Hiroomi Ogawa

Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma

Epidermal growth factor receptor (EGFR) gene mutations have been reported to be clinically significant in non-small cell lung cancer (NSCLC). However, because most previous studies focused only on adenocarcinomas, EGFR mutations in other histotypes are poorly investigated. We evaluated the frequency of EGFR gene mutations in squamous cell carcinoma (SCC) and its clinicopathological features. In total, 89 frozen tumor specimens that had been first diagnosed as SCCs, were examined for EGFR mutations in exons 19 and 21 using direct sequencing, PNA-enriched sequencing and SmartAmp2. Additionally, pathological investigation, including immunostaining for p63 and TTF-1, alcian blue staining and EGFR mutation-specific immunohistochemistry in mutation-positive samples was also performed. The frequency of EGFR mutations was 5.6% (5/89); all mutations were deletions in EGFR exon 19. Immunohistological investigation of these samples revealed that two of five were positive for p63 and TTF-1 staining, and showed production of mucin, as evidenced by alcian blue staining. Consequently, three of the samples were considered to be true SCC at final pathological diagnosis, while the remaining two samples were revised to adenosquamous carcinoma and adenocarcinoma. The final frequency of the EGFR mutations in true SCC was 3.4% (3/87). In conclusion, EGFR mutations were found in a small, but significant, number of SCC tumor samples and thus EGFR mutational analysis was useful in the accurate diagnosis of SCC. Our data demonstrate that EGFR mutational analysis should be performed not only in adenocarcinoma, but also in SCC to allow accurate diagnosis and treatment.

Three-dimensional computed tomography for analyzing the vascular anatomy in laparoscopic surgery for right-sided colon cancer.

Background: The mesenteric vessels have many branching patterns. This study clarified the anatomic relationship between the superior mesenteric vein (SMV), the right colic artery (RCA), and the ileocolic artery (ICA) using 3-dimensional computed tomography (3D-CT). The relationship between the RCA and the right colic vein (RCV) was also examined. Methods: Between April 2006 and July 2011, all patients with colorectal cancer underwent multidetector computed tomography (MDCT) before laparoscopic surgery. The 100 most recent consecutive cases were analyzed. 3D-CT images were made by combining arterial angiography, venous angiography, colonography, tumor, lymph node, and duodenal images. Results: The RCA branched from the SMA in 37 cases (37%); of these, 21 had an ICA that crossed anterior to the SMV and 16 had an ICA that crossed posterior. When the ICA crossed anterior to the SMV, all had an RCA that crossed anterior to the SMV, and no posterior RCA was seen. Furthermore, the RCV joined the SMV in 10 cases (27%) and the gastrocolic trunk in 27 cases (73%). Conclusions: Our study clarified the anatomic variety of the vessels in right-sided colon cancer. Preoperative 3D-CT is useful for understanding the anatomy to ensure a safe, precise operation.

Eprobe-mediated screening system for somatic mutations in the KRAS locus

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence ‘Eprobe’ capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05–0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.

Rapid Detection of SNP (c.309T>G) in the MDM2 Gene by the Duplex SmartAmp Method

Background Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. Methodology In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named “Duplex SmartAmp,” which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. Results and Conclusions By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

Laparoscopic resection of a paraganglioma located on the border of the thoracic and abdominal cavities using a transabdominal-transdiaphragmatic approach

We treated a 64‐year‐old woman with high blood pressure. Catecholamine metabolite levels were elevated in the blood and urine. CT revealed a densely stained tumor on the right side of the descending aorta dorsal to the inferior vena cava. PET‐CT revealed abnormal accumulation of 18F‐fluorodeoxyglucose, and 123I‐meta‐iodo‐benzylguanidine uptake was apparent on scintigraphy. The tumor was determined to be a paraganglioma located on the border between the thoracic and abdominal cavities, and laparoscopic tumorectomy was performed. The patient was placed in the left lateral position. The right lobe of the liver was turned over, and we cut the diaphragm to expose the front of the tumor. We resected the straight artery flowing in from the aorta and removed the tumor safely. Herein, we describe the removal of a paravertebral paraganglioma located in the border of the thoracic and abdominal cavities with a laparoscopic transabdominal‐transdiaphragmatic approach.

Successful surgical treatment of left atrioesophageal fistula following atrial ablation

A 69-year-old male had catheter-based ablation for atrial fibrillation. He was admitted with high fever and had neurological disorder; he was diagnosed with atrioesophageal fistula by CT scan. Intraoperative findings showed that the fistula existed adjacent to the left lower pulmonary vein with a vegetation. The esophageal fistula was repaired, and the left atrial fistula was closed. A nasogastric tube tip was placed in the esophagus for decompression and advanced into the stomach for nutritional support. After vomiting, the patient showed loss of consciousness and left hemiplegia. CT scan revealed a micro-air embolism to the brain. The nasogastric tube tip was pulled back into the esophagus. Gastrointestinal fiberscopy showed a pinhole at the fistula, and a percutaneous endoscopic gastrostomy was made. After conservative treatment, the esophageal fistula was closed and mediastinitis was improved. He was discharged with a little neurological deficit.

A novel one-step lens cleaning device using air and water flow for endoscopic surgery

In a surgical operation requiring endoscopy, it is essential to obtain a clear endoscopic view. However, it is often disturbed by the contamination on the lens during the surgery. No device can clean the lens surface simply and completely. Many surgeons are hampered by the impaired view and the distraction by the repeated cleaning of the lens. Therefore, we developed a novel endoscope cleaning device to address this problem. The device was made of 3D-printed rubber-like plastic. It contains a syringe filled with saline and an aspiration system. It would be used intraoperatively to wash the lens surface in a few seconds with rapid flow of water and air. The cleaning ability of the device was evaluated using mayonnaise with adenosine triphosphate (ATP) as a model contaminant. The gauze-wiping maneuver was selected as control. After each maneuver, the clarity of the endoscopic view was evaluated, and residual contaminants were assessed quantitatively with ATP assay. The cleaning device obtained a crisp and clear view and eliminated the contaminant on the lens every time after a single cleaning maneuver. The gauze-wiping maneuver required for the lens to be wiped at least three times to obtain a clear view, and even then, some contaminants remained. Repeated contamination and cleaning using gauze led to accumulation of contaminants on the lens, which resulted in difficulty in cleaning the lens as the operation proceeded. The cleaning device did not show such accumulation. Our novel cleaning device with air and water flow has been shown to wash out the lens contaminants completely and immediately in a simple manner. It is expected to improve the safety and cost-effectiveness of endoscopic surgery.

Prognostic role of BiP/GRP78 expression as ER stress in patients with gastric adenocarcinoma

PURPOSE The glucose-regulated protein 78 (GRP78), also referred to as immunoglobulin heavy chain binding protein (BiP) (BiP/GRP78), is a major molecular chaperone in the endoplasmic reticulum (ER) and is extensively expressed in human neoplasms. Although the enhanced expression of BiP/GRP78 has been described to be associated with poor prognosis in gastric cancer (GC), details regarding its prognostic significance remain unclear. The aim of this study was to elucidate the prognostic role of BiP/GRP78 in patients with GC. METHODS Study subjects included 328 patients who underwent surgical resection. Tumor specimens of primary tumors underwent immunohistochemical staining for BiP/GRP78. RESULTS BiP/GRP78 was highly expressed in 57% (188/328) of patients. High expression of BiP/GRP78 was significantly associated with older age, male, disease staging, T factor, lymph node metastases, differentiation, lymphatic permeation, and vascular invasion. According to univariate analysis, age, disease staging, T factor, N factor, lymphatic permeation, vascular invasion, and BiP/GRP78 expression were significant prognostic factors for OS. In particular, high BiP/GRP78 expression was proven to be a significant predictor of prognosis in patients with older age, female sex, early disease stage, T1-2 factor, well or moderately differentiated tumors, and negative vascular invasion. CONCLUSION BiP/GRP78 is significantly associated with tumor aggressiveness and progression. The increased expression of BiP/GRP78 was identified as an independent factor for predicting poor OS in patients with early stage of disease, especially T1-2 factor.

Securing the surgical field in laparoscopic pancreatectomy using a Penrose drain and Endo Close.

Introduction: We adopted the use of Penrose drains and Endo Close to secure a good surgical field during laparoscopic pancreatectomy. Methods: We used a Penrose drain with threads ligated on both ends to suspend the stomach. We then pulled the threads out of the body from the side of the trocar or from besides the xiphisternum by using Endo Close. In most cases, 2 Penrose drains were used to retract the stomach. When the greater omentum on the left side of the cardia still blocks the surgical field, we sewed the posterior wall of the stomach onto the dome of the diaphragm. Results: The use of 2 Penrose drains and Endo Close were effective to retract the stomach in most cases. However, in 3 cases, we needed to additionally sew the stomach onto the diaphragm to fully open up the field. Conclusion: This is a simple and effective method to ensure a good surgical field.

Modified Pringle Maneuver Applicable for Laparoscopic Hepatectomy.

INTRODUCTION We present a widely applicable technique of the modified Pringle maneuver to reduce blood loss for laparoscopic hepatectomy. METHODS We use a drip-infusion tube and wrap it around the hepatoduodenal ligament. In the modified Pringle maneuver ① (m-Pringle ①), we use a 60 cm long tube. Both ends of the tube are led out from the side of the umbilical port, then pulled and clipped with Pean forceps to interrupt blood flow. In the modified Pringle maneuver ② (m-Pringle ②), we use a 20 cm long tube with silk threads tied at both ends. The threads were led extraperitoneally in the same manner. RESULTS Although blood flow was sufficiently interrupted, CO2 leak occurred in 14 of 60 cases in m-Pringle ①. Blood flow was interrupted and intra-abdominal pressure was kept in all 10 patients in m-Pringle ②. CONCLUSIONS These maneuvers require no extra port, and tube pulling and releasing is readily performed from outside the body.