CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within the noncoding regions. We propose a novel, cancer-specific killing approach using CRISPR-Cas9 which exploits the requirement of a protospacer adjacent motif (PAM) for Cas9 activity. Through whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002), we identified an average of 417 somatic PAMs per tumor produced from single base substitutions. We analyzed 591 paired T-N samples from The International Cancer Genome Consortium and discovered medians of ~455 somatic PAMs per tumor in pancreatic, ~2800 in lung, and ~3200 in esophageal cancer cohorts. Finally, we demonstrated >80% selective cell death of two targeted pancreatic cancer cell lines in co-cultures using 4-9 sgRNAs, targeting noncoding regions, designed from the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs through WGS.

Analysis of ileostomy stool samples reveals dysbiosis in patients with high-output stomas.

Construction of a diverting stoma can significantly reduce the onset of severe anastomotic leakage in patients with rectal cancer. High-output stoma is one of the most important potential surgical complications after anal function-preserving surgery with ileostomy. Culture-independent techniques have revealed the interaction of the complex intestinal bacterial ecology with various diseases. Our objective was to evaluate the differences in patient characteristics and gut microbiota distribution features in patients with high-output stomas. The cases of 24 consecutive patients who underwent curative resection for rectal cancer at our hospital between November 2016 and June 2018 were reviewed, and the patients were categorized into high-output and low-output groups. Their microbiota were analyzed using next-generation sequencing of ileostomy stool samples collected on postoperative day 7. There was a significant difference in the percentage of Bacteroidetes between the high-output and low-output groups (14.8% vs 0.5%; p=0.01). The percentage of Clostridium butyricum was increased in the low-output group (p=0.01). After the exclusion of those treated with the probiotic Miya-BM, whose principal component is C. butyricum, analyses revealed no significant differences between the high-output and low-output groups. This pilot study provides the first evidence correlating gut microbiota with the pathogenesis of high- output stoma compared with low-output stoma.

The Pre-treatment Lymphocyte-to-Monocyte Ratio Predicts Efficacy in Metastatic Colorectal Cancer Treated With TAS-102 and Bevacizumab.

BACKGROUND/AIM: Our multicenter phase II TAS-CC3 study demonstrated favorable median progression-free survival (PFS) and overall survival (OS) of 32 metastatic colorectal cancer (mCRC) patients treated with TAS-102 + bevacizumab as 3rd-line treatment. PATIENTS AND METHODS: We investigated the predictive and prognostic values of pre-treatment blood inflammation-based scores, including the neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte ratio (LMR) on disease-control (DC), PFS and OS by a post-hoc analysis. RESULTS: Receiver operating characteristic curve analyses of the 3 inflammation-based scores versus DC showed the best predictive performance for LMR, followed by NLR and PLR. The high-LMR group had a significantly higher DC rate than the low group (87.5 vs. 43.8%). The high-LMR group showed significantly longer survival than the low group (4.9 vs. 2.3 m for median PFS) (21.0 vs. 6.1 m for median OS). CONCLUSION: The pre-treatment LMR is a valid predictive and prognostic biomarker for mCRC patients undergoing TAS-102 and bevacizumab treatment.

Combination of TAS-102 and bevacizumab as third-line treatment for metastatic colorectal cancer: TAS-CC3 study.

BACKGROUND: TAS-102 improved the overall survival of metastatic colorectal cancer (CRC) patients with a median progression-free survival (PFS) in the RECOURSE trial. Subsequently, the combination of TAS-102 and bevacizumab was shown to extend the median PFS (C-TASK FORCE study). However, the study included patients who received second- and third-line treatment. Our study exclusively examined patients receiving this combination as a third-line treatment to investigate the clinical impact beyond cytotoxic doublets. METHODS: This investigator-initiated, open-label, single-arm, multi-centered phase II study was conducted in Japan. Eligible CRC patients were refractory or intolerant to fluoropyrimidine, irinotecan, and oxaliplatin in first- and second-line therapy. TAS-102 (35 mg/m2) was given orally twice daily on days 1-5 and 8-12 in a 4-week cycle, and bevacizumab (5 mg/kg) was administered by intravenous infusion every 2 weeks. The primary endpoint was PFS and the secondary endpoints were time-to-treatment failure, response rate, overall survival (OS), and safety. RESULTS: Between June 2016 and August 2017, 32 patients were enrolled. All patients previously received bevacizumab. The median PFS was 4.5 months; the median overall survival was 9.3 months. Partial response was observed in two patients. The most common adverse events above grade 3 were neutropenia followed by thrombocytopenia. There were no non-hematological adverse events above grade 3 and no treatment-related deaths occurred. CONCLUSIONS: This study met its primary endpoint of PFS, which is comparable to the results of the C-TASK FORCE study. The TAS-102 and bevacizumab combination has the potential to be a therapeutic option for third-line treatment of metastatic CRC.

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. METHODS: We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. RESULTS: Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. CONCLUSIONS: Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

Current status of venous thromboembolism development during the perioperative period for colorectal cancer, its prevention with enoxaparin, and monitoring methods.

Background: There is a high incidence of venous thromboembolism (VTE) during the perioperative period for cancer. Therefore, there is an urgent need to elucidate the perioperative onset and appropriate prophylaxis for VTE. Purpose: VTE during the perioperative period for colorectal cancer was evaluated by lower limb venous ultrasonic examinations (lower limb echo) under enoxaparin prophylaxis. We also examined the relationship between hemorrhagic adverse events and anti-Xa factor activity. Patients and methods: Eighty-three subjects who underwent lower limb echo during the perioperative period for colorectal cancer were prospectively included. Enoxaparin was administered for 5 days, from day 1 to day 5 after surgery. Lower limb echo was performed before surgery and on day 5 after surgery. The activated partial thromboplastin time, D-dimer levels, and anti-Xa factor activity were measured before surgery and on days 1, 3, 5, 7, and 9 after surgery. Results: VTEs before surgery were observed on lower limb echo for 16 patients (19.2%). Three patients (3.6%) had a new thrombus during the perioperative period. The preoperative D-dimer level was an independent prognostic factor for newly formed postoperative VTEs (p=0.0036; odds ratio, 19.37). Three patients (3.6%) had hemorrhagic events; however, there was no significant trend for anti-Xa factor activity. Conclusion: VTE prevention using enoxaparin was relatively safe, and D-dimer measurements before surgery were useful for predicting perioperative VTE.

Colorectal Perforation in Patients with Connective Tissue Disease.

PURPOSE: The goal of this retrospective study was to identify prognostic factors associated with mortality after surgery for colorectal perforation among patients with connective tissue disease (CTD) and to review postoperative outcomes based on these prognostic factors. METHODS: The subjects were 105 patients (CTD group: n=26, 24.8%; non-CTD group: n=79, 75.2%) who underwent surgery for colorectal perforation at our department. Cases with iatrogenic perforation due to colonoscopic examination were excluded from the study. We retrospectively investigated perioperative clinicopathological factors in patients undergoing surgery for colorectal perforation. RESULTS: There were 7 patients (6.7%) who died within 28 days after surgery in all patients. In multivariate analysis, CTD and fecal peritonitis emerged as significant independent prognostic factors (p=0.005, odds ratio=12.39; p=0.04, odds ratio=7.10, respectively). There were 5 patients (19.2%) who died within 28 days after surgery in the CTD group. In multivariate analysis, fecal peritonitis emerged as a significant independent prognostic factor in the CTD group (p=0.03, odds ratio=31.96). The cumulative survival curve in the CTD group was significantly worse than that in the non-CTD group (p=0.006). An analysis based on the presence of fecal peritonitis indicated no significant difference in cumulative survival curves for patients without fecal peritonitis in the CTD and non-CTD groups (p=0.55) but a significant difference in these curves for patients with fecal peritonitis in the two groups (p<0.0001). CONCLUSIONS: This study demonstrated that cumulative survival in patients with CTD is significantly worse than that in patients without CTD after surgery for colorectal perforation.

[A Case of Good Quality of Life(QOL)and Favorable Response to Transarterial Chemoembolization(TACE)against Synchronous Multiple Liver Metastases].

We report a case of good quality of life(QOL)and favorable response to transarterial chemoembolization(TACE)against synchronous multiple liver metastases. An 85-year-old man was admitted to our hospital because of melena. Colonoscopy showed multiple type 2 tumors in the sigmoid colon and upper rectum. CT and EOB-MRI examinations revealed that there were multiple liver metastases. Because of his age and surgical stress, he underwent a laparoscopic Hartmann's procedure. After the resection of the primary tumor, he received tegafur/uracil for his liver metastases. However, he discontinued receiving the drugs 2 weeks later because of the development of adverse events. Instead of systemic chemotherapy, he chose to undergo TACE. He underwent TACE with irinotecan and HepaSphereTM(BioSphere Medical)8 times for his multiple liver metastases. Consequently, all multiple liver metastases disappeared. Therefore, TACE may be useful for patients who are not suitable for systemic chemotherapy.

[A Case of Systemic Chemotherapy Combined with Molecular Drug Treatment Following Biliary Stent Implantation for Obstructive Jaundice Due to Metastases of Liver Gland Lymph Node after Colorectal Cancer Surgery].

We report a case of systemic chemotherapy after biliary stent placement for obstructive jaundice due to hepatic portal lymph node metastasis after colorectal cancer surgery. The patient was a 40s woman. Laparoscopic anterior resection for rectosigmoidRS cancer was performed. The pathological diagnosis was T3N0M0PUL0R0, pStage Ⅱ according to the 8th edition of colorectal cancer handling regulations. Because multiple liver metastases were observed 8 months after the surgery, partial resection of the posterior region of the liver was performed. Multiple lung metastases were observed 1 year after hepatectomy, but she wantedto undergo follow-up observation. Jaundice was observed 1 year after the diagnosis of lung metastasis, and obstructive jaundice due to hepatic portal lymph node metastasis was diagnosed. Endoscopic retrograde biliary drainage(ERBD)was performed, and a bile duct stent was placed. After improving jaundice, 12 courses of mFOLFOX6 plus cetuximab therapy were performed. Currently, because of the exacerbation of lung metastasis, FOLFIRI plus bevacizumab therapy is being administered. Systemic chemotherapy containing a molecular-targeted drug is being administered in our case, but complications relatedto the biliary stent have not been observed. There are few reports on similar cases, andfollow - up observation with careful attention to long-term safety is necessary.

Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer.

PURPOSE: In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory. EXPERIMENTAL DESIGN: Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed. RESULTS: ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%)] with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011). CONCLUSIONS: Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.

Histological characteristics of eosinophilic myenteric ganglionitis: an under-recognised cause of chronic intestinal pseudo-obstruction.

Eosinophilic myenteric ganglionitis (EMG) is characterised by eosinophilic infiltration of the myenteric plexus. EMG has been rarely reported as a cause of chronic intestinal pseudo-obstruction (CIPO), and its histopathological features are not fully elucidated. We analysed seven patients with CIPO. Three of them were diagnosed with EMG and four patients were categorised as non-EMG. Clinicopathological features were similar in both groups. These features included subtle to mild lymphocytic infiltration at the myenteric ganglia/muscularis propria, loss of myenteric ganglions and interstitial cells of Cajal (ICC), and no significant findings in the mucosa. The exceptions were moderate to severe degree of eosinophilic infiltration at the myenteric ganglia/muscularis propria in EMG. Functional gastrointestinal obstruction may be associated with inflammatory cell infiltration at the myenteric ganglia/muscularis propria, leading to subsequent hypoganglionosis and deficiency of ICC in EMG. Pathologists and clinicians should be aware of this distinction during differential diagnosis of patients with CIPO.

A Progressive Huge Accessory Spleen in the Greater Omentum.

Huge accessory spleen (AS) is a rare condition difficult to diagnose. We recently treated a Japanese woman with a progressive huge AS. She had a history of aortic valve replacement for aortic stenosis 1 month prior. At that time, a 4-cm AS had been detected by the preoperative computed tomography (CT). This mass was a progressive tumor which grew to 7 cm over the course of 3 months. Thus, we performed surgery with a preoperative diagnosis of huge AS by CT and positron emission tomography. A laparoscopic resection was performed considering the risk of torsion, spontaneous rupture, or hemorrhage. The final pathological diagnosis was AS. This is the first reported case in the English literature of progressive AS with no symptoms at the initial presentation that was treated with laparoscopic resection.

Repeated laparoscopic resection of extra-regional lymph node metastasis after laparoscopic radical resection for rectal cancer.

Here, we report a case of repeated laparoscopic resection of extra-regional lymph node metastases in a patient after laparoscopic surgery for rectal cancer. A 72-year-old woman was diagnosed with upper rectal cancer and underwent laparoscopic low anterior resection and D3 dissection. The pathological stage was considered as T3, N2b, M0, Stage IIIC. Six months after the operation, positron emission tomography-computed tomography (PET-CT) showed fluorodeoxyglucose (FDG) accumulation in the infra-renal para-aortic lymph nodes (PALNs). Systemic chemotherapy was administered; however, chemotherapy was discontinued due to hemoptysis related to her pulmonary disease. Therefore, we performed laparoscopic PALN resection. Pathologically, one lymph node was diagnosed with a metastasis. Three months after the second operation, PET-CT identified FDG accumulation in the left lateral pelvic lymph nodes (LPLNs) and a PALN. Laparoscopic LPLN dissection and PALN resection through minilaparotomy were performed. Pathologically, lymph node metastases were diagnosed in both fields. Sixteen months after the 3rd operation, there is no recurrence.

Case report of perineal hernia after laparoscopic abdominoperineal resection.

Perineal hernia (PH) is a rare complication following laparoscopic abdominoperineal resection (APR) for rectal cancer. We present a case report of perineal hernia after laparoscopic APR and discuss its management. The patient was a 77-year-old man who was diagnosed with lower rectal cancer. He underwent laparoscopic APR and bilateral lateral lymph node dissection. Two months after the surgery, pain and bulging in the perineal region developed, and PH was diagnosed by CT. Repair with a polypropylene mesh was performed using a combination of laparoscopic abdominal and transperineal approaches. Reportedly, the incidence of secondary PH after APR has increased along with the rate of laparoscopic surgery. Treatment of secondary PH with transperineal repair alone may cause injuries to other organs because of adhesion of the pelvic viscera. In the present case, we safely repaired the hernia repair using a laparoscopy-assisted perineal approach.

Case of idiopathic and complete appendiceal intussusception.

Appendiceal intussusception is a rare disease in which the appendix invaginates into the cecum. It is often caused by organic diseases. The present case involved an appendiceal intussusception without an organic disease, and laparoscopic resection of part of the cecum was performed. Appendiceal intussusception has various causes, including malignant diseases. Therefore, diagnosis and selection of operative method are complex and could potentially lead to an excessively invasive option. By performing SILS with a multiuse single-site port, we were able to provide an appropriate, non-invasive treatment that had a good esthetic outcome.

[A Study of Treatment for Pathological T1(SM)Colorectal Cancer Patients after Endoscopic Resection at Our Department].

Approximately 10% of pathological T1(SM)colorectal cancer patients develop lymph node metastases. Therefore additional colectomy with lymph node dissection is recommended when it applies to the specific criteria in the current JSCCR guidelines. However, additional colectomy would not be done in some cases, because surgery is too invasive for some patients. Endoscopic treatment(ESD or EMR)for T1(SM)cancer was performed in 324 cases between 2008 and 2016. Of those, 231 cases had satisfied the criteria for additional colectomy. Among them, 74 cases(32.0%)did not undergo, and additional colectomy(+)groupwas 153 cases(66.2%). Between the 2 groups, no difference in prognosis could be found. We considered there was no difference, because the prognosis of SM cancer is relatively good. In consideration of patient background, the treatment policy has to be chosen according to feasibility.

[A Case of Pelvic Metastasis of Rectal Cancer That Developed Ten Years after Curative Resection].

We report a case of pelvic metastasis of rectal cancer that developed 10 years after curative resection. An 81-year-old woman underwent intersphincteric resection for lower rectal cancer 10 years previously. The tumor was pathologically diagnosed as T2N0M0, Stage Ⅰ. Nine years after the curative resection, serum carcinoembryonic antigen(CEA)levels were slightly elevated, but no recurrence was found on computed tomography(CT). Eleven months after CT, serum CEA levels elevated to 15.9 ng/mL. Pelvic metastasis in the piriformis muscle was detected on positron emission tomography(PET)-CT. Following CT-guided biopsy, she was pathologically diagnosed with metastatic rectal cancer. Radiotherapy (60 Gy/30 Fractions) was administered. Ten months after radiotherapy, PET-CT revealed no relapse in the pelvis with lung metastases.

Two Cases of Rectal Neuroendocrine Tumor Resection Combined with Dissection of the Circular Muscle Layer Using the Endoscopic Submucosal Dissection Technique.

Generally, lesions of rectal neuroendocrine tumors (NETs) 10 mm or smaller are less malignant and are indicated for endoscopic therapy. However, the vertical margin may remain positive after conventional endoscopic mucosal resection (EMR) because NETs develop in a way similar to submucosal tumors (SMTs). The usefulness of EMR with a ligation device, which is modified EMR, and endoscopic submucosal dissection (ESD) was reported, but no standard treatment has been established. We encountered 2 patients in whom rectal NETs were completely resected by combined dissection and resection of the circular muscle layer using the ESD technique. Case 1 was an 8-mm NET of the lower rectum. Case 2 was NET of the lower rectum treated with additional resection for a positive vertical margin after EMR. In both cases, the circular muscle layer was dissected applying the conventional ESD technique, followed by en bloc resection while conserving the longitudinal muscle layer. No problems occurred in the postoperative course in either case. Rectal NETs are observed in the lower rectum in many cases, and it is less likely that intestinal perforation by endoscopic therapy causes peritonitis. The method employed in these cases, namely combined dissection and resection of the circular muscle layer using the ESD technique, can be performed relatively safely, and it is possible to ensure negativity of the vertical margin. In addition, it may also be useful for additional treatment of cases with a positive vertical margin after EMR.