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109 Physician Response to Reflex Immunohistochemical (IHC) Testing for Microsatellite Instability (MSI) in Colorectal Cancers (CRC)
AGA Abstracts
diagnosed six months after a negative capsule endoscopy procedure. All neoplastic lesions were within reach of a conventional gastroduodenoscope. All patients with neoplasia were males, with mutations of different genes (MSH6, MSH2, MLH1), over 50 years of age and without a family history of small bowel cancer . Conclusions The prevalence of small bowel neoplasia in asymptomatic Lynch syndrome patients in this study was1.5%. All neoplastic lesions were localized in the duodenum and in reach of conventional gastroduodenoscopy. No neoplastic lesions were detected in patients aged under 50 years. Although capsule endoscopy has the potential to detect these neoplastic lesions at an early stage, small bowel neoplasia may still be missed.
autocrine/paracrine expression of GREM1 and other morphogens is required to maintain a stem-cell population, and that aberrant epithelial expression in sporadic lesions and cell lines, HMPS patients and Villin-Grem1 mice acts by expanding the pool of cells susceptible to subsequent somatic mutation and neoplastic transformation. 111 Altered P2X3 Receptors in Prevertebral Ganglia During Colitis Is Dependent on Mammalian Target of Rapamycin David R. Linden
109
Background: Postganglionic sympathetic neurons in the prevertebral ganglia (PVG) integrate preganglionic inputs from the CNS and viscerofugal input from the gut to reflexively coordinate gastrointestinal motor function between distant regions of the alimentary canal. In control animals, this fast synaptic transmission is purely mediated by nicotinic acetylcholine receptors. Colitis in the guinea pig is associated with an upregulation of post-synaptic P2X3 receptors in the PVG and a de novo emergence of purinergic fast synaptic transmission. Interestingly, PVG transcripts for P2X3 receptors are high in control animals and unchanged by colitis suggesting translational control of P2X3 receptor expression. Systemic inhibition of P2X receptors restores a more normal small bowel transit rate compared to delayed transit in vehicle-treated colitic animals supporting the functional significance of the colitis-induced non-cholinergic transmission. Aims: To determine whether P2X3 receptors are upregulated in mouse PVG during colitis and whether upregulation is due to signaling through mammalian target of rapamycin (mTOR). Methods: Colitis was induced in adult C57Bl/6 mice by a single enema of trinitrobenzene sulfonic acid (TNBS) dissolved in 30% EtOH. Rapamycin (1mg/kg i.p.) or vehicle (25% DMSO) was delivered 2h prior to TNBS. Total PVG protein levels of P2X3 receptors were determined using immunoblot analysis relative to GAPDH. Localization of P2X3 receptors were determined using immunohistochemistry. Evoked fast synaptic transmission was assessed by conventional intracellular electrophysiology. Results: In the mouse superior mesenteric ganglion there is the emergence of purinergic fast synaptic transmission 24h following TNBS. Immunofluorescence detected increased P2X3 receptor immunoreactivity in the PVG of mice 24h after TNBS compared to controls. Immunoblot analysis detected two distinct bands corresponding to the immature (45kDa) and glycosylated mature cell surface (56kDa) forms of the protein. Interestingly, there was a significant decrease in mature P2X3 receptors in vehicle-treated colitic mice (0.10±0.02) compared to vehicle-treated controls (0.18±0.02) that was restored by treatment with rapamycin (0.17±0.02; P,0.05 two-way ANOVA). The immature form of the P2X3 receptor was significantly increased in colitic animals treated with rapamycin (0.64±0.09) compared to vehicletreated colitic mice (0.42±0.11) and both groups of control mice. Conclusion: There is a functional upregulation of P2X3 receptors in the mouse PVG during colitis. The apparent decrease in total receptor levels may reflect activation-induced internalization and degradation of the receptor during colitis that is blocked by rapamycin suggesting upregulation of functional receptors are dependent on mTOR signaling. Supported by NIH grant DK76665.
Physician Response to Reflex Immunohistochemical (IHC) Testing for Microsatellite Instability (MSI) in Colorectal Cancers (CRC) Jeremy L. Matloff, Brijen Shah, Alexandros D. Polydorides, Lina Jandorf, Clarissa Martinez, Aimee L. Lucas, Steven H. Itzkowitz Background: Lynch Syndrome (LS) is a genetic predisposition to certain cancers, especially CRC, due to a germline mutation of a DNA mismatch repair (MMR) gene. Patients with LS often have faster adenoma-carcinoma progression, and thus require earlier and more frequent colonoscopies. Many patients are unaware that they have LS until a diagnosis of cancer has been made. The vast majority of CRCs in LS demonstrate MSI. Recent data (JAMA 308(15):1555-65) suggests that testing all CRCs for presence of MSI increases the sensitivity of identifying patients with LS. Many centers perform universal (i.e. reflex) IHC staining of all CRCs for loss of MMR protein expression as a surrogate for MSI status. However, little is known about how front-line clinicians interpret such results, or how this might affect patient care. Aim: Among a group of physicians caring for patients with CRC, we aimed to determine their awareness of reflex IHC testing and how they respond to these results. Methods: We developed and administered an IRB-approved online survey to attending physicians who have received CRC pathology reports since 2009, when our institution adopted reflex IHC testing. The results appear as an addendum to the pathology report. From pathology records, we identified clinicians as "positive" if they had received at least one CRC report with IHC suggestive of MSI, and "control" if they never received such a report. We asked whether clinicians ever received a pathology report that suggested a high likelihood of MSI-positive CRC, and how management decisions would change based on MSI status. The survey was sent to 41 gastroenterologists (GIs) and 25 colorectal, general, and oncologic surgeons who submitted CRC specimens to the Mount Sinai Pathology Department since 2009, and also to a listserv of our medical oncologists. Results: A total of 30 physicians completed the survey entirely; 13 were GIs (7 control, 6 positive), 13 were surgeons (4 control, 9 positive), and 4 were medical oncologists (all controls) who treat CRC. Of the 15 "positive" physicians, 33% (1 surgeon, 4 GIs) were unaware of ever having received an MSI-positive result. Of the 30 physicians surveyed, when encountering MSIpositive IHC results, only 5 (17%) would recommend germline testing for LS; 8 (27%) would recommend referral to a genetics counselor; 16 (53%) would inform the patient of this result; and 16 (53%) would take a more extensive family history. Discussion: Among clinicians who have received MSI-positive pathology reports based on reflex IHC testing, management is highly variable. In fact, knowledge that this test is performed is not universal, and some physicians are unaware that they have ever received MSI-positive results. Thus, for universal IHC testing to be most effective, reporting mechanisms, physician awareness, and response to positive results need to be improved.
112 Upregulation of Bile Acid Receptor (TGR5) and nNOS in the Gastric Myenteric Plexus Is Responsible for Delayed Gastric Emptying Following Chronic High Fat Feeding in Rats Hui Zhou, Shi-Yi Zhou, Yuanxu Lu, Chung Owyang Chronic high fat feeding (HF) is associated with functional dyspepsia and delayed gastric emptying. This phenomenon occurs independently of CCK's action. HF diets lead to increase in bile acid production and high concentrations of bile acid in the circulation. Recently, G protein coupled bile acid receptor 1 (GPBAR1 or TGR5) has been reported to be present in the enteric nervous system and may modulate GI motility. We hypothesize that HF feeding induces increased expression of gastric nNOS resulting in enhanced gastric relaxation and delayed gastric emptying. This is mediated by increased bile acid action on TGR5 located on nNOS gastric neurons. To test this hypothesis, we performed studies in rats fed with regular chow (11.3% kcal/g fat) or high fat diet (53% kcal/g fat). Immunohistochemical studies showed that 35% of gastric neurons expressed nNOS and 46% expressed TGR5. 35% of the nNOS containing neurons also expressed TGR5. Two wks HF caused a significant increase in nNOS (14.2+2 vs 7.5+1.3 cells/ganglion, P ,0.05) and TGR5 (15.3+2.2 vs 7.4+0.8 cells/ganglion, P,0.05) containing neurons. This was accompanied by a 3.65 and 2.13 fold increase in gene expression and a 1.6 and 1.9 fold in protein expression of nNOS and TGR5 levels in the gastric myenteric plexus. Organ bath fundic muscle strip studies (60 V, 2 ms, 1-20 Hz for 30 s) in the presence of atropine (10-6 M) and guanethidine (10-6 M) showed NANC relaxations induced by 1, 2.5, 5, 10 and 20 Hz were enhanced by 64%, 35%, 32%, 39% and 68% respectively following 2 wks HF diet (P ,0.05). This was accompanied by a 20% delay in gastric emptying of solid food following 2 wk HF feeding. To provide direct evidence that bile acid stimulates the expression of TGR5 and NOS, we showed that incubation of whole mount gastric corpus with deoxycholic acid (100 μM) for 24 hr resulted in a 46% and 92% increase in TGR5 and nNOS expression respectively. To demonstrate that the increased expression of nNOS and enhanced gastric NANC relaxation following 2 wk HF diet is mediated by increased circulatory bile acids, we performed terminal ileum resection (20 cm) to interrupt the enterohepatic circulation of bile acid. After 5 days to recover, rats were given 2 wks HF feeding. Distal ileal resection prevented increase in nNOS and TGR5 expression in the gastric myenteric plexus evoked by HF feeding and restored normal NANC relaxation. In conclusion, this is the first demonstration that HF feeding induces upregulation of nNOS and TGR5 expression in the gastric myenteric plexus resulting in enhanced NANC relaxation and delayed gastric emptying. This appears to be mediated by increased levels of circulating bile acid stimulated by HF feeding. These findings may provide a mechanism to explain functional dyspepsia and delayed gastric emptying following chronic HF diet.
110 Aberrant Epithelial Expression of the BMP Antagonist GREMLIN1 in Human Colorectal Lesions and a Mouse Model of Hereditary Mixed Polyposis Syndrome (HMPS) Acts via Promotion of a Stem-Cell Phenotype Hayley L. Davis, Pedro Rodenas Cuadrado, Chiara Bardella, Annabelle Lewis, Jaeger Emma, Sreelakshmi Mallappa, Susan K. Clark, James East, Runjan Chetty, Ian Tomlinson, Simon Leedham Background. HMPS is characterized by the generation of polyps with mixed adenomatous, hyperplastic and juvenile polyp morphology. We have shown a causative role for a 40KB duplication on chromosome 15.q13.3 in affected families resulting in ectopic epithelial expression of the BMP antagonist GREM1, a morphogen with expression usually restricted to the pericryptal fibroblasts surrounding the intestinal stem-cell niche. We generated a novel HMPS mouse model (Villin-Grem1 mouse), investigated the somatic mutation pathway of human HMPS lesions and the expression of GREM1 in sporadic polyps and cell-lines to functionally assess the consequences of epithelial GREM1 expression. Methods. The VillinGrem1 mouse construct was generated by inserting the mouse Grem1 cDNA sequence downstream of the 9KB Villin promoter. Pronuclear injection generated 4 founder lines, 3 of which shared the same phenotype with marked up-regulation of intestinal epithelial Grem1 expression. The somatic mutation burden of human HMPS lesions was determined using laser capture and candidate gene sequencing with (epi)mutations clonally ordered to establish the adenoma-carcinoma sequence. GREM1 expression was assessed in sporadic human polyps ex vivo, and colorectal cancer cell-lines and immortalised, non-oncogenic human colonic epithelial cells (HCEC) in vitro. Results. 3-month old Villin-Grem1 mice had widened, blunted villi with ectopic crypt formation. Dysplastic changes in these intravillus crypts correlated with loss of p16 expression. This progressed to a florid pan-intestinal polyposis in 7 month old animals with polyps showing a mixed adenomatous/serrated/cystic phenotype. Human lesion clonal ordering revealed KRAS and BRAF as initiating mutations with a narrow range of APC mutations subsequently selected in dyplastic lesions. Human lesions were frequently aberrantly methylated. Aberrant epithelial GREM1 expression was seen in sporadic adenomas, and in some CRC cell-lines and proliferating HCEC cells, where expression correlated with stem-cell properties and markers. Conclusions. In intestinal homeostasis a restricted paracrine gradient of GREM1 expression from the mesenchyme is important to maintain a basal crypt stem-cell niche. The formation of ectopic crypts in the villi of the Villin-Grem1 mouse indicates stem-cell migration or ectopic niche generation promoting stem-cell like properties in the transit amplifying cells of the villus. In human HMPS lesions somatic mutation is required to initiate polyp formation. We hypothesise that
AGA Abstracts
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diagnosed six months after a negative capsule endoscopy procedure. All neoplastic lesions were within reach of a conventional gastroduodenoscope. All patients with neoplasia were males, with mutations of different genes (MSH6, MSH2, MLH1), over 50 years of age and without a family history of small bowel cancer . Conclusions The prevalence of small bowel neoplasia in asymptomatic Lynch syndrome patients in this study was1.5%. All neoplastic lesions were localized in the duodenum and in reach of conventional gastroduodenoscopy. No neoplastic lesions were detected in patients aged under 50 years. Although capsule endoscopy has the potential to detect these neoplastic lesions at an early stage, small bowel neoplasia may still be missed.
autocrine/paracrine expression of GREM1 and other morphogens is required to maintain a stem-cell population, and that aberrant epithelial expression in sporadic lesions and cell lines, HMPS patients and Villin-Grem1 mice acts by expanding the pool of cells susceptible to subsequent somatic mutation and neoplastic transformation. 111 Altered P2X3 Receptors in Prevertebral Ganglia During Colitis Is Dependent on Mammalian Target of Rapamycin David R. Linden
109
Background: Postganglionic sympathetic neurons in the prevertebral ganglia (PVG) integrate preganglionic inputs from the CNS and viscerofugal input from the gut to reflexively coordinate gastrointestinal motor function between distant regions of the alimentary canal. In control animals, this fast synaptic transmission is purely mediated by nicotinic acetylcholine receptors. Colitis in the guinea pig is associated with an upregulation of post-synaptic P2X3 receptors in the PVG and a de novo emergence of purinergic fast synaptic transmission. Interestingly, PVG transcripts for P2X3 receptors are high in control animals and unchanged by colitis suggesting translational control of P2X3 receptor expression. Systemic inhibition of P2X receptors restores a more normal small bowel transit rate compared to delayed transit in vehicle-treated colitic animals supporting the functional significance of the colitis-induced non-cholinergic transmission. Aims: To determine whether P2X3 receptors are upregulated in mouse PVG during colitis and whether upregulation is due to signaling through mammalian target of rapamycin (mTOR). Methods: Colitis was induced in adult C57Bl/6 mice by a single enema of trinitrobenzene sulfonic acid (TNBS) dissolved in 30% EtOH. Rapamycin (1mg/kg i.p.) or vehicle (25% DMSO) was delivered 2h prior to TNBS. Total PVG protein levels of P2X3 receptors were determined using immunoblot analysis relative to GAPDH. Localization of P2X3 receptors were determined using immunohistochemistry. Evoked fast synaptic transmission was assessed by conventional intracellular electrophysiology. Results: In the mouse superior mesenteric ganglion there is the emergence of purinergic fast synaptic transmission 24h following TNBS. Immunofluorescence detected increased P2X3 receptor immunoreactivity in the PVG of mice 24h after TNBS compared to controls. Immunoblot analysis detected two distinct bands corresponding to the immature (45kDa) and glycosylated mature cell surface (56kDa) forms of the protein. Interestingly, there was a significant decrease in mature P2X3 receptors in vehicle-treated colitic mice (0.10±0.02) compared to vehicle-treated controls (0.18±0.02) that was restored by treatment with rapamycin (0.17±0.02; P,0.05 two-way ANOVA). The immature form of the P2X3 receptor was significantly increased in colitic animals treated with rapamycin (0.64±0.09) compared to vehicletreated colitic mice (0.42±0.11) and both groups of control mice. Conclusion: There is a functional upregulation of P2X3 receptors in the mouse PVG during colitis. The apparent decrease in total receptor levels may reflect activation-induced internalization and degradation of the receptor during colitis that is blocked by rapamycin suggesting upregulation of functional receptors are dependent on mTOR signaling. Supported by NIH grant DK76665.
Physician Response to Reflex Immunohistochemical (IHC) Testing for Microsatellite Instability (MSI) in Colorectal Cancers (CRC) Jeremy L. Matloff, Brijen Shah, Alexandros D. Polydorides, Lina Jandorf, Clarissa Martinez, Aimee L. Lucas, Steven H. Itzkowitz Background: Lynch Syndrome (LS) is a genetic predisposition to certain cancers, especially CRC, due to a germline mutation of a DNA mismatch repair (MMR) gene. Patients with LS often have faster adenoma-carcinoma progression, and thus require earlier and more frequent colonoscopies. Many patients are unaware that they have LS until a diagnosis of cancer has been made. The vast majority of CRCs in LS demonstrate MSI. Recent data (JAMA 308(15):1555-65) suggests that testing all CRCs for presence of MSI increases the sensitivity of identifying patients with LS. Many centers perform universal (i.e. reflex) IHC staining of all CRCs for loss of MMR protein expression as a surrogate for MSI status. However, little is known about how front-line clinicians interpret such results, or how this might affect patient care. Aim: Among a group of physicians caring for patients with CRC, we aimed to determine their awareness of reflex IHC testing and how they respond to these results. Methods: We developed and administered an IRB-approved online survey to attending physicians who have received CRC pathology reports since 2009, when our institution adopted reflex IHC testing. The results appear as an addendum to the pathology report. From pathology records, we identified clinicians as "positive" if they had received at least one CRC report with IHC suggestive of MSI, and "control" if they never received such a report. We asked whether clinicians ever received a pathology report that suggested a high likelihood of MSI-positive CRC, and how management decisions would change based on MSI status. The survey was sent to 41 gastroenterologists (GIs) and 25 colorectal, general, and oncologic surgeons who submitted CRC specimens to the Mount Sinai Pathology Department since 2009, and also to a listserv of our medical oncologists. Results: A total of 30 physicians completed the survey entirely; 13 were GIs (7 control, 6 positive), 13 were surgeons (4 control, 9 positive), and 4 were medical oncologists (all controls) who treat CRC. Of the 15 "positive" physicians, 33% (1 surgeon, 4 GIs) were unaware of ever having received an MSI-positive result. Of the 30 physicians surveyed, when encountering MSIpositive IHC results, only 5 (17%) would recommend germline testing for LS; 8 (27%) would recommend referral to a genetics counselor; 16 (53%) would inform the patient of this result; and 16 (53%) would take a more extensive family history. Discussion: Among clinicians who have received MSI-positive pathology reports based on reflex IHC testing, management is highly variable. In fact, knowledge that this test is performed is not universal, and some physicians are unaware that they have ever received MSI-positive results. Thus, for universal IHC testing to be most effective, reporting mechanisms, physician awareness, and response to positive results need to be improved.
112 Upregulation of Bile Acid Receptor (TGR5) and nNOS in the Gastric Myenteric Plexus Is Responsible for Delayed Gastric Emptying Following Chronic High Fat Feeding in Rats Hui Zhou, Shi-Yi Zhou, Yuanxu Lu, Chung Owyang Chronic high fat feeding (HF) is associated with functional dyspepsia and delayed gastric emptying. This phenomenon occurs independently of CCK's action. HF diets lead to increase in bile acid production and high concentrations of bile acid in the circulation. Recently, G protein coupled bile acid receptor 1 (GPBAR1 or TGR5) has been reported to be present in the enteric nervous system and may modulate GI motility. We hypothesize that HF feeding induces increased expression of gastric nNOS resulting in enhanced gastric relaxation and delayed gastric emptying. This is mediated by increased bile acid action on TGR5 located on nNOS gastric neurons. To test this hypothesis, we performed studies in rats fed with regular chow (11.3% kcal/g fat) or high fat diet (53% kcal/g fat). Immunohistochemical studies showed that 35% of gastric neurons expressed nNOS and 46% expressed TGR5. 35% of the nNOS containing neurons also expressed TGR5. Two wks HF caused a significant increase in nNOS (14.2+2 vs 7.5+1.3 cells/ganglion, P ,0.05) and TGR5 (15.3+2.2 vs 7.4+0.8 cells/ganglion, P,0.05) containing neurons. This was accompanied by a 3.65 and 2.13 fold increase in gene expression and a 1.6 and 1.9 fold in protein expression of nNOS and TGR5 levels in the gastric myenteric plexus. Organ bath fundic muscle strip studies (60 V, 2 ms, 1-20 Hz for 30 s) in the presence of atropine (10-6 M) and guanethidine (10-6 M) showed NANC relaxations induced by 1, 2.5, 5, 10 and 20 Hz were enhanced by 64%, 35%, 32%, 39% and 68% respectively following 2 wks HF diet (P ,0.05). This was accompanied by a 20% delay in gastric emptying of solid food following 2 wk HF feeding. To provide direct evidence that bile acid stimulates the expression of TGR5 and NOS, we showed that incubation of whole mount gastric corpus with deoxycholic acid (100 μM) for 24 hr resulted in a 46% and 92% increase in TGR5 and nNOS expression respectively. To demonstrate that the increased expression of nNOS and enhanced gastric NANC relaxation following 2 wk HF diet is mediated by increased circulatory bile acids, we performed terminal ileum resection (20 cm) to interrupt the enterohepatic circulation of bile acid. After 5 days to recover, rats were given 2 wks HF feeding. Distal ileal resection prevented increase in nNOS and TGR5 expression in the gastric myenteric plexus evoked by HF feeding and restored normal NANC relaxation. In conclusion, this is the first demonstration that HF feeding induces upregulation of nNOS and TGR5 expression in the gastric myenteric plexus resulting in enhanced NANC relaxation and delayed gastric emptying. This appears to be mediated by increased levels of circulating bile acid stimulated by HF feeding. These findings may provide a mechanism to explain functional dyspepsia and delayed gastric emptying following chronic HF diet.
110 Aberrant Epithelial Expression of the BMP Antagonist GREMLIN1 in Human Colorectal Lesions and a Mouse Model of Hereditary Mixed Polyposis Syndrome (HMPS) Acts via Promotion of a Stem-Cell Phenotype Hayley L. Davis, Pedro Rodenas Cuadrado, Chiara Bardella, Annabelle Lewis, Jaeger Emma, Sreelakshmi Mallappa, Susan K. Clark, James East, Runjan Chetty, Ian Tomlinson, Simon Leedham Background. HMPS is characterized by the generation of polyps with mixed adenomatous, hyperplastic and juvenile polyp morphology. We have shown a causative role for a 40KB duplication on chromosome 15.q13.3 in affected families resulting in ectopic epithelial expression of the BMP antagonist GREM1, a morphogen with expression usually restricted to the pericryptal fibroblasts surrounding the intestinal stem-cell niche. We generated a novel HMPS mouse model (Villin-Grem1 mouse), investigated the somatic mutation pathway of human HMPS lesions and the expression of GREM1 in sporadic polyps and cell-lines to functionally assess the consequences of epithelial GREM1 expression. Methods. The VillinGrem1 mouse construct was generated by inserting the mouse Grem1 cDNA sequence downstream of the 9KB Villin promoter. Pronuclear injection generated 4 founder lines, 3 of which shared the same phenotype with marked up-regulation of intestinal epithelial Grem1 expression. The somatic mutation burden of human HMPS lesions was determined using laser capture and candidate gene sequencing with (epi)mutations clonally ordered to establish the adenoma-carcinoma sequence. GREM1 expression was assessed in sporadic human polyps ex vivo, and colorectal cancer cell-lines and immortalised, non-oncogenic human colonic epithelial cells (HCEC) in vitro. Results. 3-month old Villin-Grem1 mice had widened, blunted villi with ectopic crypt formation. Dysplastic changes in these intravillus crypts correlated with loss of p16 expression. This progressed to a florid pan-intestinal polyposis in 7 month old animals with polyps showing a mixed adenomatous/serrated/cystic phenotype. Human lesion clonal ordering revealed KRAS and BRAF as initiating mutations with a narrow range of APC mutations subsequently selected in dyplastic lesions. Human lesions were frequently aberrantly methylated. Aberrant epithelial GREM1 expression was seen in sporadic adenomas, and in some CRC cell-lines and proliferating HCEC cells, where expression correlated with stem-cell properties and markers. Conclusions. In intestinal homeostasis a restricted paracrine gradient of GREM1 expression from the mesenchyme is important to maintain a basal crypt stem-cell niche. The formation of ectopic crypts in the villi of the Villin-Grem1 mouse indicates stem-cell migration or ectopic niche generation promoting stem-cell like properties in the transit amplifying cells of the villus. In human HMPS lesions somatic mutation is required to initiate polyp formation. We hypothesise that
AGA Abstracts
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