Analysis of colorectal cancer‐related mutations by liquid biopsy: Utility of circulating cell‐free DNA and circulating tumor cells

We recruited 56 colorectal cancer patients and compared the mutational spectrum of tumor tissue DNA, circulating cell‐free DNA (ccfDNA) and circulating tumor cell (CTC) DNA (ctcDNA) to evaluate the potential of liquid biopsy to detect heterogeneity of cancer. Tumor tissue DNA, ccfDNA, and ctcDNA were extracted from each patient and analyzed using next‐generation sequencing (NGS) and digital PCR. To maximize yields of CTC, three antibodies were used in the capture process. From 34 untreated patients, 53 mutations were detected in tumor tissue DNA using NGS. Forty‐seven mutations were detected in ccfDNA, including 20 not detected in tissues. Sixteen mutations were detected in ctcDNA, including five not detected in tissues. In 12 patients (35.3%), mutations not found in tumor tissues were detected by liquid biopsy: nine (26.5%) in ccfDNA only and three (8.8%) in ctcDNA only. Combination analysis of the two liquid biopsy samples increased the sensitivity to detect heterogeneity. From 22 stage IV patients with RAS mutations in their primary tumors, RAS mutations were detected in 14 (63.6%) ccfDNA and in eight (36.4%) ctcDNA using digital PCR. Mutations not detected in primary tumors can be identified in ccfDNA and in ctcDNA, indicating the potential of liquid biopsy in complementing gene analysis. Combination analysis improves sensitivity. Sensitivity to detect cancer‐specific mutations is higher in ccfDNA compared with ctcDNA.     

Association of gBRCA1/2 mutation locations with ovarian cancer risk in Japanese patients from the CHARLOTTE study

Whether germline (g) breast cancer susceptibility gene (BRCA) mutations are located within or outside the ovarian cancer cluster region (OCCR) (1380‐4062 bp for gBRCA1, and between 3249‐5681 bp and 6645‐7471 bp for gBRCA2) may influence risk variations for ovarian cancers. This ad hoc analysis of the CHARLOTTE epidemiological study in Japan assessed the distribution of gBRCA1/2 mutations in patients with newly diagnosed ovarian cancer, and investigated an association between gBRCA1/2 mutation locations and ovarian cancer risk. Differences in patient background and clinical characteristics in subgroups stratified by gBRCA1/2 mutation locations were also evaluated. We analyzed the data of 93 patients (14.7%) from the CHARLOTTE study who were positive for gBRCA1/2 mutations. After excluding 16 cases with L63X founder mutation, 28 (65.1%) of gBRCA1 mutations were within the OCCR. Of 30 gBRCA2 mutations, 15 (50.0%) were within the OCCR. Of 27 patients (one patient excluded for unknown family history) with gBRCA1 mutations located in the OCCR, 11 (40.7%) had a family history of ovarian cancer; the proportion of patients with a family history of ovarian cancer and gBRCA1 mutations outside the OCCR was lower (13.3%). Sixty percent of patients with gBRCA1 mutations outside the OCCR had a family history of breast cancer; the proportion of patients with a family history of breast cancer and gBRCA1 mutations within the OCCR was relatively lower (33.3%). Understanding the mutation locations may contribute to more accurate risk assessments of susceptible individuals and early detection of ovarian cancer among gBRCA mutation carriers.     

Idiopathic myointimal hyperplasia of mesenteric veins: Rare case of ischemic colitis mimicking inflammatory bowel disease

Idiopathic myointimal hyperplasia of mesenteric veins (IMHMV) is a rare and poorly understood ischemic colitis that occurs in the rectosigmoid colon of predominantly young, previously healthy, male patients. A 76‐year‐old Japanese man presented to our hospital with a 1‐year history of worsening diarrhea, lower abdominal pain, and weight loss (−6 kg). Laboratory evaluation revealed white blood cell count of 13 200/μL, C‐reactive protein level of 2.0 mg/dL (normal range, 0.0–0.3), and negative results for stool culture (including Clostridium difficile). Colonoscopy showed circumferential and edematous narrowing of the sigmoid colon with deep longitude ulceration. Biopsy was done and examination of the specimen demonstrated no specific ischemia. The patient was treated with bowel rest, antibiotics, and i.v. fluids; however, his symptoms worsened. Finally, sigmoidectomy was carried out. Histological examination demonstrated significant myointimal hyperplasia of mesenteric veins leading to thickening and stenosis of the venous lumen. Therefore, the final diagnosis was IMHMV. Three months following sigmoidectomy, he was asymptomatic.     

Nicotine does not affect stem cell properties requisite for suicide gene therapy against glioma

ABSTRACT

Objective: 

Glioblastoma is one of the most lethal tumors in adult central nervous system with a median survival of a year and half and effective therapeutic strategy is urgently needed. For that reason, stem cell-based suicide gene therapies have attracted much interest because of potent tumor tropism of stem cells and bystander effect. In this current clinical situation, stem cells are promising delivery tool of suicide genes for glioma therapy. Since habitual cigarette smoking still prevails worldwide, we investigated the effect of nicotine on stem cell tropism toward glioma and gap junctional intercellular communication (GJIC) function between glioma and stem cells, both of which are important for suicide gene therapies. Methods: Mouse induced pluripotent stem cell-derived neural stem cells (iPS-NSCs) and human dental pulp mesenchymal stem cells (DPSCs) were used. The effect of nicotine on tumor tropism to glioma-conditioned medium (CM) at a non-cytotoxic concentration was assessed with Matrigel invasion assay. Nicotine effect on GJIC was assessed with the scrape loading/dye transfer (SL/DT) assay for co-culture of glioma and stem cells and the parachute assay among glioma cells using high-content analysis. Results: Tumor tropism of iPS-NSCs toward GL261-CM and DPSCs toward U251-CM was not affected by nicotine (0.1 and 1 µM). Nicotine at the concentrations equivalent to habitual smoking (1 µM) did not affect GJIC of iPS-NSC/GL261 and DPSC/U251 and GJIC among each glioma cells. Conclusions: The study demonstrated that non-cytotoxic concentrations of nicotine did not significantly change the stem cell properties requisite for stem cell-based suicide gene therapy.     

Innovations and techniques for balloon-enteroscope-assisted endoscopic retrograde cholangiopancreatography in patients with altered gastrointestinal anatomy

Endoscopic retrograde cholangiopancreatography (ERCP) remains challenging in patients who have undergone surgical reconstruction of the intestine. Recently, many studies have reported that balloon-enteroscope-assisted ERCP (BEA-ERCP) is a safe and effective procedure. However, further improvements in outcomes and the development of simplified procedures are required. Percutaneous treatment, Laparoscopy-assisted ERCP, endoscopic ultrasound-guided anterograde intervention, and open surgery are effective treatments. However, treatment should be noninvasive, effective, and safe. We believe that these procedures should be performed only in difficult-to-treat patients because of many potential complications. BEA-ERCP still requires high expertise-level techniques and is far from a routinely performed procedure. Various techniques have been proposed to facilitate scope insertion (insertion with percutaneous transhepatic biliary drainage (PTBD) rendezvous technique, Short type single-balloon enteroscopes with passive bending section, Intraluminal injection of indigo carmine, CO₂ inflation guidance), cannulation (PTBD or percutaneous transgallbladder drainage rendezvous technique, Dilation using screw drill, Rendezvous technique combining DBE with a cholangioscope, endoscopic ultrasound-guided rendezvous technique), and treatment (overtube-assisted technique, Short type balloon enteroscopes) during BEA-ERCP. The use of these techniques may allow treatment to be performed by BEA-ERCP in many patients. A standard procedure for ERCP yet to be established for patients with a reconstructed intestine. At present, BEA-ERCP is considered the safest and most effective procedure and is therefore likely to be recommended as first-line treatment. In this article, we discuss the current status of BEA-ERCP in patients with surgically altered gastrointestinal anatomy.     

Condyloma acuminatum of the anal canal,treated with endoscopic submucosal dissection

Condyloma acuminatum(CA) is a common sexually transmitted disease caused by human papilloma virus infection. Not all individuals develop persistent, progressive disease, but careful follow up is required with moderate-to-severe dysplasia to prevent progression to malignancy. Standard therapies include surgical treatments(trans-anal resection and transanal endoscopic microsurgery) and immunotherapeutic and topical methods(topical imiquimod); however, local recurrence remains a considerable problem. Here, we report a case with superficial CA of the anal canal, treated with endoscopic submucosal dissection(ESD). A 28-year-old man presented with a chief complaint of hematochezia. Digital exam did not detect a tumor. Screening colonoscopy revealed 10-mm long, whitish condyles extending from the anal canal to the lower rectum. The lesion covered almost the whole circumference, and only a small amount of normal mucosa remained. Magnifying endoscopy with narrow band imaging showed brownish hairpin-shaped, coiled capillaries. Although histopathological diagnosis by biopsy revealed CA, accurate histological differentiation between CA, papilloma, and squamous cell carcinoma can be difficult with a small specimen. Therefore, weperformed diagnostic ESD, which provides a complete specimen for precise histopathological evaluation. The pathological diagnosis was CA, with moderate dysplasia(anal intraepithelial neoplasia 2). There was no recurrence at 16 mo after the initial ESD. Compared to surgical treatment, endoscopic diagnosis and resection could be performed simultaneously and the tumor margin observed clearly with a magnifying chromocolonoscopy, resulting in less recurrence. These findings suggest that endoscopic resection may be an alternative method for CA that prevents recurrence.