Jean Christophe Saurin

Variants in the Netrin-1 Receptor UNC5C Prevent Apoptosis and Increase Risk of Familial Colorectal Cancer

BACKGROUND & AIMS Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk of colorectal cancer (CRC). METHODS We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, United States, and Finland). Almost all cases from France and the United States had familial CRC; of the Finnish cases, 92 of 984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis to determine the origin of this variant. RESULTS Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio, 8.8; Walds 95% confidence interval, 1.47-52.93; P = .03) and in 2 families in the US cohort (odds ratio, 1.9; P = .6) but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109-kilobase identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in white subjects (14 generations; 95% confidence interval, 6-36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared with wild-type UNC5C, measured in an assay of active caspase-3. CONCLUSIONS Inherited mutations in UNC5C prevent apoptosis and increase risk of CRC.

Oxaliplatin sensitizes human colon cancer cells to TRAIL through JNK-dependent phosphorylation of Bcl-xL.

BACKGROUND & AIMS Oxaliplatin sensitizes drug-resistant colon cancer cell lines to tumor necrosis factor-related apoptosis inducing ligand (TRAIL), a death receptor ligand that is selective for cancer cells. We investigated the molecular mechanisms by which oxaliplatin sensitizes cancer cells to TRAIL-induced apoptosis. METHODS We incubated the colon cancer cell lines HT29 and V9P, which are resistant to TRAIL, with TRAIL or with oxaliplatin for 2 hours, followed by TRAIL. Annexin V staining was used to measure apoptosis; RNA silencing and immunoblot experiments were used to study the roles of apoptosis-related proteins. Site-directed mutagenesis experiments were used to determine requirements for phosphorylation of Bcl-xL; co-immunoprecipitation experiments were used to analyze the interactions among Bcl-xL, Bax, and Bak, and activation of Bax. RESULTS Oxaliplatin-induced sensitivity to TRAIL required activation of the mitochondrial apoptotic pathway; reduced expression of Bax, Bak, and caspase-9, and stable overexpression of Bcl-xL, reduced TRAIL-induced death of cells incubated with oxaliplatin. Mitochondrial priming was induced in cells that were sensitized by oxaliplatin and required signaling via c-Jun N-terminal kinase and phosphorylation of Bcl-xL. Mimicking constitutive phosphorylation of Bcl-xL by site-directed mutagenesis at serine 62 restored sensitivity of cells to TRAIL. Co-immunoprecipitation experiments showed that oxaliplatin-induced phosphorylation of Bcl-xL disrupted its ability to sequestrate Bax, allowing Bax to interact with Bak to induce TRAIL-mediated apoptosis. CONCLUSIONS Oxaliplatin facilitates TRAIL-induced apoptosis in colon cancer cells by activating c-Jun N-terminal kinase signaling and phosphorylation of Bcl-xL. Oxaliplatin-induced sensitivity to TRAIL might be developed as an approach to cancer therapy.

Challenges and Future of Wireless Capsule Endoscopy

In 2015, capsule endoscopy was introduced as the main investigation method for small bowel mucosal diseases, and its role in colonic diseases has been gradually revealed. Future challenges for capsule endoscopy, besides improvements of image quality and visualization of each part of the small bowel and colonic mucosa, include the development of gastric capsules, the capacity to perform histological examination of the mucosa, and maybe in the future, some capsule endoscopy-driven therapeutics. The aim of this review was to evaluate the clinical demands and feasibility of achieving the aforementioned objectives.

Surveillance of duodenal adenomas in familial adenomatous polyposis patients: medical objectives and technical requirements.

Patients with classical familial adenomatous polyposis (FAP) present with hundreds of colorectal adenomas requiring (sub)total colectomy at around age 20 years, and frequently duodenal adenomas. Duodenal polyposis is a progressive disease. The burden of duodenal adenomas evolves more slowly as compared to colorectal adenomas, but increases with age to the highest stage of duodenal polyposis, stage IV according to the Spigelman’s classification, with an expected cumulative frequency of 50 % at age 70 years 1. Although the surveillance recommendations are relatively clear and homogeneous in the different countries, with the first duodenal surveillance between 20 and 25 years, the modalities of treatment are much less well defined. Duodenal adenomas either ampullary or peri-ampullary present usually as flat, whitish, small lesions and slightly elevated as compared to the surrounding mucosa. There is a majority of small (  1 cm) lesions as these have been shown to contain foci of high-grade dysplasia in up to 50 % of cases and have been associated with a risk of cancer development.2 3 The role of the endoscopist during duodenal surveillance is to 1) detect any existing cancer (complete evaluation of the duodenum and firsts jejunal loops); and 2i) evaluate the burden of duodenal adenomas in order to estimate the risk of advanced neoplastic evolution and the adapted surveillance interval. The only available criterion nowadays is the Spigelman’s classification, which includes the number (  20), the size (  10 mm) and the histology (low- or high-grade dysplasia) of duodenal adenomas. Optimal endoscopic examination of the duodenum, with lateral viewing and axial viewing at the same time, using a long endoscope to visualize the proximal jejunum and indigo-carmine to improve visualization of the mucosa, although relatively consensual for expert centers, has not really been validated through prospective studies. At least, indigo-carmine dye has been shown to increase the number of adenomas detected in two series and to increase the Spigelman’s score.4 5 The question addressed in the paper by Pittayanon and colleagues 6 is that of the diagnostic value of new imaging methods (narrow band imaging and confocal microscopy) as regards the identification of duodenal adenomas. The main clinical question in this disease should be: What are the limitations of present surveillance modalities, what is the reference method of examination, and do we need new methods to improve surveillance? The main limitation of the usual surveillance of duodenal polyposis would be to overlook major neoplastic area that would evolve into cancer during the recommended surveillance interval (2 – 3 years). Some old retrospective series suggest such a limitation, but these are mainly based on a largely obsolete methodology (axial viewing, old endoscopes, no sedation) 3. In contrast, recent prospective series, even in patients with severe duodenal polyposis, do not suggest a high frequency of overlooked precancerous lesions 7. Finally, the recent progress of usual endoscopes into high-definition endoscopy will probably replace, based on our experience, even indigo-carmine dye, given the excellent visualization of even tiny adenomas. And this evolution is still underway, given the new generation of endoscopes that are under development or research. Thus, there will probably be no clinical need for new technologies to identify more duodenal adenomas in FAP patients. On the other hand, is it important to differentiate duodenal adenomas from other duodenal polyps, as proposed by Pittayanon and colleagues? There is a real concern regarding the duodenal bulb as some lesions can mimic duodenal adenomas in this area, including gastric metaplasia and Brunner glands. There is also a question regarding the identification of adenomatous tissue on the duodenal papilla, especially because biopsies, although with a very low frequency, can induce pancreatitis. In the latter case, however, it would be important to determine whether microscopic (macroscopically invisible) ampullary adenoma represent a danger for patients even on a long-term basis, given the very slow evolution of duodenal adenomas 8. In our experience, we usually leave in place small, flat, visible ampullary adenomas for years. Regarding the numerous small duodenal adenomas observed throughout the proximal and distal duodenum of patients with FAP, there is no recommendation to biopsy and thus confirm the diagnosis of adenomas, as there is almost no differential diagnosis in this area. Practically, we never take numerous biopsies of these lesions, but simply count the number of adenomas to get a satisfying evaluation of the Spigelman’s score. For these reasons, identifying exactly the nature of small whitish lesions in the proximal and distal duodenum is probably of low clinical relevance. Moreover, if this was of any clinical relevance, any new diagnostic method should be compared with simple, white-light imaging, and probably to the reference method, i.e indigo carmine dye. Finally, what is important for patients with FAP regarding duodenal examination? Three measures: 1) beginning relatively early surveillance, around age 20 years, as we observe severe polyposis in a low number of young patients; 2) following recommended modalities of examination, including anesthesia, lateral and axial viewing, examination of the proximal jejunum to identify all possible areas of advanced neoplasia; and 3) being very cautious with large (> 1 cm) adenomas, which should be removed because they represent a significant risk of high-grade dysplasia or cancer. We should keep in mind that this evaluation needs to be done at regular intervals, is time consuming, and should remain relatively simple so that gastroenterologists can keep on doing an excellent and regular evaluation of this significant risk of duodeno-jejunal cancer in patients with FAP.

Why should we systematically specify the clinical relevance of images observed at capsule endoscopy

In 2014, capsule endoscopy is the reference procedure for examining the small bowel of patients with obscure digestive bleeding. The diagnostic yield has long been shown to be greater than 50 % in most series 1 2 3, and to be up to 90 % in emergency settings in patients with overt obscure bleeding 1. In the case of a patient with obscure bleeding, capsule endoscopy identifies a majority of telangiectases (65 %) and ulcers (18 %) and relatively few tumors (2 – 8 %) and other diagnoses, such as portal hypertension. The sensitivity of capsule endoscopy is very high in most series that include long-term follow-up. Long-term follow-up is important to rule out missed diagnoses and to clarify the clinical relevance of lesions identified by capsule endoscopy. In fact, the main question regarding capsule endoscopy findings is not ‘Did we miss a diagnosis?’. This happens infrequently in clinical practice. Rather, the main question is ‘What is the clinical relevance of our findings?’. The relevance of the findings is the predicted significance of the observed lesions in a patient with obscure bleeding (including anemia and overt bleeding). We all have had the experience of discovering small-bowel telangiectasia at capsule endoscopy and considering it to be the cause of bleeding, with a secondary diagnosis made several weeks later – for example, a missed colonic or endometrial tumor. In this example, the significance of the telangiectasia was equal to zero. This could have been suspected immediately after the diagnosis of telangiectasia if the lesion was very small and not bleeding. There are also published cases of surgical interventions scheduled on the basis of irrelevant small-bowel lesions detected at capsule endoscopy 4. This is why in 2003 we proposed a simple classification for the relevance of lesions (P0, P1, P2), allowing the capsule reader to estimate the relevance of each lesion detected at capsule endoscopy. We consider that such relevance has a major impact on patient management and should be taken into account, especially before any therapeutic intervention (by enteroscopy in most cases) is scheduled 3. This classification has been validated by the following: (i) lesions were stated to be highly relevant (P2) by both readers in blind tandem readings in 100 % of cases, compared with 73 % and 27 % of cases, respectively, for P1 and P0 lesions; (ii) the therapeutic impact proved to be 61 % for P2 lesions versus 23 % for P1 or P0 lesions; and (iii) the follow-up of patients 5. Misinterpretation of the clinical relevance of small-bowel lesions can lead to unjustified aggressive therapy; in a tandem reading study with expert review of discordant cases, a 50 % error rate of experienced readers was finally stated for discordant cases (13 of 25 discordant results), which corresponded in 5 of the 13 errors to the “overclassification” of an irrelevant abnormality 6. Another comparative study showed an “overclassification” of such irrelevant abnormalities in about 10 % of capsule endoscopy readings 7. Thus, stating the relevance of each lesion detected at capsule endoscopy may help readers to improve the quality of their reading and their decision making, and to decide whether enteroscopy or other treatments should be attempted, although this remains to be scientifically proven. Indeed, we all have had the experience, when reviewing the capsule film before performing enteroscopy, of deciding that the procedure should be cancelled because the capsule findings did not justify an aggressive approach. In the rare situations in which tumor detection is the main objective of capsule endoscopy, such as in patients with Lynch syndrome or familial adenomatous polyposis, the relevance of the lesions observed at capsule endoscopy is of even greater importance, considering the risk and difficulty involved in accessing the small bowel of patients who have undergone multiple [SJ1] surgeries; in these cases, only highly relevant lesions justify further investigations 8 9. The relevance of lesions is one important point in the paper by Sato and colleagues published in this issue. The study deals with the interesting question of the usefulness of virtual chromoendoscopy (flexible spectral imaging color enhancement, or FICE) for the detection of clinically significant lesions in patients with various clinical problems and small-bowel involvement. The informatics and capsule system used are those of Given Imaging (Yokneam, Israel). The paper is one of the first to suggest the superiority of a specific FICE setting for the detection of vascular and erosive lesions in the small bowel of patients with miscellaneous clinical indications. This may be of importance based on the fact that only “clinically relevant” lesions (P1 or P2) were considered in the study, in which the P0 – P2 classification was used. However, the question then is that of the clinical usefulness of the setting, as 4 % of lesions of each type (vascular and ulcerative) are detected only with use of the FICE setting, but the highest sensitivity for tumor corresponds to white light imaging. The Cornelian dilemma is thus, What mode is optimal for reading the small-bowel capsule film, as nobody will accept a two-mode, time-consuming reading protocol? Indeed, detecting all lesions is one aim, but detecting all high-risk lesions (and especially tumors) may be even more important. Probably the answer could come from a large prospective study with only one clinical indication, probably the most important one for capsule endoscopy – that is, obscure digestive bleeding.

A neural network algorithm for detection of GI angiectasia during small-bowel capsule endoscopy

BACKGROUND AND AIMS GI angiectasia (GIA) is the most common small-bowel (SB) vascular lesion, with an inherent risk of bleeding. SB capsule endoscopy (SB-CE) is the currently accepted diagnostic procedure. The aim of this study was to develop a computer-assisted diagnosis tool for the detection of GIA. METHODS Deidentified SB-CE still frames featuring annotated typical GIA and normal control still frames were selected from a database. A semantic segmentation images approach associated with a convolutional neural network (CNN) was used for deep-feature extractions and classification. Two datasets of still frames were created and used for machine learning and for algorithm testing. RESULTS The GIA detection algorithm yielded a sensitivity of 100%, a specificity of 96%, a positive predictive value of 96%, and a negative predictive value of 100%. Reproducibility was optimal. The reading process for an entire SB-CE video would take 39 minutes. CONCLUSIONS The developed CNN-based algorithm had high diagnostic performances, allowing detection of GIA in SB-CE still frames. This study paves the way for future automated CNN-based SB-CE reading softwares.

4625 Endoscopic ultrasound and intraductal ultrasonography are complementary for the management of ampullary tumors.

Whipples resection is the gold standard treatment of the ampullary tumors. Nevertheless endoscopic snare resection (ESR), which is less invasive, can be applied in a curative intent when some criteria are respected : -1- no risk of metastatic lymph node e.g. benign ampullomas or early carcinomas respecting the submucosae -2- no tumoral extension inside the pancreatic and the bile ducts. While endoscopic ultrasound (EUS) stages, with a good accuracy, an ampullary tumor as respecting the muscularis propria (uT1), intraductal ultrasonography (IDUS) is very promising to appreciate the involvement of the submucosae and the existence of an intraductal extension. The combination of the two methods could then allow optimizing the therapeutic choice. AIM: to estimate the clinical impact of EUS +/- IDUS in the management of ampullary tumors PATIENTS AND METHODS: between Jan 99 and Nov 99, 10 patients with ampullary tumors had a pretherapeutic staging. A radial EUS (GFUM20, Olympus Co) was systematically performed. When the staging was uT1 an IDUS (UM-G20-29R, Olympus Co) was done. A Whipples resection was proposed when final staging was -1- lesion infiltrating the submucosae or more ; -2- tumor extension inside the biliary or pancreatic ducts. An ESR was proposed in the other cases. RESULTS: EUS concluded to a tumor >uT1 in 4 cases. Pathology (Whipples resection) confirmed the staging (3 pT3; 1 pT2). EUS conclude to a tumor uT1 in 6 cases and IDUS was performed. - In 4 cases tumors were considered without intraductal involvement and respecting the submucosae. ESR was always curative (severe dysplasia; resection margins free of tumor). – In 2 cases IDUS evidenced involvement of the submucosae. 1 with a tumor extension inside the ducts was operated on and diagnosed as pT2. 1 refused surgery and an ESR was performed confirming the involvement of the submucosae (positive resection margins). A moderate pancreatitis was the only ESR complication. CONCLUSION: EUS and IDUS togheter allow an accurate T staging of ampullary tumors. The clinical impact is major: endoscopic snare resection can be applied in a curative intent in early tumors, Whipples resection in other cases or when ESR is not complete.

3532 Endoscopic mucosal resection of superficial neoplastic esophageal lesions: macroscopic and histological results.

Endoscopic mucosal resection (EMR) is a new endoscopic method for the treatment of superficial neoplastic lesions of the digestive tract. The goal of this study is to assess the feasibility and results of EMR in esophageal dysplastic lesions and superficial cancers. Patients and methods: 31 EMR were performed in 27 patients (pts) (23 men, 4 women, median age: 67 years). The 31 lesions were 3 squamous cell high grade dysplasias (HGD), 4 HGD on Barrett, 20 squamous cell cancers and 4 adenocarcinomas. Eight pts had been previously treated using photodynamic therapy (PDT). Following lugol or indigocarmine dying, median lesion diameter was 15mm (range: 10-40 mm). Lesions were classified uT0N0 or uT1N0 on standard EUS (13 cases) or uTmuqN0 on 20MHz miniprobe EUS (18). Inoue method (transparent cap at the tip of the endoscope and asymetrical snare) was used in all pts. Immediate results: resection was in bloc in 23 cases or by fragments (2-6) in 8 cases. Five lesions (HGD on Barrett: 1, squamous cell carcinoma: 4) were not found on the specimen in pts previously treated using PDT. Concerning the 26 lesions found on the specimen, resection was considered macroscopically complete by the endoscopist in 24 cases. However, on histology, resection was in sano in only 4 cases. Indeed, 1- analysis of the specimen margins was not possible due to coagulation artifacts in 5 cases; 2- resection was non in sano laterally alone in 10 cases and non in sano both in depth and laterally in 4 cases. Submucosal carcinomatous invasion was detected in 6 cases. Follow-up: due to their age and submucosal invasion or incomplete resection, 6 pts had a complementary radiochemotherapy (4 cases) or a surgical resection (2 cases). 1 pt died from another cancer. 18 pts were reexamined endoscopically (mean: 6 months) without lesions and 1 had a persistant lesion treated by complementary PDT. Side-effects: One out of 3 bleedings required an endoscopic clipping. No symptomatic perforation was observed despite muscular resection in 2 pts. Conclusion: These results demonstrate the feasibility and safety of the method and the advantage of EMR to provide a precise histological diagnosis. Endoscopic assessment of results is satisfactory; however, histology shows that the section frequently passes on the margins of the lesion especially laterally and technical improvements are necessary to enlarge the resection.