Comparison of two primer-probe sets of Fusobacterium nucleatum using droplet digital polymerase chain reaction for the detection of colorectal neoplasia from faecal samples.

BACKGROUND: Although faecal DNA testing of Fusobacterium nucleatum (Fn) is expected to be useful for colorectal neoplasia detection, there is no standardized quantification method of Fn. We performed this study to establish a possible standardized method. METHODS: In this study, 322 participants including 71 subjects without colorectal neoplasia (control group), 31 patients with non-advanced colorectal adenoma, 93 patients with advanced colorectal adenoma, and 127 patients with colorectal cancer were enrolled. Faecal Fn were quantified by droplet digital PCR (ddPCR) using two PCR primer-probe sets reported previously that are tentatively named Fn1 and Fn2. Fn1 has been used in ddPCR by us and Fn2 has been widely used in quantitative real-time PCR. RESULTS: The Fn copy number using Fn1 was five times higher than that using Fn2, with a linear relationship shown between them. Receiver operating characteristic curve analysis showed the area under the curve (AUC) to be almost the same between Fn1 and Fn2 in discriminating between the control group and the colorectal cancer group (AUC = 0.81 and 0.81, respectively), and between the control/non-advanced colorectal adenoma group and the advanced colorectal adenoma/colorectal cancer group (AUC = 0.74 and 0.74, respectively). CONCLUSIONS: As the diagnostic performance was quite similar between Fn1 and Fn2, ddPCR-based Fn testing using Fn1 and Fn2 could be a possible standardized method for a colorectal neoplasia screening test, considering that Fn levels quantified by Fn1 are about five times higher than those by Fn2.

Fecal DNA Testing of TWIST1 Methylation Identifies Patients With Advanced Colorectal Adenoma Missed by Fecal Immunochemical Test for Hemoglobin.

INTRODUCTION: We have reported previously that fecal DNA testing of TWIST1 methylation in combination with the fecal immunochemical test for hemoglobin (FIT) (combination test) is useful for colorectal neoplasia screening. In this study, using larger sample sizes, we studied the clinical performance of the combination test for the detection of colorectal neoplasia and, especially, advanced colorectal adenoma. METHODS: We performed a prospective study in which FIT, fecal DNA testing of TWIST1 methylation, and colonoscopy were performed on 372 patients with colorectal neoplasia and 71 subjects without colorectal neoplasia. We assessed the individual clinical performance of each of FIT and fecal DNA testing of TWIST1 methylation and of the combination test for the detection of colorectal neoplasia including advanced adenoma based on morphologic subtypes. RESULTS: The FIT alone had a sensitivity of 7.5% (3/40) for nonadvanced adenoma, 32.3% (41/127) for advanced adenoma, and 93.7% (192/205) for colorectal cancer and a specificity of 87.3% (62/71). The combination test had a sensitivity of 35.0% (14/40) for nonadvanced adenoma, 68.5% (87/127) for advanced adenoma, and 95.6% (196/205) for colorectal cancer and a specificity of 80.3% (57/71). For morphological subtypes of advanced adenoma, the sensitivity of FIT was only 28.2% (20/71) for polypoid type and 16.1% (5/31) for nonpolypoid type, whereas the combination test increased the sensitivities to 64.8% (46/71) and 71.0% (22/31), respectively. DISCUSSION: The combination of the fecal DNA test with FIT seemed to be useful to detect colorectal neoplasia and, especially, advanced adenoma of the nonpolypoid type.

Objective Assessment of the Utility of Chromoendoscopy with a Support Vector Machine.

PURPOSE: The utility of chromoendoscopy for early gastric cancer (GC) was determined by machine learning using data of color differences. METHODS: Eighteen histopathologically confirmed early GC lesions were examined. We prepared images from white light endoscopy (WL), indigo carmine (Indigo), and acetic acid-indigo carmine chromoendoscopy (AIM). A border between cancerous and non-cancerous areas on endoscopic images was established from post-treatment pathological findings, and 2000 pixels with equivalent luminance values were randomly extracted from each image of cancerous and non-cancerous areas. Each pixel was represented as a three-dimensional vector with RGB values and defined as a sample. We evaluated the Mahalanobis distance using RGB values, indicative of color differences between cancerous and non-cancerous areas. We then conducted diagnosis test using a support vector machine (SVM) for each image. SVM was trained using the 100 training samples per class and determined which area each of 1900 test samples per class came from. RESULTS: The means of the Mahalanobis distances for WL, Indigo, and AIM were 1.52, 1.32, and 2.53, respectively and there were no significant differences in the three modalities. Diagnosability per endoscopy technique was assessed using the F1 measure. The means of F1 measures for WL, Indigo, and AIM were 0.636, 0.618, and 0.687, respectively. AIM images were better than WL and Indigo images for the diagnosis of GC. CONCLUSION: Objective assessment by SVM found AIM to be suitable for diagnosis of early GC based on color differences.

Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias.

Although methylated TWIST1 is a biomarker of colorectal neoplasia, its detection from serum samples is very difficult by conventional bisulfite-based methylation assays. Therefore, we have developed a new methylation assay that enables counting of even one copy of a methylated gene in a small DNA sample amount without DNA bisulfite treatment. We performed this study to evaluate the sensitivity and specificity of serum DNA testing by the new methylation assay in combination with and without the fecal immunochemical test for hemoglobin for the detection of colorectal neoplasia. This study comprised 113 patients with colorectal neoplasia and 25 control individuals. For the new methylation assay, DNA was treated in two stages with methylation-sensitive restriction enzymes, followed by measurement of copy numbers of hTERT and methylated TWIST1 by multiplex droplet digital PCR. The fecal immunochemical test had a sensitivity of 8.0% for non-advanced adenoma, 24.3% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 88.0%. The new assay had a sensitivity of 36.0% for non-advanced adenoma, 30.0% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 92.0%. Combination of the both tests increased the sensitivity to 40.0%, 45.7%, and 72.2% for the detection of non-advanced adenoma, advanced adenoma, and colorectal cancer, respectively, and resulted in a specificity of 84.0%. Combination of both tests may provide an alternative screening strategy for colorectal neoplasia including potentially precancerous lesions and colorectal cancer.

Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population.

BACKGROUND: Accumulating evidence shows an overabundance of Fusobacterium nucleatum in colorectal tumor tissues. However, the correlation between the absolute copy number of F. nucleatum in colorectal cancer tissues and colorectal cancer progression is unclear from previous reports. Therefore, we performed a study to compare the abundance of F. nucleatum in colorectal tissues with clinicopathologic and molecular features of colorectal cancer. METHODS: We collected 100 colorectal cancer tissues and 72 matched normal-appearing mucosal tissues. Absolute copy numbers of F. nucleatum were measured by droplet digital PCR. RESULTS: The detection rates of F. nucleatum were 63.9% (46/72) in normal-appearing mucosal tissues and 75.0% (75/100) in CRC tissue samples. The median copy number of F. nucleatum was 0.4/ng DNA in the normal-appearing colorectal mucosa in patients with colorectal cancer and 1.9/ng DNA in the colorectal cancer tissues (P = 0.0031). F. nucleatum copy numbers in stage IV colorectal cancer tissues were significantly higher than those in the normal-appearing mucosa in patients with colorectal cancer (P = 0.0016). The abundance of F. nucleatum in colorectal cancer tissues correlated with tumor size and KRAS mutation and was significantly associated with shorter overall survival times; this trend was notable in the patients with stage IV colorectal cancer. Focusing on normal-appearing mucosa in the patients with colorectal cancer, the F. nucleatum copy number was significantly higher in the patients with stage IV rather than stages I-III. CONCLUSION: These results suggest that determining F. nucleatum levels may help predict clinical outcomes in colorectal cancer patients. Further confirmatory studies using independent datasets are required to confirm our findings.

[Case with gradually enlarging lymphoepithelial cyst of the pancreas that necessitated enucleation].

A 54-year-old man exhibited a pancreatic mass on abdominal ultrasonography. Diagnostic imaging with endoscopic ultrasonography, computed tomography, and magnetic resonance imaging revealed that the mass comprised various internal structures and was not connected to the pancreatic duct. Over a period of 4 years and 2 months, the mass increased from 22mm to 32mm in diameter. Laparoscopic enucleation was performed, and a histopathological diagnosis of a lymphoepithelial cyst (LEC) of the pancreas was obtained. LEC is rare and seldom reported in the literature. Although it is considered to be benign, most case reports indicate that they tend to increase in size. This indicates that LEC should be carefully monitored if surgery is not performed after diagnosis.

[Is the incidence of postoperative pulmonary thromboembolism in Japan lower than that in the western countries?].

BACKGROUND: The Japanese Society of Anesthesiologists (JSA) has investigated the incidence of postoperative pulmonary thromboembolism (PTE) for several years. METHODS: According to the JSA project, we surveyed the annual incidence of postoperative PTE in Kanazawa Medical University Hospital in 2008. RESULTS: PTE was diagnosed radiographically in 6 out of 2339 patients, who were > or =15 years of age and had undergone surgical procedures under general anesthesia. The incidence was 0.257% and approximately tenfold that reported by the JSA. All the patients were subjected to lower extremity surgery. Two patients went into shock after they had first walked postoperatively. Symptoms of the other four patients were nonspecific but surgeons in charge suspected PTE. CONCLUSIONS: Our data suggest that the actual incidence of postoperative PTE in Japan may be far greater than that reported by the JSA.