Gastric Mucosa-associated Lymphoid Tissue Lymphoma Research Articles

A Rare Case of Helicobacter Pylori-Negative Gastric- MALT Lymphoma, Disseminated to the Small bowel, Colon, and Lung

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (EMZL-MALT) have been linked to chronic immune stimulation due to infection or autoimmune stimuli. <i>Helicobacter Pylori</i> induced gastric MALT lymphoma is the most extensively documented and well understood. However, in those where <i>H. pylori</i> is not detected, data is less established. Its treatment and prognosis are based on histological evaluation, immunophenotyping, and the use of fluorescence in situ hybridization (FISH), or polymerase chain reaction (PCR) to identify chromosomal translocation and/or unbalanced aberrations.

299P - Efficacy of salvage therapy of helicobacter pylori-positive gastric mucosa-associated lymphoid tissue lymphoma

299P - Efficacy of salvage therapy of helicobacter pylori-positive gastric mucosa-associated lymphoid tissue lymphoma

MECHANISMS OF INTERRACTION OF HELICOBACTER PYLORI WITH EPITHELIUM OF GASTRIC MUCOSA. I. PATHOGENIC FACTORS PROMOTING SUCCESSFUL COLONIZATION

H. pylori is a Gram-negative, crimp and motile bacterium that colonizes the hostile microniche of the human stomach roughly one half of the human population. Then persists for the host’s entire life, but only causes overt gastric disease in a subset of infected hosts. To the reasons contributing to the development of diseases, usually include: concomitant infections of the gastrointestinal tract, improper sterilization of medical instruments, usually endoscopes, nonobservance of personal hygiene rules, prolonged contact with infected or carriers, including family members and a number of other factors. Clinically, H. pylori plays a causative role in the development of a wide spectrum of diseases including chronic active gastritis, peptic and duodenal ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Due to the global distribution of H. pylori, we are able to conclude that smart strategies are contributing to adaptation of the bacterium in an aggressive environment of a stomach and lifelong permanent circulation in its host. Thirty-four years after the discovery of this bacterium, there are still many unanswered questions. For example, which strategies help the bacterium to survive in this inhospitable conditions? Understanding the mechanisms governing H. pylori persistence will improve identification of the increased risk of different gastric diseases in persons infected with this bacterium. A well-defined and long-term equilibrium between the human host and H. pylori allows bacterial persistence in the gastric microniche; although this coexistence leads to a high risk of severe diseases the diseases which are listed above. In this review, we discuss the pathogenesis of this bacterium and the mechanisms it uses to promote persistent colonization of the gastric mucosa, with a focus on recent insights into the role of some virulence factors like urease, LPS, outer membrane proteins, cytotoxins, factors, promoting invasion. Information on the mechanisms related to H. pylori persistence can also provide the direction for future research concerning effective therapy and management of gastroduodenal disorders. The topics presented in the current review are important for elucidating the strategies used by H. pylori to help the bacterium persist in relation to the many unfavorable features of living in the gastric microniche.

Excellent Outcomes after Reduced-Dose Intensity Modulated Radiation Therapy for Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma

Excellent Outcomes after Reduced-Dose Intensity Modulated Radiation Therapy for Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma

Involved-Site Radiation Therapy for H. Pylori-Independent Gastric MALT Lymphoma: 26 Years of Experience with 178 Patients

Gastric mucosa associated lymphoid tissue lymphoma (GML) is often associated with Helicobacter pylori (H. pylori) infection. Eradication of H. pylori is effective for up to 80% of patients. For H. pylori-independent GML, treatment options include surgery, immunotherapy, chemotherapy and radiation therapy (RT). The purpose of this study is to investigate the efficacy and safety of involved site radiation therapy and routine endoscopic surveillance, hypothesizing that radiation is highly effective for H. pylori–independent GML and most patients are curable with RT alone. A single institution database at a tertiary referral cancer center was queried for patients with H. pylori-independent GML treated with radiation between 1991 and 2017. Prior to treatment, pathology was confirmed by hematopathology experts and patients underwent staging with either computed tomography scan, positron emission tomography, endoscopic ultrasound, or a combination of techniques. All patients received involved site radiation therapy to the stomach. The clinical treatment volume (CTV) included the stomach and first part of the duodenum. Perigastric lymph nodes and other parts of the duodenum were also included in CTV if involved with disease. Response was assessed by follow up endoscopies (EGDs) within 10-12 weeks following RT with multiple biopsies at frequent intervals, as well as appropriate radiographic imaging. Survival analysis was calculated using the Kaplan Meier estimator and toxicity was graded according to CTCAEv4 criteria. There were 178 patients identified (56% female). Median age was 63 years (range 25-91 years). Eighty-eight percent of patients presented with stage I disease; 6% were stage II and 6% were stage IV. Median RT dose was 3000 cGy over 20 fractions. Post treatment EGD with biopsy was obtained in 173 patients, of which 95% demonstrated a complete pathologic response. Intriguingly, 4 patients had abnormal posttreatment EGD biopsies that spontaneously resolved. One patient experienced grade 3 toxicity. Over a median follow up of 9 years, 8% experienced local failures and 10% developed distant sites of disease. Eighty-three percent of patients remained lymphoma free. Five and 10-year overall survival estimates from all causes were 97% and 84%, respectively. RT is a highly effective, safe treatment for GML with excellent overall survival and very rare acute or late treatment related toxicities. Spontaneous resolution of abnormal post treatment biopsies offers an intriguing window into histologic responses to radiation. Favorable outcomes from this large retrospective sample of patients provide credible and compelling support for the use of RT for H. pylori-independent GML as standard of care.

Sequential H. pylori eradication and radiation therapy with reduced dose compared to standard dose for gastric MALT lymphoma stages IE &amp; II1E: a prospective randomized trial

In Helicobacter pylori (H. pylori) positive stage I gastric low-grade MALT lymphoma, eradication is the accepted first-line therapy. The role of eradication therapy in lymphoma > stage IE is still unclear. However, about 20% of patients show persistent lymphoma following successful eradication or primary H. pylori-negative lymphoma. A prospective study for salvage radiation therapy with standard 36Gy in comparison to a reduced dose of 25.2Gy is still missing. A prospective, multicentre study investigated the efficacy of eradication in H. pylori-positive gastric low-grade MALT lymphoma stages IE and II1E (HELYX I). Refractory lymphoma or H. pylori-negative patients were treated in a prospective, randomised, multicentre, phase II study to receive either 25.2Gy or 36Gy radiotherapy (HELYX II). 102 patients (3 drop outs) were included in HELYX I: 75/99 (75.8%) showed complete remission after a median of 2.8months. 18 (18.2%) had partial remission (PR) and 6 (6.0%) no change (NC). 29 patients (7 drop outs) were randomized in HELYX II (7 primarily H. pylori-negative, 15 patients from HELYX I with refractory disease after eradication). All patients achieved stable CR irrespective of radiation dose. Both presence of the t(11,18) translocation (OR 9.0, p = 0.01) and monoclonality of the tumour cells (OR 6.3, p = 0.006) were predictors for persistant lymphoma after eradication therapy. Most H. pylori-positive low grade gastric MALT lymphoma stage IE and II1E respond with stable CR after eradication therapy. In patients with refractory disease or H. pylori negative low grade gastric MALT lymphoma a dosage-reduced radiation therapy with 25.2Gy is an effective standard dose in stage IE and II1E. ClinicalTrials.gov: NCT00154440.

Place de la radiothérapie pour les lymphomes du mucosa-associated lymphoid tissue gastriques : résultats d’une étude rétrospective

PurposeWe aimed to evaluate therapeutic results of radiotherapy for gastric mucosa-associated lymphoid tissue (MALT) lymphomas. Patients and methodsWe reviewed retrospectively the records of 11 patients presenting with gastric MALT lymphoma treated between 1993 and 2014. Patients with low-grade lymphoma in failure after helicobacter eradication had exclusive gastric external radiotherapy. Chemotherapy followed by radiotherapy were indicated in case of high grade lymphoma. Radiotherapy doses range between 30 and 40Gy (2Gy per fraction, five fractions per week). ResultsAll tumours were IE stage. Seven patients with low-grade lymphoma had radiotherapy. Four patients with high-grade lymphoma received chemotherapy then radiotherapy. Ten patients are in complete remission after treatment achievement. Five and 10 years disease-free survival are 100%. No severe toxicity was seen. ConclusionEradication of Helicobacter pylori is the mainstay of treatment of gastric MALT. External irradiation is an effective and well-tolerated treatment modality in case of resistance to helicobacter eradication.

An Unusual Presentation of HCV-Associated Gastric MALT Lymphoma

Extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT Lymphoma) is a form of non-Hodgkin's lymphoma(NHL), commonly found in the gastrointestinal tract. The average age of diagnosis is between fifth and seventh decade. MALT Lymphomas are well documented to be associated with chronic inflammation of either autoimmune or infectious etiology. Chronic Helicobacter Pylori (H. Pylori) is associated with approximately 90% of the affected gastric MALT Lymphoma. Treatment of H. Pylori with regimen of antibiotics and PPI associated gastric Maltomas has an approximately 70% cure rate. While there are no current guidelines for management of patient who are H. Pylori negative, recognition and resolution of the underlying inflammatory process is paramount. 34-year-old man with no medical history presented after a syncopal event with tachycardia, hypotension, and black stool on exam. Reported fatigue for several hours preceding the event. Labs were notable for Cr of 1.6, total protein 7.3, albumin 2.5, and Hgb of 3.1. TSH and free T4 were both within normal limits. CT revealed a gastric ulcer 1.2x1.0 cm. Once patient was stabilized, EGD revealed a 4cm non-bleeding necrotic gastric ulcer and a 1cm clean based gastric ulcer. Pathology results read as MALT lymphoma with negative H. Pylori on immunochemistry. Subsequent lab work revealed Hepatitis C viral load of 744,000 IU/ml. Unfortunately, patient left the hospital against medical advice prior to finalized pathology report. A year later he presented again after another syncopal episode. Repeat EGD revealed a 10cm Gastric H. Pylori Negative MALT Lymphoma. The patient left against medical advice again. Gastric MALT lymphomas present with non-specific symptoms including abdominal pain, loss of appetite, and weight loss. Occult blood loss is a reported symptom in less than 20% of cases. In our case, gastric MALT lymphoma is diagnosed on EGD as part evaluation of these non-specific complaints. Given negative histology for H. Pylori from two separate EGDs, gastric MALT lymphoma was likely secondary to chronic HCV.His age, HCV association, negative H.Pylori, and presenting complaint of syncope are all very atypical for gastric MALT Lymphoma. While antibiotics are curative in H. Pylori positive MALT Lymphoma, treatment of active HCV in our patient may not be curative treatment of gastric MALT lymphoma. Additional conventional approaches like surgery, chemotherapy and radiotherapy may be required.2611_A Figure 1. Necrotic Gastric Ulcer2611_B Figure 2. 10cm Gastric MALT Lymphoma

Beyond the Borders of the Gastric Lumen: A Rare Case of Colonic MALT Lymphoma

Colonic mucosa-associated lymphoid tissue (MALT) lymphoma is a rare subtype of marginal zone B-cell non-Hodgkin's Lymphoma (NHL) owing to 2.5% of cases. It also requires more aggressive measures than that of its gastric variant, as seen in the case below. A 56 year old asymptomatic male with a medical history of diabetes, hypertension, hyperlipidemia, and family history positive for colorectal cancer presented to his outpatient gastroenterologist for a surveillance colonoscopy. His last was 5 years prior and notable for two benign tubular adenomas. Repeat colonoscopy revealed two 5 and 10 mm sessile polyps in the transverse and sigmoid colon respectively, which were resected and sent for analysis. Pathology demonstrated atypical lymphoid proliferation, with extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT lymphoma) localized to the sigmoid colon. FISH testing was negative for rearrangement involving MALT1. He was subsequently referred to oncology for further workup to assess the extent of his disease. MALT lymphoma, an uncommon variant of marginal zone B-cell NHL, arise from multiple extra-nodal locations, with the gastrointestinal tract serving as the most common site. Gastric MALT lymphomas have been well documented in the literature, consisting of approximately 60-75% of cases with colonic MALT lymphoma representing a mere 2.5%. Unlike our patient who was asymptomatic on presentation, the majority have abdominal pain as their leading complaint. While management of gastric MALToma has been well-established with treatment consisting of antibiotic therapy targeting H. pylori, there is currently no standard of care for its colonic counterpart. Debates on treatment modalities for both localized versus disseminated disease demonstrate a need for standardization of therapy. In addition, an overall poor prognosis of non-gastric MALToma with five and ten year survival rates of approximately 50 and 20%, respectively, also calls for a more aggressive approach-- such as prompt workup to assess the extent of disease and ensuring regular surveillance as seen in our patient. We present this case to raise physician awareness of this disease to aid in prompt diagnosis and management of this rare entity with the hopes of improving survival.1473_A Figure 1. Sessile polyp, Sigmoid Colon1473_B Figure 2. Sessile polyp, Sigmoid Colon

Efficacy of compound Lactobacillus acidophilus tablets combined with quadruple therapy for Helicobacter pylori eradication and its correlation with pH value in the stomach: a study protocol of a randomised, assessor-blinded, single-centre study

IntroductionHelicobacter pylori (Hp) is an important pathogenic factor for chronic gastritis, peptic ulcer, gastric cancer, gastric mucosa-associated lymphoid tissue lymphoma and other diseases. In China, the Hp infection rate is...

Value of 18F-FDG PET/CT in the staging, interim therapeutic and prognostic evaluation of mucosa-associated lymphoid tissue lymphoma

Objective To assess the value of 18F-fluorodeoxyglucose (FDG) PET/CT in the staging, interim therapeutic and prognostic evaluation of mucosa-associated lymphoid tissue (MALT) lymphoma. Methods Thirty-six MALT lymphoma patients (20 males, 16 females; average age: 61.7 years) confirmed by pathology from January 2008 to January 2018 were retrospectively analyzed.18F-FDG PET/CT were performed before chemotherapy and radiotherapy for staging. The detective sensitivity was evaluated. The staging results of gastric MALT lymphoma and extragastric MALT lymphoma by PET/CT were compared with Fisher exact probability method. PET/CT was performed in 17 of 36 patients after 4 courses of chemotherapy, and 17 patients were divided into positive group (≥4) and negative group (<4) according to scores of Deauville 5-point scale. The progression-free survival (PFS) was evaluated using Kaplan-Meier survival analysis. Results FDG-positive lesions were found in 31 of 36 patients with the sensitivity of 86.1%(31/36). The results of PET/CT were negative in stageⅠpatients. In stageⅡ-Ⅳ patients, the results of 18F-FDG PET/CT combined with bone marrow biopsy were in accordance with the results of clinical staging. The accuracy of PET/CT in staging of gastric MALT lymphoma patients was 9/17, which was significantly lower than that of extragastric MALT lymphoma patients (17/19; P=0.025). The PFS of negative group evaluated by interim PET/CT was longer than that of positive group (χ2=4.16, P<0.05). The 2-year PFS rates of the 2 groups were (85.7±13.2)% and (27.8 ±21.3)%, respectively. The PFS of patients with low expression of Ki-67 was significantly longer than that of patients with high Ki-67 expression(χ2=4.22, P<0.05). Conclusions In stageⅡ-Ⅳ MALT patients, 18F-FDG PET/CT combined with bone marrow biopsy can improve the staging accuracy. The staging accuracy of PET/CT in extragastric MALT lymphoma is significantly higher than that of gastric MALT lymphoma. PET/CT and Ki-67 can provide effective information on the prognostic evaluation for patients with MALT lymphoma. Key words: Lymphoma, B-cell, marginal zone; Neoplasm staging; Prognosis; Positron-emission tomography; Tomography, X-ray computed; Deoxyglucose

MALT Lymphoma, Stress Ulcer and Cholinergic Nerves from the Viewpoint of Bilateral and Unilateral Truncal Vagotomy and Substance P.

Vagal nerve plays an important role in the stomach function. The cholinergic nerves are the most abundantly distributed nerves in the gastric tissue. It has recently been reported that the vagal nerve is significantly related to both gastric cancer development and progression. However, its relation to the mesenchymal tumor, including MALT lymphoma, is not known. In this study, we investigated the effect of unilateral truncal vagotomy on gastric MALT lymphoma development by using Helicobacter heilmannii-infected mouse model as well as that of bilateral truncal vagotomy on stress-induced ulcer formation. In the first part of this study, the distribution of the cholinergic nerves in the rat gastric mucosa and the effect of bilateral truncal vagotomy, as well as various kinds of agents acting on autonomic nerves in rats, were investigated by the histochemical and macroscopic method. In the second part, we employed MALT lymphoma formation in C57BL/6NCrl mice that were infected with Helicobacter heilmannii. A total of 38 infected mice underwent unilateral vagotomy under microscopy. The mice were randomized into 4 groups from which samples were collected; 2, 3, 4 and 6 months after infection. Both the anterior and posterior sides of the stomachs were sampled from each mouse for pathological and immunohistochemical analyses. The bilateral truncal vagotomy significantly suppressed the restraint-induced gastric ulcer formation in rats, while bethanechol, and 6-hydroxydopamine led to an increase of the gastric ulcer formation. In the unilateral truncal vagotomy study using MALT lymphoma, the thickness of the gastric mucosa was reduced in the vagotomized side compared to the non-vagotomized side. Furthermore, the gastric MALT lymphoma was more prominently found in the vagotomized anterior side of stomach compared with that in the non-vagotomized posterior side of stomach. Substance P-immunoreactive nerves markedly increased surrounding the MALT lymphoma and the neurokinin-1 receptor immunoreactive lymphocytes increased within the MALT lymphoma in the vagotomized side. In conclusion, vagotomy enhanced gastric MALT lymphoma development possibly through the substance P-neurokinin-1 receptor pathway.

DWI-MRI vs CT in gastric MALT lymphoma-preliminary results in 19 patients.

To assess the diagnostic performance of [F18] fluoro-2-desoxy-D-glucose positron emission tomography/CT [(18F) FDG-PET/CT] compared to diffusion-weighted imaging (DWI)-MRI of lesion detection in patients with non-FDG avid gastric mucosa-associated lymphoid tissue (MALT) lymphoma. 19 patients with histologically proven gastric MALT lymphoma were included in this prospective Institutional Review Board-approved study. Patients underwent [18F]-FDG-PET/CT and consecutive MRI/DWI. Images were evaluated for the presence of gastric lesions in two anatomically defined groups (region 1: cardia, body, fundus; region 2: antrum, pyloric region) by two senior board-certified radiologists, in an observer-blinded manner. Overall accuracy relative to the reference standard (histology obtained by biopsy) was calculated for each reader and a consensus rating. We found a statistically significant higher accuracy of lesion detection for lesions in region 1 (p = 0.030) and 2 (p = 0.070) for DWI-MRI (100%/78.9%) than for CT (68.4%/42.1%). DWI-MRI seems to be superior accurate to CT for lesion detection in non-FDG avid gastric MALT lymphoma. DWI-MRI seems to be an alternative reliable imaging method for locoregional disease evaluation of non-FDG avid gastric MALT lymphoma.

The fourth Mexican consensus on Helicobacter pylori.

Important advances have been made since the last Mexican consensus on the diagnosis and treatment of Helicobacter pylori (H. pylori) infection was published in 2007. Therefore, the Asociación Mexicana de Gastroenterología summoned 20 experts to produce "The Fourth Mexican Consensus on Helicobacter pylori". From February to June 2017, 4 working groups were organized, a literature review was performed, and 3 voting rounds were carried out, resulting in the formulation of 32 statements for discussion and consensus. From the ensuing recommendations, it was striking that Mexico is a country with an intermediate-to-low risk for gastric cancer, despite having a high prevalence of H. pylori infection. It was also corroborated that peptic ulcer disease, premalignant lesions, and histories of gastric cancer and mucosa-associated lymphoid tissue lymphoma should be considered clear indications for eradication. The relation of H. pylori to dyspeptic symptoms continues to be controversial. Eradication triple therapy with amoxicillin, clarithromycin, and a proton pump inhibitor should no longer be considered first-line treatment, with the following 2 options proposed to take its place: quadruple therapy with bismuth (proton pump inhibitor, bismuth subcitrate, tetracycline, and metronidazole) and quadruple therapy without bismuth (proton pump inhibitor, amoxicillin, clarithromycin, and metronidazole). The need for antimicrobial sensitivity testing when 2 eradication treatments have failed was also established. Finally, the promotion of educational campaigns on the diagnosis and treatment of H. pylori for both primary care physicians and the general population were proposed.

Gastric malt lymphoma mickmicking a submucosal tumor with complete response after chemotherapy

Gastric malt lymphoma mickmicking a submucosal tumor with complete response after chemotherapy

Gastric MALT Lymphoma in a Sleeve Gastrectomy Specimen: Case Report and Literature Review

In the last years, there has been a rise in the use of sleeve gastrectomy (SG) as primary surgical option in the management of morbid obesity. As result, SG specimens (SGSs) are commonly received f...

P-063 - The clinical outcomes and the pathogenetic background of gastric MALT lymphoma in Korea

P-063 - The clinical outcomes and the pathogenetic background of gastric MALT lymphoma in Korea

Treatment of Helicobacter pylori-associated gastritis using traditional Chinese medicine

Abstract Helicobacter pylori is a pathogenic bacterium with strong pathogenicity, which can induce chronic gastritis and peptic ulcer in addition to causing gastric mucosal atrophy and intestinal metaplasia after longterm infection. It is even associated closely with the occurrence of gastric cancer and mucosa-associated lymphoid tissue lymphoma. Recently, the incidence of H.pylori-associated gastritis has increased rapidly worldwide, especially in China. The prevention and treatment of H. pylori infection has become an urgent issue to be resolved by the medical community. Traditional Chinese medicine and the combination of traditional Chinese medicine and Western medicine have recently shown unique advantages in the treatment of H.pylori–associated gastritis. The progress in this area is summarized in this review.

Update on the management of Helicobacter pylori infection. Position paper from the Catalan Society of Digestology

More than 30 years after its discovery, Helicobacter pylori (H. pylori) infection remains the most common cause of gastric and duodenal diseases. H. pylori is the leading cause of chronic gastritis, peptic ulcer, gastric MALT lymphoma and gastric adenocarcinoma. Several consensuses have recently been published on the management of H. pylori infection. The general guidelines of the Spanish consensus, the Toronto Consensus and the Maastricht V Consensus of 2016 are similar but concrete recommendations can vary significantly. In addition, the recommendations of some of these consensuses are decidedly complex. This position paper from the Catalan Society of Digestology is an update of evidence-based recommendations on the management and treatment of H. pylori infection. The aim of this document is to review this information in order to make recommendations for routine clinical practice that are simple, specific and easily applied to our setting.

Anti-Helicobacter pylori therapy in localized gastric mucosa-associated lymphoid tissue lymphoma: A prospective, nationwide, multicenter study in Japan.

Background Helicobacter pylori eradication therapy was approved in Japan for the first‐line, standard treatment of H. pylori‐positive gastric mucosa‐associated lymphoid tissue (MALT) lymphoma. Although several retrospective studies or small‐scale single‐center studies have been reported, a prospective, large‐scale, nationwide, multicenter study has not been reported from Japan.Materials and MethodsWe conducted a prospective, nationwide, multicenter study to evaluate the clinical efficacy of rabeprazole‐based triple H. pylori eradication therapy for patients with localized gastric MALT lymphoma in practice‐based clinical trial. A total of 108 H. pylori‐positive patients with stage I/II 1 gastric MALT lymphoma underwent H. pylori eradication therapy. The primary endpoints were complete remission (CR) rate and the rate of transfer to secondary treatment. The secondary endpoints were CR maintenance duration and overall survival (OS).Results CR of lymphoma was achieved in 84 of 97 patients (86.6%), during the period 2.0‐44.7 months (median, 5.3 months) after starting H. pylori eradication treatment. CR was maintained in 77 of 81 patients (95.1%) for 0.4‐53.2 months (median, 33.1 months). Secondary treatments (radiotherapy, rituximab, or gastrectomy) for gastric MALT lymphoma were needed in 10 of the 97 patients (10.31%). During follow‐up, OS rate was 96.9% (94/97) and the causes of 3 deaths were not related to lymphoma.ConclusionsRabeprazole‐based H. pylori eradication therapy demonstrated a high CR rate, long CR maintenance, and a good OS for patients with localized gastric MALT lymphoma in this prospective, practice‐based, multicenter study.