Determination of esophageal squamous cell carcinoma and gastric adenocarcinoma on raw tissue using Raman spectroscopy

BACKGROUND Cancer detection is a global research focus,and novel,rapid,and label-free techniques are being developed for routine clinical practice.This has led to the development of new tools and techniques from the bench side to routine clinical practice.In this study,we present a method that uses Raman spectroscopy(RS)to detect cancer in unstained formalin-fixed,resected specimens of the esophagus and stomach.Our method can record a clear Raman-scattered light spectrum in these specimens,confirming that the Raman-scattered light spectrum changes because of the histological differences in the mucosal tissue.AIM To evaluate the use of Raman-scattered light spectrum for detecting endoscopically resected specimens of esophageal squamous cell carcinoma(SCC)and gastric adenocarcinoma(AC).METHODS We created a Raman device that is suitable for observing living tissues,and attempted to acquire Raman-scattered light spectra in endoscopically resected specimens of six esophageal tissues and 12 gastric tissues.We evaluated formalin-fixed tissues using this technique and captured shifts at multiple locations based on feasibility,ranging from six to 19 locations 200 microns apart in the vertical and horizontal directions.Furthermore,a correlation between the obtained Raman scattered light spectra and histopathological diagnosis was performed.RESULTS We successfully obtained Raman scattered light spectra from all six esophageal and 12 gastric specimens.After data capture,the tissue specimens were sent for histopathological analysis for further processing because RS is a label-free methodology that does not cause tissue destruction or alterations.Based on data analysis of molecular-level substrates,we established cut-off values for the diagnosis of esophageal SCC and gastric AC.By analyzing specific Raman shifts,we developed an algorithm to identify the range of esophageal SCC and gastric AC with an accuracy close to that of histopathological diagnoses.CONCLUSION Our technique provides qualitative information for real-time morphological diagnosis.However,further in vivo evaluations require an excitation light source with low human toxicity and large amounts of data for validation.

Submucosal tunnel endoscopy:Peroral endoscopicmyotomy and peroral endoscopic tumor resection

Peroral endoscopic myotomy(POEM) is an innovative,minimally invasive, endoscopic treatment for esophageal achalasia and other esophageal motility disorders, emerged from the natural orifice transluminal endoscopic surgery procedures, and since the first human case performed by Inoue in 2008, showed exciting results in international level, with more than 4000 cases globally up to now. POEM showed superior characteristics than the standard 100-year-old surgical or laparoscopic Heller myotomy(LHM), not only for all types of esophageal achalasia [classical(Ⅰ), vigorous(Ⅱ), spastic(Ⅲ), Chicago Classification], but also for advanced sigmoid type achalasia(S1 and S2), failed LHM, or other esophageal motility disorders(diffuse esophageal spasm, nutcracker esophagus or Jackhammer esophagus). POEM starts with a mucosal incision, followed by submucosal tunnel creation crossing the esophagogastric junction(EGJ) and myotomy. Finally the mucosal entry is closed with endoscopic clip placement. POEM permitted relatively free choice of myotomy length and localization. Although it is technically demanding procedure, POEM can be performed safely and achieves very good control of dysphagia and chest pain. Gastroesophageal reflux is the most common troublesome side effect, and is well controllable with proton pump inhibitors. Furthermore, POEM opened the era of submucosal tunnel endoscopy, with many other applications. Based on the same principles with POEM, in combination with new technological developments, such as endoscopic suturing, peroral endoscopic tumor resection(POET), is safely and effectively applied for challenging submucosal esophageal, EGJ and gastric cardia tumors(submucosal tumors), emerged from muscularis propria. POET showed up to know promising results, however, it is restricted to specialized centers. The present article reviews the recent data of POEM and POET and discussed controversial issues that need further study and future perspectives.

Endoscopic features of early-stage signet-ring-cell carcinoma of the stomach

AIM: To identify the features of early signet ring cell gastric carcinoma using magnification endoscopy with narrow band imaging(NBI).METHODS: A retrospective review was conducted of 12 cases of early signet ring cell gastric carcinoma who underwent treatment in a single institution between January 2009 and April 2013. All patients had magnification endoscopy with NBI and indigo carmine contrast to closely examine the mucosal architecture, including the microvasculature and arrangement of gastric pits. Histologic examination of the final endoscopic submucosal dissection or gastrectomy specimen was performed and compared with the endoscopic findings to identify patterns specific to signet ring cell carcinoma.RESULTS: Twelve patients with early signet ring cell gastric carcinoma were identified; 75% were male, and average age was 61 years. Most of the lesions were stage T1a(83%), while the remainder were T1b(17%). The mean lesion size was 1.4 cm2. On standard endoscopy, all 12 patients had a pale, flat lesion without any evidence of mucosal abnormality such as ulceration, elevation, or depression. On magnification endoscopywith NBI, all of the patients had irregularities in the glands and microvasculature consistent with early gastric cancer. In addition, all 12 patients exhibited the "stretch sign", an elongation or expansion of the architectural structure. Histologic examination of the resected specimens demonstrated an expanded and edematous mucosal layer infiltrated with tumor cells.CONCLUSION: The "stretch sign" appears to be specific for signet ring cell carcinoma and may aid in the early diagnosis and treatment of this aggressive pathology.

In vivo gastric mucosal histopathology using endocytoscopy

AIM:To study the ability of endocytoscopy to identify normal gastric mucosa and to exclude Helicobacter pylori(H.pylori) infection.METHODS:Endocytoscopic examination of the gastric corpus and antrum was performed in 70 consecutive patients.Target biopsy specimens were also obtained from the assessed region and multiple H.pylori tests were performed.The normal endocytoscopy patterns of the corpus and antrum were divided into the normal pit-dominant type(n-Pit) or the normal papilladominant type(n-Pap), respectively characterized as either regular pits with capillary networks or round, smooth papillary structures with spiral capillaries.On the other hand, normal mucosa was defined as mucosa not demonstrating histological abnormalities, including inflammation and atrophy.RESULTS:The sensitivity and specificity of n-Pit for normal mucosa in the gastric corpus were 94.4%and 97.1%,respectively,whereas those of n-Pap for normal mucosa in the antrum were 92.0%and 86.7%,respectively.The positive predictive values of n-Pit and n-Pap for H.pylori-negative tissue were 88.6%and 93.1%,respectively,and their negative predictive values for H.pylori-negative tissues were 42.9%and41.5%,respectively.The inter-observer agreement for determining n-Pit and n-Pap for normal mucosa were0.857 and 0.769,respectively,which is considered reliable.CONCLUSION:N-Pit and n-Pap,seen using EC,are considered useful predictors of normal mucosa and theabsence of H.pylori infection.

Endocytoscopic visualization of squamous cell islands within Barrett's epithelium

AIM: To study the endocytoscopic visualization of squamous cell islands within Barrett's epithelium. METHODS: Endocytoscopy (ECS) has been studied in the surveillance of Barrett's esophagus, with controversial results. In initial studies, however, a soft catheter type endocytoscope was used, while only methylene blue dye was used for the staining of Barrett's mucosa. Integrated type endocytoscopes (GIF-Q260 EC, Olympus Corp, Tokyo, Japan) have been recently developed, with the incorporation of a high-power magnifying endocytoscope into a standard endoscope together with narrow-band imaging (NBI). Moreover, double staining with a mixture of 0.05% crystal violet and 0.1% of methylene blue (CM) during ECS enables higher quality images comparable to conventional hematoxylin eosin histopathological images.RESULTS: In vivo endocytoscopic visualization of papillary squamous cell islands within glandular Barrett's epithelium in a patient with long-segment Barrett's esophagus is reported. Conventional white light endoscopy showed typical long-segment Barrett's esophagus, with small squamous cell islands within normal Barrett's mucosa, which were better visualized by NBI endoscopy. ECS after double CM staining showed regular Barrett's esophagus, while higher magnification (×480) revealed the orifices of glandular structures better. Furthermore, typical squamous cell papillary protrusion, classified as endocytoscopic atypia classification (ECA) 2 according to ECA, was identified within regular glandular Barrett's mucosa. Histological examination of biopsies taken from the same area showed squamous epithelium within glandular Barrett's mucosa, corresponding well to endocytoscopic findings. CONCLUSION: To our knowledge, this is the first report of in vivo visualization of esophageal papillary squamous cell islands surrounded by glandular Barrett's epithelium.

Microvasculature of the esophagus and gastroesophageal junction: Lesson learned from submucosal endoscopy

Advanced therapeutic endoscopy,in particular endoscopic mucosal resection,endoscopic submucosal dissection,per-oral endoscopic myotomy,submucosal endoscopic tumor resection opened a new era where direct esophageal visualization is possible.Combining these information with advanced diagnostic endoscopy,the esophagus is organized,from the luminal side to outside,into five layers(epithelium,lamina propria with lamina muscularis mucosa,submucosa,muscle layer,adventitia).A specific vascular system belonging to each layer is thus visible: Mucosa with the intra papillary capillary loop in the epithelium and the sub-epithelial capillary network in the lamina propria and,at the lower esophageal sphincter(LES) level with the palisade vessels; submucosa with the drainage vessels and the spindle veins at LES level; muscle layer with the perforating vessels; periesophageal veins in adventitia.These structures are particularly important to define endoscopic landmark for the gastro-esophageal junction,helpful in performing submucosal therapeutic endoscopy.

Raman spectroscopy for the diagnosis of unlabeled and unstained histopathological tissue specimens

AIM To investigate the possibility of diagnosing gastric cancer from an unstained pathological tissue using Raman spectroscopy, and to compare the findings to those obtained with conventional histopathology. METHODS We produced two consecutive tissue specimens from areas with and without cancer lesions in the surgically resected stomach of a patient with gastric cancer. One of the two tissue specimens was stained with hematoxylin and eosin and used as a reference for laser irradiation positioning by the spectroscopic method. The other specimen was left unstained and used for Raman spectroscopy analysis. RESULTS A significant Raman scattering spectrum could be obtained at all measurement points. Raman scattering spectrum intensities of 725 cm^(-1) and 782 cm^(-1), are associated with the nucleotides adenine and cytosine, respectively. The Raman scattering spectrum intensity ratios of 782 cm^(-1)/620 cm^(-1), 782 cm^(-1)/756 cm^(-1), 782 cm^(-1)/1250 cm^(-1), and 782 cm^(-1)/1263 cm^(-1) in the gastric adenocarcinoma tissue were significantly higher than those in the normal stomach tissue.CONCLUSION The results of this preliminary experiment suggest the feasibility of our spectroscopic method as a diagnostic tool for gastric cancer using unstained pathological specimens.