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12 Gene Pancreatitis Susceptibility Panel: Clinical Validation of a Targeted, High-Fidelity, Next Generation Sequencing Approach
AGA Abstracts
significant effects on cell viability, apoptosis or ER stress. However, E+CSE treatment significantly induced upregulation of CHOP and cell death, as determined by propidium iodide staining, and these effects were associated with a marked decrease in XBP1s expression. Moreover, pharmacological inhibition of XBP1s recapitulated the toxic effects of E+CSE. E+CSE also induced cell death in R122H acini, but the effect was greater in R122H than in WT acini. Conclusions: These data support the relevance of alcohol abuse, smoking and dysregulated trypsinogen activation as causative factors in CP. The R122H mouse model provides a platform for studying the interactions between genetic and environmental factors relevant to humans in CP. Funding: DoD W81XWH-15-1-0257 to Ji and Pandol
Tu1377 ESTABLISHMENT OF INDIVIDUALIZED TREATMENT FOR PANCREATIC CANCER PATIENTS BY ANALYZING CANCER GENOMES OF PATIENTORIGIN CANCER CELL LINES Hee Seung Lee, Seungmin Bang, Moon Jae Chung Background: Pancreatic cancer is a highly lethal malignancy and the application of comprehensive next-generation sequencing (NGS) and the genome analysis of patients with pancreatic cancer is proposed to improve patient outcomes. One of the greatest challenges in pancreatic cancer research has been generation of stable cancer cell lines from primary tumors. Conditional reprogramming cells (CRCs) technology is a novel cell-culture system facilitating the generation of stable cultures. CRCs could be grown indefinitely under defined conditions without using genetic immortalization technique. Our objective is to utilize CRCs technology with human pancreatic cancer specimens to develop cancer cell lines, which phenotypically represent the native tumor. Methods: Blood samples (10ml) and tissue specimens (<1x1x1cm, 500mg) were obtained and CRCs culture was done for patients with pancreatic cancer at Severance Hospital, South Korea. We obtained the tumor specimens from resection specimens for patients with operable pancreatic cancer or EUS-guided biopsy for patients with inoperable pancreatic cancer. Cancer cells were co-cultured with irradiated feeder cells and Rho-associated kinase inhibitor, Y-27632. To enable the rational design and testing of patient-origin cancer cell lines for patients with pancreatic cancer, we analyzed the CRCs at a genetic level using whole exome sequencing. In vivo, NOD/SCID mice (5weeks-old Female) were injected with CRCs (2 x 106) in the right and left flank. After sacrifice, tumor size was measured and implanted tumor tissue was fixed in paraformaldehyde for histology documentation. Results: 16 (34.8%) CRCs were established from 46 pancreatic cancer patients. 7 significantly mutated genes, including KRAS, GNAQ, TP53, BRAF, AKT1, NTRK1, and SMAD4 were found in 8 patients with pancreatic cancer. Key oncogenic mutations, KRAS was found in original pancreatic cancer tissue (7/8, 87.5%). Especially, mutation profiles were 100% concordant between original pancreatic cancer tissue and CRCs for 4 patients with pancreatic cancer. In vivo, 2 CRCs was implanted in NOD/SCID mice and the implanted tumor tissue shared a similar morphology to the parent tumor tissue when compared by routine haematoxylin and eosin histology. Conclusions: The ability to generate rapidly patient-origin cancer cells from small tumor specimens may establish individualized treatment system using CRCs for patients with pancreatic cancer. Characteristics of patients and tumors
Tu1375 RAB7 LOCALIZES TO ZYMOGEN GRANULE MEMBRANE IN PANCREATIC ACINAR CELLS AND CONTRIBUTES TO MATURATION OF ZYMOGEN GRANULES BUT NOT TO EXOCYTOSIS Kenichi Takahashi, Hirosato Mashima, Kouichi Miura, Takahsi Goto, Hirohide Ohnishi [Background and Aim] Rab7 is a member of ras-related GTPase Rab protein family and plays crucial roles in late steps of autophagy and endocytosis in various types of cells. However, its physiological role in pancreatic aicnar cells is still uncertain. We therefore analyzed rab7 function in pancreatic acinar cells using pancreas-specific Rab7 knock out mice. [Methods] Pancreas-specific Rab7 knockout mice (Rab7∆pan) were generated by crossing Rab7flox/flox mice with ptf1a-Cre mice. The littermates without ptf1a-Cre alleles were used for the control. The localization of Rab7 in pancreatic acinar cells was analyzed with immunofluorescence confocal microscopy and Western blotting. Regulated exocytosis was examined by measuring amylase secretion from isolated mouse pancreatic acini prepared by collagenase digestion. The diameter of zymogen granules was measured by electron microscopy. [Results] HE staining of pancreas showed no historogical change between control and Rab7∆pan. Confocal immunofluorescence microscopy of pancreas using antiRab7 and anti-amylase antibodies showed that Rab7 localized to apical region of acini and colocalized with amylase. Western blotting of purified zymogen granule membrane fraction of pancreas showed that Rab7 was expressed on zymogen granule membrane of control but not Rab7∆pan. We then examined the effect of Rab7 deficiency on amylase secretion. Both basal and cholecystokinin (~100pM)-stimulated amylase secretion from pancreatic acini were not significantly changed in Rab7∆pan compared with those in control Next, we evaluated the function of Rab7 expressed in zymogen granule maturation. Electron microscopy revealed that diameter of zymogen granules in pancreatic acinar cells of Rab7∆pan were significantly smaller than in those of control (0.54µm vs. 0.75µm; P<0.01). [Conclusion] Rab7 Localizes to zymogen granule membrane in pancreatic acinar cells. Furthermore, Rab7 on zymogen granule membrane plays an important role in the maturation of zymogen granules but not in exocytosis.
Tu1376 12 GENE PANCREATITIS SUSCEPTIBILITY PANEL: CLINICAL VALIDATION OF A TARGETED, HIGH-FIDELITY, NEXT GENERATION SEQUENCING APPROACH David C. Whitcomb, Jessica LaRusch, Mark Haupt Background: Recurrent acute & chronic pancreatitis (CP) are inter-related complex disorders with features overlapping other known & idiopathic conditions of the pancreas & abdomen. At least 12 genes increase susceptibility and/or modify severity of pancreatic disorders: CaSR, CEL, CFTR, CTRC, CPA1, GGT1, PRSS1/2, SBDS, SPINK1, UBR1 & a CLDN2 risk haplotype (PDSG). Precision medicine for pancreatic disorders allows integration of demographics, other risk factors, biomarkers & clinical context. We hypothesize high-quality genetic data will improve patient classification & management. PancreasDx® is a new advanced next generation sequencing technology (NGS) evaluating 12 pancreatic disease susceptibility genes (PDSG). Objective: Evaluate PancreasDx performance identifying PDSG DNA variants in deeply phenotyped patients with known PDSG variants. Methods: PancreasDx was designed using Thunderstorm® (RainDance Technologies, Billerica, MA). The 1680 targets & sequencing method were technically validated as a laboratory-developed test under CLIA/CAP protocols at HudsonAlpha Institute of Biotechnology Clinical Services Laboratory (Huntsville, AL). De-identified genomic DNA acquired in IRB-approved protocols was obtained. Coded amplicons were sequenced with MiSeq (Illumina, San Diego, CA). NextGene (Softgenetics, State College, PA) aligned sequence files and determined genotypes. Variant calls required read depth >100, minor allele frequency 25-75% for heterozygotes, & high quality score. Mutation reports were compared. Results: 36 subjects (35 pancreatitis, 1 control), mean age at sample collection of 40 yrs (range 6-81), were included. Most common TIGAR-O classification were genetic (>50%), alcohol and idiopathic. Mean DNA sequence depth was 1,404. PancreasDx correctly identified all known, clinically significant variants (CSV). Variants in all genes except PRSS2 were identified, most commonly in CFTR, PRSS1 & SPINK1. Previously unrecognized CSV were seen in 8 patients, including novel CFTR & CTRC variants. CSV occurred in all idiopathic CP patients & changed classification of 5 patients with alcohol and hyperlipidemic etiology to atypical cystic fibrosis. Variants with predicted functional effects were noted in all genes, including GGT1, CaSR & CEL. A significant enrichment of functional CaSR variants (> 50% of patients) were also identified and linked to a specific pathogenic pathway. Conclusion: PancreasDx accurately identifies CSV in an established pancreatitis population. The broad coverage & deep sequencing method detected a likely etiologic mechanism in subjects classified as idiopathic pancreatitis. PancreasDx identified new mutations in patients with prior genetic testing. Clinical utility was demonstrated by identification of multiple clinically significant mechanistic variants, allowing for reclassifying patients by mechanistic etiology.
AGA Abstracts
S-900
significant effects on cell viability, apoptosis or ER stress. However, E+CSE treatment significantly induced upregulation of CHOP and cell death, as determined by propidium iodide staining, and these effects were associated with a marked decrease in XBP1s expression. Moreover, pharmacological inhibition of XBP1s recapitulated the toxic effects of E+CSE. E+CSE also induced cell death in R122H acini, but the effect was greater in R122H than in WT acini. Conclusions: These data support the relevance of alcohol abuse, smoking and dysregulated trypsinogen activation as causative factors in CP. The R122H mouse model provides a platform for studying the interactions between genetic and environmental factors relevant to humans in CP. Funding: DoD W81XWH-15-1-0257 to Ji and Pandol
Tu1377 ESTABLISHMENT OF INDIVIDUALIZED TREATMENT FOR PANCREATIC CANCER PATIENTS BY ANALYZING CANCER GENOMES OF PATIENTORIGIN CANCER CELL LINES Hee Seung Lee, Seungmin Bang, Moon Jae Chung Background: Pancreatic cancer is a highly lethal malignancy and the application of comprehensive next-generation sequencing (NGS) and the genome analysis of patients with pancreatic cancer is proposed to improve patient outcomes. One of the greatest challenges in pancreatic cancer research has been generation of stable cancer cell lines from primary tumors. Conditional reprogramming cells (CRCs) technology is a novel cell-culture system facilitating the generation of stable cultures. CRCs could be grown indefinitely under defined conditions without using genetic immortalization technique. Our objective is to utilize CRCs technology with human pancreatic cancer specimens to develop cancer cell lines, which phenotypically represent the native tumor. Methods: Blood samples (10ml) and tissue specimens (<1x1x1cm, 500mg) were obtained and CRCs culture was done for patients with pancreatic cancer at Severance Hospital, South Korea. We obtained the tumor specimens from resection specimens for patients with operable pancreatic cancer or EUS-guided biopsy for patients with inoperable pancreatic cancer. Cancer cells were co-cultured with irradiated feeder cells and Rho-associated kinase inhibitor, Y-27632. To enable the rational design and testing of patient-origin cancer cell lines for patients with pancreatic cancer, we analyzed the CRCs at a genetic level using whole exome sequencing. In vivo, NOD/SCID mice (5weeks-old Female) were injected with CRCs (2 x 106) in the right and left flank. After sacrifice, tumor size was measured and implanted tumor tissue was fixed in paraformaldehyde for histology documentation. Results: 16 (34.8%) CRCs were established from 46 pancreatic cancer patients. 7 significantly mutated genes, including KRAS, GNAQ, TP53, BRAF, AKT1, NTRK1, and SMAD4 were found in 8 patients with pancreatic cancer. Key oncogenic mutations, KRAS was found in original pancreatic cancer tissue (7/8, 87.5%). Especially, mutation profiles were 100% concordant between original pancreatic cancer tissue and CRCs for 4 patients with pancreatic cancer. In vivo, 2 CRCs was implanted in NOD/SCID mice and the implanted tumor tissue shared a similar morphology to the parent tumor tissue when compared by routine haematoxylin and eosin histology. Conclusions: The ability to generate rapidly patient-origin cancer cells from small tumor specimens may establish individualized treatment system using CRCs for patients with pancreatic cancer. Characteristics of patients and tumors
Tu1375 RAB7 LOCALIZES TO ZYMOGEN GRANULE MEMBRANE IN PANCREATIC ACINAR CELLS AND CONTRIBUTES TO MATURATION OF ZYMOGEN GRANULES BUT NOT TO EXOCYTOSIS Kenichi Takahashi, Hirosato Mashima, Kouichi Miura, Takahsi Goto, Hirohide Ohnishi [Background and Aim] Rab7 is a member of ras-related GTPase Rab protein family and plays crucial roles in late steps of autophagy and endocytosis in various types of cells. However, its physiological role in pancreatic aicnar cells is still uncertain. We therefore analyzed rab7 function in pancreatic acinar cells using pancreas-specific Rab7 knock out mice. [Methods] Pancreas-specific Rab7 knockout mice (Rab7∆pan) were generated by crossing Rab7flox/flox mice with ptf1a-Cre mice. The littermates without ptf1a-Cre alleles were used for the control. The localization of Rab7 in pancreatic acinar cells was analyzed with immunofluorescence confocal microscopy and Western blotting. Regulated exocytosis was examined by measuring amylase secretion from isolated mouse pancreatic acini prepared by collagenase digestion. The diameter of zymogen granules was measured by electron microscopy. [Results] HE staining of pancreas showed no historogical change between control and Rab7∆pan. Confocal immunofluorescence microscopy of pancreas using antiRab7 and anti-amylase antibodies showed that Rab7 localized to apical region of acini and colocalized with amylase. Western blotting of purified zymogen granule membrane fraction of pancreas showed that Rab7 was expressed on zymogen granule membrane of control but not Rab7∆pan. We then examined the effect of Rab7 deficiency on amylase secretion. Both basal and cholecystokinin (~100pM)-stimulated amylase secretion from pancreatic acini were not significantly changed in Rab7∆pan compared with those in control Next, we evaluated the function of Rab7 expressed in zymogen granule maturation. Electron microscopy revealed that diameter of zymogen granules in pancreatic acinar cells of Rab7∆pan were significantly smaller than in those of control (0.54µm vs. 0.75µm; P<0.01). [Conclusion] Rab7 Localizes to zymogen granule membrane in pancreatic acinar cells. Furthermore, Rab7 on zymogen granule membrane plays an important role in the maturation of zymogen granules but not in exocytosis.
Tu1376 12 GENE PANCREATITIS SUSCEPTIBILITY PANEL: CLINICAL VALIDATION OF A TARGETED, HIGH-FIDELITY, NEXT GENERATION SEQUENCING APPROACH David C. Whitcomb, Jessica LaRusch, Mark Haupt Background: Recurrent acute & chronic pancreatitis (CP) are inter-related complex disorders with features overlapping other known & idiopathic conditions of the pancreas & abdomen. At least 12 genes increase susceptibility and/or modify severity of pancreatic disorders: CaSR, CEL, CFTR, CTRC, CPA1, GGT1, PRSS1/2, SBDS, SPINK1, UBR1 & a CLDN2 risk haplotype (PDSG). Precision medicine for pancreatic disorders allows integration of demographics, other risk factors, biomarkers & clinical context. We hypothesize high-quality genetic data will improve patient classification & management. PancreasDx® is a new advanced next generation sequencing technology (NGS) evaluating 12 pancreatic disease susceptibility genes (PDSG). Objective: Evaluate PancreasDx performance identifying PDSG DNA variants in deeply phenotyped patients with known PDSG variants. Methods: PancreasDx was designed using Thunderstorm® (RainDance Technologies, Billerica, MA). The 1680 targets & sequencing method were technically validated as a laboratory-developed test under CLIA/CAP protocols at HudsonAlpha Institute of Biotechnology Clinical Services Laboratory (Huntsville, AL). De-identified genomic DNA acquired in IRB-approved protocols was obtained. Coded amplicons were sequenced with MiSeq (Illumina, San Diego, CA). NextGene (Softgenetics, State College, PA) aligned sequence files and determined genotypes. Variant calls required read depth >100, minor allele frequency 25-75% for heterozygotes, & high quality score. Mutation reports were compared. Results: 36 subjects (35 pancreatitis, 1 control), mean age at sample collection of 40 yrs (range 6-81), were included. Most common TIGAR-O classification were genetic (>50%), alcohol and idiopathic. Mean DNA sequence depth was 1,404. PancreasDx correctly identified all known, clinically significant variants (CSV). Variants in all genes except PRSS2 were identified, most commonly in CFTR, PRSS1 & SPINK1. Previously unrecognized CSV were seen in 8 patients, including novel CFTR & CTRC variants. CSV occurred in all idiopathic CP patients & changed classification of 5 patients with alcohol and hyperlipidemic etiology to atypical cystic fibrosis. Variants with predicted functional effects were noted in all genes, including GGT1, CaSR & CEL. A significant enrichment of functional CaSR variants (> 50% of patients) were also identified and linked to a specific pathogenic pathway. Conclusion: PancreasDx accurately identifies CSV in an established pancreatitis population. The broad coverage & deep sequencing method detected a likely etiologic mechanism in subjects classified as idiopathic pancreatitis. PancreasDx identified new mutations in patients with prior genetic testing. Clinical utility was demonstrated by identification of multiple clinically significant mechanistic variants, allowing for reclassifying patients by mechanistic etiology.
AGA Abstracts
S-900