Fluorescent imaging of superficial head and neck squamous cell carcinoma using a γ-glutamyltranspeptidase-activated targeting agent: a pilot study.

BACKGROUND: Detecting superficial head and neck squamous cell carcinoma (HNSCC) by endoscopy is challenging because of limited morphological hallmarks, and iodine cannot be applied to head and neck lesions due to severe mucosal irritation. γ-glutamyltranspeptidase (GGT), a cell surface enzyme, is overexpressed in several cancers, and it has been reported that γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), a fluorescent targeting agent which can be enzymatically activated and becomes fluorescent after cleavage of a GGT-specific sequence, can be activated within a few minutes after application to animal models. We investigated whether early HNSCC can be detected by applying gGlu-HMRG to clinical samples. METHODS: gGlu-HMRG was applied to four HNSCC cell lines, and fluorescence was observed by fluorescence microscopy and flow cytometry. Immunohistological examination was performed in three recent cases of endoscopic submucosal dissection (ESD) to investigate GGT expression. Fluorescence imaging with gGlu-HMRG in eight clinical samples resected by ESD or surgery was performed, and fluorescence intensity of tumor and normal mucosa regions of interest (ROI) was prospectively measured. RESULTS: All four gGlu-HMRG-applied cell lines emitted green fluorescence. Immunohistological examination demonstrated that GGT was highly expressed in HNSCC of the recent three ESD cases but barely in the normal mucosa. Fluorescence imaging showed that iodine-voiding lesions became fluorescent within a few minutes after application of gGlu-HMRG in all eight resected tumors. Tumor ROI fluorescence intensity was significantly higher than in the normal mucosa five minutes after gGlu-HMRG application. CONCLUSIONS: Fluorescence imaging with gGlu-HMRG would be useful for early detection of HNSCC.

Technical difficulty according to location, and risk factors for perforation, in endoscopic submucosal dissection of colorectal tumors.

BACKGROUND: Colorectal endoscopic submucosal dissection (ESD) is a widely accepted treatment for colorectal tumors, but is technically more difficult and has a higher risk of complications such as perforation than gastric ESD. Few studies have investigated the factors associated with technical difficulty and perforation in colorectal ESD. This study aimed to evaluate the technical difficulty according to location, and the risk factors for perforation, in colorectal ESD. METHODS: This retrospective study included 134 consecutive colorectal tumors treated by ESD in 122 patients at the Division of Endoscopy of Hokkaido University Hospital and the Department of Gastroenterology of Kitami Red Cross Hospital from November 2011 to February 2013. To evaluate the technical difficulty of performing ESD for colorectal tumors at specific locations, the en bloc R0 resection rate, specimen diameter, procedure speed, and procedure time were compared among tumor locations using the χ (2) test or analysis of variance. Risk factors for perforation were identified by multiple logistic regression analysis. RESULTS: The en bloc R0 resection rate was 86.6 % (116/134), the mean tumor diameter was 27.1 mm, and the mean procedure time was 63.5 min. The mean speed of procedures was significantly slower in the sigmoid colon (24.7 min/cm(2)) than in other areas. Perforation occurred in nine cases (6.7 %). Submucosal fibrosis was the only factor independently associated with perforation (odds ratio 5.684, 95 % confidence interval 1.307-24.727). CONCLUSIONS: ESD was slower for sigmoid colon tumors than for tumors in other areas, suggesting that ESD was technically more difficult in the sigmoid colon than in other colorectal areas. Submucosal fibrosis was independently associated with perforation during colorectal ESD.

[H. pylori-negative gastric cancer].

Broad category of H. pylori-negative gastric cancer includes true gastric cancer without history of H. pylori infection, gastric cancer after successful eradication of H. pylori, and H. pylori-negative gastric cancer with history of H. pylori infection. The frequency of gastric cancer without history of H. pylori infection was less than 1% in Japan. Although preventive effect for gastric cancer of H. pylori eradication can be expected, risk of gastric cancer incidence continues after eradication of H. pylori. The frequency of gastric cancer after successful eradication has been increasing, since eradication treatment was widely spread in Japan. The features of H. pylori-negative gastric cancer were reported to be different from conventional H. pylori-positive gastric cancer. Endoscopic screening of gastric cancer requires to understand the characteristics of gastric cancer based on status of H. pylori infection.

Endoscopic diagnosis of early neoplasia of the esophagus with narrow band imaging: correlations among background coloration and iodine staining findings.

BACKGROUND AND AIM: It was previously reported that high-grade intraepithelial neoplasia of the esophagus turns pink within a few minutes after iodine staining (pink-color sign; PCS); however, iodine staining is uncomfortable. By using narrow band imaging (NBI), color change in the area between the intraepithelial papillary capillary loop (background coloration; BGC) is often observed within the brownish area. The diagnostic usefulness of BGC findings for differentiating high-grade intraepithelial neoplasia from low-grade intraepithelial neoplasia was evaluated. METHODS: In a prospective observational study from September 2010 to August 2012, 285 patients who were in a high-risk group for esophageal squamous cell carcinoma underwent endoscopic examination. Lesions with both endoscopic findings of dilated intraepithelial papillary capillary loop on NBI and iodine-unstained areas were studied, in which endoscopic biopsy or endoscopic resection was subsequently performed. The esophageal background mucosa was also evaluated on the basis of the iodine staining pattern (uniform type: Group U, scattered type: Group S). RESULTS: One hundred three esophageal lesions in 87 patients were studied. When BGC was used as the differentiation index, sensitivity was 93.8%, specificity was 88.2%, and accuracy was 91.3%. When PCS was used, sensitivity was 97.9%, specificity was 88.2%, and accuracy was 93.2% (P = 0.79). In Group U (n = 54), BGC had an accuracy of 93.8%, and PCS had an accuracy of 92.3% (P = 1.0). On the other hand, in Group S (n = 33), BGC had an accuracy of 86.8%, while PCS had an accuracy of 94.7% (P = 0.27). CONCLUSIONS: Diagnosis using BGC on NBI may substitute for diagnosis based on PCS in many patients.

Endoscopic in vivo cellular imaging of superficial squamous cell carcinoma of the head and neck by using an integrated endocytoscopy system (with video).

BACKGROUND: Recently, some patients have been found to have superficial squamous cell carcinoma (SCC) of the head and neck region during GI endoscopy; however, endoscopic biopsy from a lesion in the head and neck region is troublesome. An endocytoscopy system has been reported to enable optical biopsy of an esophageal lesion. OBJECTIVE: To evaluate the feasibility of in vivo cellular imaging with an integrated endocytoscopy system for patients with superficial SCC of the head and neck. DESIGN: Experimental pilot study. SETTING: University hospital. PATIENTS: This study involved 12 patients who were found to have superficial SCC of the head and neck during GI endoscopy. INTERVENTION: Endocytoscopic images were taken of each lesion and of the surrounding mucosa. The images were later reviewed by 1 pathologist and 2 endoscopists who were unaware of any other findings. MAIN OUTCOME MEASUREMENTS: Correlation between endocytoscopy diagnosis and histologic diagnosis. RESULTS: Adequate endocytoscopic images could be obtained in 11 of the 12 patients (15 of 16 lesions). For endocytoscopic images of 15 lesions and 12 areas of surrounding mucosa, the overall accuracy of endocytoscopic diagnosis in differentiating between nonmalignant and malignant histopathology by the pathologist, endoscopist 1, and endoscopist 2 were 96%, 96%, and 96%, respectively. The kappa value for interobserver agreement was 0.77. LIMITATIONS: Single-center experience, small number of patients. CONCLUSION: An endocytoscopy system has the potential to be used as an optical biopsy for superficial head and neck lesions.

Identification of a high risk gastric cancer group using serum pepsinogen after successful eradication of Helicobacter pylori.

BACKGROUND AND AIM: Pepsinogen (PG) method is widely used to identify high risk groups of gastric cancer. It is very useful before Helicobacter pylori eradication, but after eradication the method becomes useless because the PGI, PGII, PGI/II ratios change. Therefore, we aimed to identify a high risk group for gastric cancer using serum pepsinogen after successful eradication of H. pylori. METHODS: A total of 261 participants were enrolled after successful eradication of H. pylori in Hokkaido University Hospital from 1995 to 2010. Participants with renal failure, taking proton pump inhibitors, and those with advanced gastric cancer were excluded. Serum levels of PGI and II were measured using chemiluminescent immunoassay method. RESULTS: Receiver operating characteristic curves using cancerous and non-cancerous data in post-eradication determined the optimal cut-off value of PGI/II as 4.5. The sensitivity and the specificity were 65.9% and 79.3%, respectively. The usual PG method includes 48.9% of cancer cases, and the PGI/II ≤ 4.5 in post-eradication includes 65.9% of them, and it includes approximately half of the high risk group of diffuse type cancer. PGI/II ≤ 4.5 in post-eradication included many gastric cancer cases detected after eradication (12/16 = 75%). CONCLUSION: In the identification of a high risk group for gastric cancer, we suggest that the optimal cut-off value of PGI/II after successful eradication of H. pylori is 4.5. PGI/II ≤ 4.5 in post-eradication includes more gastric cancer cases compared with the traditional PG method, and 75% of gastric cancer cases detected after eradication.

Comparison of gastric relaxation and sensory functions between functional dyspepsia and healthy subjects using novel drinking-ultrasonography test.

BACKGROUND: Functional dyspepsia (FD) is a heterogeneous disease characterized by various upper abdominal symptoms. The major mechanism of FD includes impaired fundic accommodation, delayed gastric emptying and visceral hypersensitivity. We developed a novel drinking-ultrasonography test to combine a drink test with ultrasonography to assess gastric motility and sensory function of FD patients. METHOD: Subjects were 20 healthy volunteers and 26 successive FD patients according to the Rome III criteria. The subjects ingested 200 ml of water at 2-min intervals 4 times (total 800 ml) through a straw. The maximum cross section of the proximal stomach was visualized before water intake, after each water intake, and 5 and 10 min after the completion of drinking using extracorporeal ultrasonography. Abdominal symptoms were evaluated using the visual analog scale (VAS) a total of 5 times. RESULTS: The mean cross-sectional area of the fornix after 800 ml of water intake was significantly lower in the FD group compared with the control group. In the FD group, marked abdominal symptoms developed immediately after initiation of water intake, and VAS score differed significantly (p < 0.01) between the control and FD groups at each time point. CONCLUSION: We developed the novel drinking-ultrasonography test which revealed abnormalities in gastric accommodation and sensation in patients with FD compared with healthy controls. This approach can be readily performed and allows the simultaneous evaluation of gastric accommodation, emptying and sensation.

Outcomes and predictive factors of "not self-completion" in gastric endoscopic submucosal dissection for novice operators.

BACKGROUND: Endoscopic submucosal dissection (ESD) has become the standard endoscopic treatment for gastric neoplasms because of its safety and high rate of curability; however, it is not easy for novice operators to learn the technique of ESD. In this study, predictive factors of gastric neoplasms in which novices could not finish ESD by self-completion were evaluated. METHODS: Eighty consecutive ESD procedures performed by four novice operators were retrospectively analyzed. Standard ESD procedures were performed using an insulation-tipped (IT) knife under supervision. Self-completion rates, procedure time, and complete resection rates were evaluated, and predictive factors for "not self-completion" were assessed. RESULTS: The overall self-completion rate and en bloc plus R0 resection rate were 87.5% (70/80) and 95.7 % (67/70), respectively. In "not self-completion" cases (n = 10), the procedure time was longer and resected specimens were larger than those in self-completion cases (83.7 ± 47.3 min vs. 189.5 ± 106.8 min, p < 0.05; 641.2 ± 487.8 vs. 1,116 ± 480.4 mm(2), p < 0.01). Predictive factors of "not self-completion" were tumor location in the middle or upper third of the stomach or in the greater curvature and size of resected specimens larger than 900 mm(2). The self-completion rate of ESD was decreased in cases with more than two predictive factors. CONCLUSIONS: For novice operators, tumor location and resected areas are predictive factors for failure to finish gastric ESD by self-completion. Selection of cancer lesions could be a key factor for effectiveness of ESD training.

Endoscopic resection (endoscopic mucosal resection/ endoscopic submucosal dissection) for superficial esophageal squamous cell carcinoma: current status of various techniques.

Endoscopic resection (ER) has been widely accepted as an effective and minimally invasive treatment for patients with superficial esophageal squamous cell carcinoma (SCC). Techniques of conventional endoscopic mucosal resection (EMR) were first developed for ER. There are three representative methods of conventional EMR: endoscopic esophageal mucosal resection (EEMR)-tube method, EMR using a cap-fitted endoscope (EMRC) method and two-channel EMR method. In the past decade, techniques of endoscopic submucosal dissection (ESD) have become established as standard methods of ER. ESD allows en bloc resection of a lesion, irrespective of the size and shape of the lesion. Recently, results of retrospective cohort studies confirming that ESD is superior to EMR as a curative treatment for superficial esophageal SCC have been reported. Representative knives that are now frequently used in esophageal ESD include Hook knife, Triangle tip knife, IT knife nano, Flush knife-BT, Dual knife, SB knife, and so on. Although there are various knives developed for ESD, the basic techniques for safe and effective ESD are the same.

Long-term clinical outcome of gastric MALT lymphoma after eradication of Helicobacter pylori: a multicentre cohort follow-up study of 420 patients in Japan.

OBJECTIVE: A multicentre cohort follow-up study of a large number of patients with gastric mucosa-associated lymphoid tissue (MALT) lymphoma was conducted to elucidate the long-term outcome of the disease after Helicobacter pylori eradication. METHODS: 420 patients with gastric low-grade MALT lymphoma who had undergone successful H pylori eradication and been followed up for at least 3 years were registered from 21 participating institutes. Responders to treatment were defined as patients whose post-treatment biopsies showed complete histological response (ChR) or probable minimal residual disease (pMRD). Treatment failure was defined as the status of progressive disease or lymphoma relapse after ChR/pMRD. RESULTS: 323 patients (77%) responded to H pylori eradication. A logistic regression analysis showed that absence of H pylori, submucosal invasion determined by endoscopic ultrasonography and t(11;18)/API2-MALT1 were independent predictors of resistance to H pylori eradication. During the follow-up periods ranging from 3.0 to 14.6 years (mean 6.5 years, median 6.04 years), the disease relapsed in 10 of 323 responders (3.1%) while progressive disease was found in 27 of 97 non-responders (27%). Thus, 37 of 420 patients (8.8%) were regarded as treatment failures. Of these 37 patients, transformation into diffuse large B cell lymphoma occurred in nine patients. Among the non-responders and relapsed patients, 17 patients were subjected to a 'watch and wait' strategy while 90 patients underwent second-line treatments including radiotherapy (n=49), chemotherapy (n=26), surgical resection (n=6), chemoradiotherapy (n=5), antibiotic treatment (n=2), rituximab monotherapy (n=1) or endoscopic resection (n=1). Probabilities of freedom from treatment failure, overall survival and event-free survival after 10 years were 90%, 95% and 86%, respectively. Cox multivariate analysis revealed endoscopic non-superficial type to be an independent prognostic factor for adverse freedom from treatment failure, overall survival and event-free survival. CONCLUSIONS: The excellent long-term outcome of gastric MALT lymphoma after H pylori eradication was confirmed by this large-scale follow-up study.

Efficacy of carbon dioxide-insufflating colonoscopy in patients with irritable bowel syndrome: a randomized double-blind study.

BACKGROUND AND AIM: Colonoscopy has the disadvantage of pain and discomfort for patients. It has been shown in randomized controlled trials that carbon dioxide (CO(2) ) insufflations significantly reduce pain and discomfort in patients undergoing colonoscopy. However, there have been no studies in which CO(2) insufflation in colonoscopy of patients with irritable bowel syndrome (IBS) was investigated. METHODS: Randomized double-blind controlled study was conducted to assess the suffering from colonoscopy in patients with IBS and the efficacy of CO(2) insufflation in colonoscopy for patients with IBS. Patients with IBS and controls who received colonoscopy were randomized into an air or CO(2) insufflation group. Patients' symptoms such as distension and pain were compared using a 10-cm visual analog scale (VAS). RESULTS: There were 18 patients in the IBS/air group, 19 patients in the IBS/CO(2) group, 25 patients in the control/air group and 26 patients in the control/CO(2) group. The mean value of severity of distension after colonoscopy and the mean value of severity of pain from during examination to one hour after the examination were higher in the IBS group than in the control group. The severity of these symptoms was reduced earlier in the CO(2) group. CO(2) insufflation in colonoscopy was more effective in the IBS group than in the control group from 15 min to one hour after the examination. CONCLUSION: Regarding colonoscopy-related suffering, IBS patients showed significant differences from non-IBS patients. CO(2) insufflation in colonoscopy is effective for IBS patients, particularly for patients who commence activities after colonscopy.

Frequency of Helicobacter pylori -negative gastric cancer and gastric mucosal atrophy in a Japanese endoscopic submucosal dissection series including histological, endoscopic and serological atrophy.

BACKGROUND: The definition of Helicobacter pylori-negative gastric cancer depends on the accuracy of diagnosis of H. pylori infection. The aim of this study was to determine the frequency of H. pylori-negative gastric cancer and to clarify relationships with histological atrophy, endoscopic atrophy, and serological atrophy. METHODS: A total of 240 early gastric cancers were included in this study. The status of H. pylori infection was determined from the rapid urease test, (13)C-urea breath test, H. pylori culture, histopathological examination and examination of IgG antibodies. In H. pylori-negative gastric cancer, histological atrophy and intestinal metaplasia, endoscopic atrophy and serological atrophy were assessed by pepsinogen. RESULTS: The rate of H. pylori infection was 77.9% and 19 patients (7.9%) had a history of eradication. 34 patients (14.2%) were diagnosed with H. pylori-negative gastric cancer using diagnostic tools of H. pylori. However, most of the patients with H. pylori-negative gastric cancer had histological atrophy and intestinal metaplasia. Only 1 gastric cancer (0.42%) occurred in the mucosa without histological atrophy, endoscopic atrophy or serological atrophy. CONCLUSION: Early gastric cancers in the Japanese endoscopic submucosal dissection series were strongly related to current or past infection with H. pylori and to gastric mucosal atrophy.

Target biopsy using magnifying endoscopy in clinical management of gastric mucosa-associated lymphoid tissue lymphoma.

BACKGROUND AND AIM: We have reported the characteristics of magnified endoscopic images of gastric mucosa-associated lymphoid tissue (MALT) lymphoma before and after treatment. In this study, we investigated the diagnostic efficacy of magnified endoscopic images for target biopsy and evaluation of clinical remission. METHODS: Twenty-one patients diagnosed with localized gastric MALT lymphoma were enrolled. Magnified endoscopy was performed prior to treatment and at a mean period of 1.8 months (1-6 months) after therapy (Helicobacter pylori eradication in 19 patients and radiation therapy in two patients). Microstructural pattern and abnormal vessels in the lesions were assessed, and corpus mucosa without lymphoma was divided into H. pylori-negative mucosa and H. pylori-positive mucosa. Biopsy was the gold standard in this study. RESULTS: Nonstructural areas with abnormal vessels were observed in all patients before treatment. Fifteen patients achieved pathological complete remission. Disappearance of nonstructural areas and abnormal vessels after therapy was associated with pathological remission. Sensitivities of these findings for diagnosis were 76.9% and 85.7%, respectively, and the specificities were 87.5% and 85.7%, respectively. H. pylori eradication therapy was invalid in three patients with H. pylori-negative mucosa in magnified images. CONCLUSIONS: Magnifying endoscopy may be useful for target biopsy of superficial gastric MALT lymphoma in clinical management.

[The significance of H. pylori eradication in NSAIDs ulcer].

The significance of H. pylori eradication for NSAIDs induced gastroduodenal ulcer has not been clarified. NSAIDs and H. pylori infection are independent causal factors for gastroduodenal mucosal injuries. However, the interaction between these two factors is complicated. H. pylori eradication can reduce the risk of NSAIDs induced ulcer in NSAIDs naive patients. However, H. pylori eradication is not recommended in NSAIDs user because of no ulcer suppression and ulcer healing delay. In prevention of NSAIDs induced ulcer recurrence, H. pylori eradication plus PPI treatment is necessary.

Low-grade dysplasia component in early invasive squamous cell carcinoma of the esophagus.

BACKGROUND AND AIMS: It has not been determined whether low-grade squamous dysplasia (LGD) of the esophagus is a precancerous lesion or not. If LGD progresses to squamous cell carcinoma, early carcinoma lesions that have such a natural history might contain a remaining LGD component. METHODS: The lesions in the 68 patients with early invasive squamous cell carcinoma who underwent endoscopic mucosal resection were examined for the presence of an LGD component. If LGD components were observed, the degrees of architectural and cytological abnormalities of LGD components and those of tumor invasive fronts in the same lesions were studied. The degrees of abnormalities of 28 small LGD lesions were also studied. RESULTS: Histological examination of resected specimens confirmed LGD components in 43% of the squamous cell carcinoma lesions. The lesions of lamina propria mucosae (m2) cancer contained a significantly broader area of LGD component than did the lesions of muscularis mucosae (m3) and submucosal layer (sm) cancer (P = 0.037). Mean score for the degrees of cytological abnormalities of LGD component was similar to that of tumor invasive front (P = 0.457) and significantly higher than that of small LGD lesions (P < 0.001). CONCLUSION: Our results indicate the possibility that the lesion was formed by a combination of small lesions that arose as a multicentric occurrence of squamous cell carcinoma and dysplasia. Our results also suggest that an LGD component would transform to carcinoma along with tumor progression. However, the concept of 'basal cell layer type carcinoma in situ' may be suitable for squamous cell lesions with a high degree of cytological abnormalities confined to the lower half of the epithelium.

Long-term treatment of localized gastric marginal zone B-cell mucosa associated lymphoid tissue lymphoma including incidence of metachronous gastric cancer.

BACKGROUND AND AIM: According to a few recent reports on the long-term clinical outcome of gastric marginal zone B-cell mucosa associated lymphoid tissue lymphoma (MALT lymphoma); localized gastric MALT lymphoma generally has a favorable prognosis. However, the risk of metachronous gastric cancer has not been evaluated. In this study, we analyzed long-term outcomes of localized gastric MALT lymphoma including the incidence of metachronous gastric cancer. METHODS: Between April 1996 and May 2008, 60 patients (31 men and 29 women; mean age 58.1 years) with localized gastric MALT lymphoma (stage I and II(1) according to Lugano classification) were analyzed retrospectively. RESULTS: Forty-eight patients (82.6%) achieved complete remission by eradication therapy. Radiation therapy was conducted on eight patients as second-line treatment, and all of them achieved remission. The median follow-up period was 76 months (range, 12-157 months). One patient had local relapse after remission for 5 years and three patients developed early gastric cancer without recurrence of lymphoma (5%). All of the three gastric cancers appeared in the same areas where MALT lymphoma had been eradicated. CONCLUSION: Eradication therapy and radiation therapy for localized gastric MALT lymphoma have a favorable long-term outcome, though regular follow-up endoscopy should be performed for detecting metachronous early gastric cancer.

Lafutidine prevents low-dose aspirin and loxoprofen induced gastric injury: a randomized, double-blinded, placebo controlled study.

BACKGROUND AND AIM: The concomitant use of non-steroidal anti-inflammatory drugs is a risk factor for low-dose aspirin (LDA)-associated upper gastrointestinal toxicity. Lafutidine is an H2-receptor antagonist with gastroprotective activity, produced by acting on capsaicin-sensitive afferent neurons. To evaluate the preventive effect of lafutidine on gastric damage caused by LDA alone and by the combination of both LDA and loxoprofen, we conducted a clinical study using healthy volunteers. METHODS: A randomized, double-blinded, placebo-controlled, crossover study was carried out. Sixteen healthy volunteers without Helicobacter pylori infection were randomly assigned to two groups. Both groups received 81 mg of aspirin once daily for 14 days (on days 1 to 14) and 60 mg of loxoprofen three times daily for the last 7 days (on days 8 to 14). Placebo or 10 mg of lafutidine was administered twice daily for 14 days in each group. After a 2-week washout period, placebo and lafutidine were crossed over. Endoscopic findings of gastric mucosal damage were evaluated according to the modified Lanza score. RESULTS: The mean modified Lanza score was 2.19 ± 1.06 (SD) for aspirin plus placebo as compared with 0.50 ± 0.77 for aspirin plus lafutidine (P < 0.001), and 3.00 ± 1.56 for aspirin plus loxoprofen and placebo as compared with 1.25 ± 1.37 for aspirin plus loxoprofen and lafutidine (P < 0.01). CONCLUSIONS: The addition of loxoprofen to LDA increases gastric mucosal damage. Standard-dose lafutidine significantly prevents gastric mucosal damage induced by LDA alone or LDA plus loxoprofen in H. pylori-negative volunteers. Larger controlled studies are needed to strengthen these findings.