some lung cancer patients after surgical treatment.

Neurophysiologic investigation of anal function following double stapling anastomosis.

BACKGROUND AND AIM: Manual dilatation of the anal sphincter and transanal introduction of the circular stapling device are required for intraluminal stapling anastomosis. This procedure has been regarded as one of the causes of postoperative evacuatory disorder in low anterior resection. However, there has been no evidence of this matter. Therefore, we conducted this study to clarify the impact of the procedure of stapling anastomosis on postoperative anal function. METHODS: Twenty-five cases with sigmoid colon cancer underwent potentially curative sigmoid colectomy with stapling anastomosis (ST group) and 20 cases with hand-sewn anastomosis (non-ST group). The patients were questioned regarding the daily frequency of bowel movement, the presence of urgency and soiling, and Wexner's incontinence score. Anorectal manomatry and pudendal nerve terminal motor latency were also evaluated. The patients' questionnaire and physiologic examinations were prospectively obtained before, and 1 and 6 months after the operation. RESULTS: Postoperative bowel habit was graded as satisfied in 92% (23/25 patients) in the ST group and 90% (18/20 patients) in the non-ST group. There was no significant difference between the 2 groups in terms of presence of fecal incontinence, discrimination of gas and stool, and daily frequency of bowel movement. In anal manometry, there was no significant difference between the 2 groups regarding the resting and squeezing anal canal sphincter pressures at 1 and 6 months postoperatively. Pudendal nerve terminal motor latency showed their latency from 2.0 to 2.5 ms throughout the periods, and there was no difference between the 2 groups before, and 1 and 6 months after the operation. CONCLUSION: Stapling anastomosis does not affect anal function in the early postoperative period.

Surgical indications and procedures for bleeding peptic ulcer.

Recent important insights into the surgical treatment of bleeding peptic ulcer are reviewed in this article. Although the widespread use of endoscopic treatment and interventional radiology has reduced the number of surgical cases, surgery still plays a pivotal role in managing bleeding peptic ulcer. Failure to stop the bleeding by endoscopy and/or interventional radiology is the most important indication for emergency surgery. An early elective/planned surgery after the initial endoscopic control to prevent life-threatening rebleeding seems justified in patients who have risk factors for rebleeding, although its true efficacy still remains controversial. The surgical procedures in emergency situations should be limited to safe hemostasis. The addition of acid-reduction surgery may be unnecessary as a result of the increasing utilization of proton pump inhibitors. Angiographic embolization may be a less invasive alternative to surgery, and may further enhance endoscopic hemostasis.

Quality assurance of pelvic autonomic nerve-preserving surgery for advanced lower rectal cancer--preliminary results of a randomized controlled trial.

BACKGROUND: Pelvic autonomic nerve-preserving (PANP) lateral node dissection (LLND) has been introduced in rectal cancer surgery in Japan, but quality assurance of this approach has not been tested by randomized controlled trials. METHODS: Patients with advanced lower rectal cancer were randomized either to complete PANP + LLND surgery combined with intraoperative radiotherapy (preserved group; n = 28) or to pelvic autonomic nerve resection + LLND surgery (resected group; n = 27). Operation-related parameters were compared statistically. RESULTS: Patient and tumor characteristics were well comparable. The incidence of anastomotic breakdown, intrapelvic abscess, and small bowel obstruction was not different between the two groups. In the preserved group, no patients had ureteral stenosis, pelvic bone fractures, or peripheral neuropathy due to intraoperative radiotherapy. Sphincter-preserving operation was possible with similar ratio in both groups. Adjuvant chemotherapy was given with similar ratio in both groups. The average operation time was 513 minutes in the preserved group and 409 minutes in the resected group, with a significant difference between the two groups. The average amount of hemorrhage was not different significantly between the preserved group (996 ml) and the resected group (970 ml). Circumferential resection margin status and operative curability were similar between the two groups. The average number of harvested and metastatic nodes in the mesentery and pelvic sidewall was not different significantly between the two groups. CONCLUSIONS: This study revealed, for the first time, that the surgical quality of PANP + LLND is the same as pelvic autonomic nerve resection + LLND.

Rationale of pelvic autonomic nerve preservation in rectal cancer surgery based on immunohistochemical study.

PURPOSE: Previous studies revealed that the incidence of cancer cell involvement along the pelvic autonomic nerves ranged from 4 to 14%. However, patients' profiles and methodologies differed among the studies. This study was conducted to clarify the incidence of cancer cell involvement in and around the pelvic autonomic nerves immunohistochemically. METHODS: Immunohistochemical staining was performed on pelvic autonomic nerve specimens resected from 17 patients with p-Stage I-III lower rectal cancers. Antibodies used were pan-cytokeratin (AE1/AE3) for staining cancer cells, S-100 for autonomic nerves, and D2-40 for lymphatic vessels. Lymphatic permeation around the pelvic autonomic nerves was defined as present when AE1/AE3-positive cells were detected in D2-40-stained lymphatic vessels. The presence of metastasis to the interstitial tissue or contaminants was also recorded. RESULTS: TNM staging was stage I in 1, stage II in 5, and stage III in 11 cases, respectively. No cases had lymphatic permeation or metastasis to the interstitial tissue in and around the pelvic autonomic nerves. Cancer cell contaminants were seen in four cases (23%). In three cases (18%), metastatic nodes were located at the root of the middle rectal artery, very close to the pelvic autonomic nerves. CONCLUSIONS: Cancer cell involvement was not seen in and around the pelvic autonomic nerves, suggesting that complete pelvic autonomic nerve preservation may be feasible, unless nerves are invaded by the tumor. In some cases, however, metastatic nodes were seen very close to the nerves. Meticulous lymph node dissection along the pelvic autonomic nerves is mandatory.

Advanced ampullary carcinoma showing complete response to S-1: report of a case.

We report a case of advanced ampullary carcinoma with para-aortic lymph node metastasis, which showed a complete response to S-1. The patient underwent cholecystectomy and Roux-en-Y choledochojejunostomy, and was then given S-1 orally 80 mg daily for 14 days, followed by 7 days of rest. After four cycles of the S-1 chemotherapy, both the tumor and the swollen paraaortic lymph node had completely disappeared. An additional six cycles were given at the request of the patient. No adverse effects were seen during the S-1 chemotherapy, and the patient has been free of the disease for the 27 months since its completion. Thus, S-1 monotherapy may be considered as a chemotherapeutic strategy for unresectable ampullary carcinoma, although large-scale studies will be required to confirm its true efficacy.

Comparison of functional and clinical outcomes: colonic J-pouch vs. coloplasty in patients with low rectal cancer.

BACKGROUND/AIMS: Coloplasty has been reported as substitution to colonic J-pouch when it is difficult to fashion. However, previous studies showed conflicting results with reference to functional outcome. METHODOLOGY: Forty-seven patients with low rectal cancer were involved in this study from July 2000 to December 2006. Coloplasty was performed when colonic J-pouch was hard to construct due to technical difficulty such as short masenterium and narrow pelvis. Clinical and functional evaluations were performed before the operation and 12 months after stoma closure. RESULTS: Colonic J-pouch was abandoned in 12 of 37 cases (26%) due to short colon and mesenterium in 8 cases and narrow pelvis in 4 cases. Frequency of daily bowel movement was significantly increased in both groups but no difference between the groups. Anal sphincter tones were maintained even after the operation. Moreover, no difference was noted between the groups. Anal canal length and sensory factor were also maintained. Volumetric factors such as maximum tolerable volume and neo-rectal capacity showed significant changing before and after the operation. However, there was no significant difference between the groups. CONCLUSIONS: Coloplasty could be a possible substitution to colonic J-pouch in patients with low rectal cancer from functional point of view.

Single-port endoscopic cholecystectomy: a bridge between laparoscopic and translumenal endoscopic surgery.

BACKGROUND AND OBJECTIVE: The intentional puncture of the normal viscera is likely the most important issue limiting the widespread use of natural orifice translumenal endoscopic surgery (NOTES). We developed a new procedure for cholecystectomy using a flexible endoscope via a single port placed in the abdominal wall without visceral puncture (single-port endoscopic cholecystectomy; SPEC) as a bridge between laparoscopic surgery and NOTES. This study aimed to evaluate the technical feasibility of SPEC. METHODS: Five pigs were subjected to SPEC. An endoscope was inserted through a 12-mm port placed in the right upper abdomen. After grasping and retracting the gallbladder using a 2-mm retractor that was directly introduced into the peritoneal cavity, gallbladder excision with ligation of the cystic artery and duct using endoclips was carried out. RESULTS: A complete gallbladder excision was carried out easily and safely in all cases. No major adverse events occurred. The mean operating time was 67 min (range 52-84 min). CONCLUSIONS: SPEC is a technically feasible procedure. It is simpler, easier, and safer than NOTES cholecystectomy. SPEC could be a less invasive alternative to the conventional four-port laparoscopic cholecystectomy.

Modified liver hanging maneuver with extraparenchymal isolation of the middle hepatic vein in left hepatectomy.

BACKGROUND/PURPOSE: The liver hanging maneuver has been employed mostly for right hepatectomy. After the space between the inferior vena cava and the liver parenchyma is bluntly dissected, a tape is passed through the space, with the upper end of the tape between the right hepatic vein and middle hepatic vein (MHV). We devised a modified maneuver with tape repositioning between the MHV and left hepatic vein (LHV) after extraparenchymal isolation of the MHV, in left hepatectomy with the caudate lobe and without the MHV. METHODS: The ligamentum venosum is divided. Traction of the cranial stump of the ligament in the cranial and left direction shows an avascular plane between the LHV and MHV, and enables the taping of each MHV and LHV. Finally, the upper end of the hanging tape is repositioned. RESULTS: This maneuver was successfully performed in three patients. CONCLUSIONS: The present maneuver is feasible and useful in left hepatectomy.

Endoscopic full-thickness resection with laparoscopic assistance as hybrid NOTES for gastric submucosal tumor.

BACKGROUND AND OBJECTIVE: Laparoscopic wedge resection using a linear stapler is widely accepted as a treatment for gastric submucosal tumor (SMT). Although this surgery is simple, it can lead to excessive normal tissue removal. To avoid the latter, we have introduced endoscopic full-thickness resection with laparoscopic assistance, known as laparoscopy-assisted endoscopic full-thickness resection (LAEFR). Herein, we present the preliminary results of LAEFR for gastric SMT patients. METHODS: Four patients with gastric SMT underwent LAEFR. LAEFR consists of four major procedures: (1) a circumferential incision as deep as the submucosal layer around the lesion by the endoscopic submucosal dissection technique, (2) endoscopic full-thickness (from the muscle layer to the serosal layer) incision around the three-fourths or two-thirds circumference on the above-mentioned submucosal incision under laparoscopic supervision, (3) completion of the full-thickness incision laparoscopically from inside the peritoneal cavity, and (4) handsewn closure of the gastric-wall defect. RESULTS: LAEFR was successfully carried out without any intraoperative or postoperative adverse events. Mean operating time and estimated blood loss were 201 min and 27 mL, respectively. Contrast roentgenography on postoperative day 3 showed neither gastric deformity nor disturbance of gastric emptying in all the patients. CONCLUSIONS: LAEFR may be considered one of the so-called hybrid natural orifice transluminal endoscopic surgery (NOTES) techniques because a peroral endoscope advances into the peritoneal cavity. LAEFR enabled whole-layer excision as small as possible with an adequate margin. LAEFR is a safe and minimally invasive treatment for patients with gastric SMT, and could be a more reasonable and economical alternative to other laparoscopic procedures.

Falciform ligament in pancreatoduodenectomy for protection of skeletonized and divided vessels.

BACKGROUND/PURPOSE: Pancreatic fistula, which is one of the main causes of late postpancreatectomy hemorrhage (PPH), is a common complication of pancreatoduodenectomy (PD). It may erode the anastomosis site and vascular wall in its vicinity, resulting in pseudoaneurysm formation and/or the rupture of major vessels. To protect the vessels near the area for pancreaticojejunostomy from potential pancreatic fistula, we have adopted a surgical option by which such vessels are separated from the pancreaticojejunostomy using a pedicled falciform ligament. We reviewed 36 patients who underwent PD that included this option. METHODS: After the PD was completed (before reconstructions), the pedicled falciform ligament was spread widely on the major vessels exposed during resection, and was fixed to the surrounding retroperitoneal connective tissue. These procedures enabled the complete separation of these vessels from the pancreaticojejunostomy. RESULTS: The mobilization and placement of the falciform ligament in the space between the pancreaticojejunostomy and the major vessels were successfully carried out without any complications. Although ten (28%) patients developed pancreatic fistula and three (8%) developed intraabdominal infection, none of the patients developed late PPH. CONCLUSIONS: The present surgical option is technically simple and easy, and may be an effective prophylactic measure against late PPH following PD.

Pancreatic duct holder for facilitating duct-to-mucosa pancreatojejunostomy after pancreatoduodenectomy.

Duct-to-mucosa pancreatojejunostomy after pancreatoduodenectomy may be technically difficult, particularly in cases in which the remnant pancreas is soft with a small main pancreatic duct. We devised a pancreatic duct holder for duct-to-mucosa pancreatojejunostomy. The holder has a cone-shaped tip. A one-third circle of the tip is cut away, which makes a slit. As the tip is inserted gently into the pancreatic duct, the duct can be adequately expanded. The holder provides a good surgical field for anastomosis. A slit of the tip allows needle insertion. The holder facilitates stitches of the jejunum also. Twelve patients underwent pancreatoduodenectomy, followed by duct-to-mucosa pancreatojejunostomy using the holder. The holder allowed 8 or more stitches in duct-to-mucosa anastomosis, even in patients with a small pancreatic duct. No patients developed prolonged pancreatic leakage or pancreatic fistula postoperatively. In conclusion, the pancreatic duct holder is a simple and useful tool for facilitating duct-to-mucosa pancreatojejunostomy.