- Email: [email protected]
Plasma Levels of Pirnas as Biomarkers for Prognosis and Predicting Tumor Recurrence in Colorectal Cancer Patients
AGA Abstracts
observed in patients infected with clarithromycin-resistant strains of H. pylori. Vonoprazan improved the eradication rates of H. pylori with no increase in the incidence of adverse events.
701 MICROBIOME AND METABOLOME RESPONSES TO FECAL MICROBIOTA TRANSPLANTATION FOR RECURRENT CLOSTRIDIUM DIFFICILE INFECTION IN PEDIATRIC PATIENTS Richard Kellermayer, Miriam Balderas, Dorottya Nagy-Szakal, Ruth Ann Luna, Faith Ihekweazu, Karen Queliza, Claire Bocchini, Jennifer Spinler, Numan Oezguen, Robert J. Shulman, James Versalovic, Tor C. Savidge Background & Aims: Fecal microbiota transplantation (FMT) is the most effective treatment for recurrent (antibiotic refractory) Clostridium difficile (C. difficile) infection (rCDI). Special considerations for FMT must be taken with pediatric rCDI, given a higher asymptomatic carriage rate and more frequent underlying clinical conditions when compared to adults. We performed fecal microbiome and metabolome analyses in a cohort of pediatric rCDI patients who received FMT. Methods: Eighteen rCDI patients (1.5-18 years old) received filtered, frozen-thawed fecal preparation from screened, self-designated (1 case) or universal donors (2 donors, 8 and 9 cases, respectively). Eight patients had no underlying disease. Five patients had inflammatory bowel disease (IBD: 4 ulcerative colitis [UC], 1 Crohn's disease [CD]), 1 had a heart transplant, 2 had significant neurologic impairment, and 2 had intestinal dysmotility as underlying conditions. The fecal microbiome and metabolome (in 4 responder cases) was analyzed using unbiased global high throughput methods and was compared to healthy age matched controls. Results: All 8 patients without underlying disease (44.4%) had resolution of their symptoms following a single FMT. One to 3 FMTs cleared C. difficile in all 5 IBD patients, but symptomatic improvement was observed only in 1 (20%) of these cases. The 3 patients without clearance of C. difficile were deemed as carriers of the bacterium based on their post-FMT course. The 2 remaining patients (heart transplant, and mild dysmotility) responded to 2 and 1 FMT, respectively. Post-FMT recipient microbiomes in the responders clustered closer to the standardized donors than to healthy age matched controls in most cases, and up to 16 months after the treatment. Microbiome changes in the non-responders varied. Post-FMT metabolomes in 4 responders with uncomplicated rCDI clustered with that of healthy children. Conclusions: This is the largest reported pediatric FMT cohort with high throughput microbiome and metabolome analyses utilizing age matched controls. FMT is highly effective for the treatment of pediatric rCDI in patients without complicating clinical disorders. Prolonged donor derived tax onomic changes lead to metabolic recovery in recipient microbiomes. Special attention should be taken when considering FMT for rCDI in pediatric patients with underlying conditions.
677 INFLUENCE OF CYP3A4/5 GENOTYPE STATUS FOR OUTCOME OF VONOPRAZAN-CONTAINED HELICOBACTER PYLORI ERADICATION THERAPY IN JAPANESE Mitsushige Sugimoto, Hiromitsu Ban, Taketo Otsuka, Osamu Inatomi, Shigeki Bamba, Akira Andoh Introduction: Potent acid inhibition with acid inhibitory drugs is crucial to the success of eradication for Helicobacter pylori infection. In 2015, vonoprazan, which competitively inhibits the binding of potassium ion to H+/K+-ATPase in gastric parietal cells, has been clinically available in Japan. Vonoprazan inhibits H+/K+-ATPase activity at 400-fold more potent than that of lansoprazole, proton pump inhibitor (PPI). Therefore, the eradication regimen contained vonoprazan has advantage for increasing the eradication rate, more than 90%, compared with a PPI-based therapy, irrespective with infection of clarithromycin-resistant H. pylori strain. Although vonoprazan is mainly metabolized by CYP3A4/5, not CYP2C19, it is unclear whether its acid inhibitory effect and outcome of H. pylori eradication differ among CYP3A4/5 genotypes. Our aim was to clarify the influence of CYP3A4/5 genotypes on outcome of H. pylori eradication including vonoprazan in Japanese. Methods: We investigated the influence of CYP3A4/5 and CYP2C19 genotypes and susceptibility to antimicrobial agents for outcome of vonoprazan-contained eradication regimen for 7 days in 120 Japanese: (1) with amoxicillin 750mg and clarithromycin 20mg twice daily (bid) as the first-line treatment (n=76); (2) with amoxicillin 750mg and metronidazole 250mg bid as the secondline (n=29); and (3) with amoxicillin 500mg qid and sitafloxacin 100mg bid as the thirdline (n=15). Eradication status was assessed at eight weeks via a 13C-urea breath test. CYP3A4*22, CYP3A5*3 and CYP2C19*2/*3 were genotyped for all patients. Results: Eradication rate on intention-to-treat analysis was 82.9% (95% confidence interval: 72.5%-90.6%, 63/76) in the first-line treatment, 93.1% (77.2%-99.1%, 27/29) in the second-line and 80.0% (51.9%-95.7%, 12/15) in the third-line. None with CYP3A4*22 was observed. 38.3% of patients (46/120) were CYP3A5 *1/*3 type and 55.0% (66/120) were *3/*3 type. In naive patients, the prevalence of clarithromycin-resistant strain was 42.4% (14/33). Eradication rate in patients with CYP3A5 *1 carrier type (CYP3A5 *1/*1 and *1/*3 types) was 72.7% (54.5%-86.7%, 24/33), which was significantly lower than that in the *3/*3-type (90.7%, 77.9%-97.4%, 39/43, p=0.039) in the first-line treatment. However, no significant differences of clinical outcome in the second-line and third-line therapies were seen among CYP3A4/ 5 genotypes. Conclusions: Eradication rates of vonoprazan-based eradication therapy can be achieved high compared with PPI-based therapy, irrespective with eradication history and infection of clarithromycin-resistant strain. However, because CYP3A5*3 genotype may be one of determinate for outcome of eradication regimen including vonoprazan in Japan, genotyping of CYP3A5*3 will be required to be paid attention for clinical outcome before treatment.
708 PLASMA LEVELS OF PIRNAS AS BIOMARKERS FOR PROGNOSIS AND PREDICTING TUMOR RECURRENCE IN COLORECTAL CANCER PATIENTS Tsuyoshi Ozawa, Yuji Toiyama, Naoki Takahashi, Takeshi Nagasaka, Toshiyoshi Fujiwara, Yasuhide Yamada, Masato Kusunoki, Toshiaki Watanabe, Ajay Goel Background: Accumulating evidence in recent years indicates that small non-coding RNAs (sncRNAs), such as miRNAs, lncRNAs, snoRNAs and piwi-interacting RNAs (piRNAs), play a central role in many diseases, including cancer. In this regard, although the mechanistic role piRNAs play in cancer pathogenesis continues to evolve, it is believed that they may function just like miRNAs in causing transcriptional repression of gene targets or by inducing hypermethylation of specific tumor suppressor genes. Considering their small size and stability in biological fluids, and lack of any reports for their role as disease biomarkers in cancer, we undertook this study to systematically and comprehensively evaluate the piRNA expression patterns and their biomarker potential in tissue and plasma specimens from colorectal cancer (CRC) patients. Methods: We performed a discovery phase by performing statistical analysis on RNA-Seq data from TCGA, to identify differentially expressed piRNAs between early (stage I/II) vs. late (stage IV) CRCs. Thereafter we evaluated the clinical significance of these piRNAs in 405 surgically resected tissue and 145 plasma samples from two, independent patient cohorts (testing: n=202, validation: n=203) by quantitative PCR, and analyzed our results with various clinicopathological features and patient survival. Results: We identified a panel of 3 piRNAs (piR61919, piR30652, and piR31111), which were significantly up-regulated in cancer vs. matched normal tissues in CRC patients. KaplanMeier curves revealed that high piR61919 and piR30652 expression were significantly associated with poor overall survival (p < 0.001 and 0.016, respectively), and these results were subsequently validated in an independent validation cohort (p = 0.016 and 0.005, respectively). More importantly, high levels of piR61919 and piR30652 in plasma specimens significantly associated with poor recurrence free survival (p = 0.029 and < 0.001, respectively) in stage II-III CRC patients. A Cox regression model which included combined expression levels of piR61919 and piR30652 revealed a robust AUC value of 0.779 to predict 5 years' recurrence in stage II-III CRC patients. Conclusions: We for the first time demonstrate that piR61919 and piR30652 expression levels in tissue, but more intriguingly in plasma, may serve as novel and clinically useful biomarkers to predict prognosis and tumor recurrence in CRC.
700 USE OF THE MICROBIOME AS A BIOMARKER TO IDENTIFY PATIENTS WITH COLORECTAL POLYPS Patrick M. Gillevet, Ezzat Dadkhah, Masoumeh Sikaroodi, Swati Dalmet, Louis Y. Korman, Robert Hardi, Jeffrey H. Baybick, David K. Hanzel, Greg Kuehn, Thomas Kuehn Background: Alterations in the colonic microbiota are likely to play a role in colonic neoplasia. Characterization of these complex changes could be used as a non-invasive biomarker to screen patients for pre-malignant lesions. A prospective study of patients undergoing screening or surveillance colonoscopy was performed to determine if a unique microbiome pattern could be used to identify patients with colorectal polyps (hyperplastic and adenoma). Methods: Home collected stool samples (HS), rectal swabs (SS), and sigmoid biopsies (BS) were obtained from 232 subjects. The cohort consisted of 87 females and 131 males of which 193 were Caucasian, 21 African American, and 4 Asian Americans. The microbiome in these samples were interrogated using Multitag™ sequencing on an Ion Torrent PGM Next Generation Sequencing System. We analyzed 504 samples (168 HS, 211 SS, 125 BS) that met depth of coverage criteria based on rarefaction analysis with three different operational taxonomic units (OTUs) clustering techniques (UPARSE, UPGMA, and UCLUST). Nonparametric statistical methods (Metastats, LEfSe, Kruskal Wallis, Indicator) were used to identify the taxa that were significantly different between subjects with polyps and subjects without polyps. These informative OTUs were then used to build classifying predictors for the presence of polyps using a Naïve Bayes, Support Vector Machine (SVM), and Neural Network models. The prediction power of classifiers was highest when the informative UPARSE OTUs from the HS samples were used in the model. Results: Polyps were found in 59% of the colonoscopies; one or more adenomas were found in 42% and advanced adenomas in 10%. Microbiome exploratory modeling and analysis resulted in classification accuracy for a Naïve Bayes model of 79%, the SVM 68% and the Neural Network 81%. A naïve holdout analysis was performed using these models and we observed an average false positive rate (FPR) of 12% and an average false negative rate (FNR) of 11.5% over all the models. Furthermore, only 7 out of 19 false negatives were wrong with all three models and, of these, only two were advanced adenomas. Conclusion: These results indicate that microbiome analysis combined with advanced machine learning methods could be used: as a biomarker to screen patients for polyps, to optimize the use of colonoscopy, and to reduce the associated health care costs. Additional studies with larger populations need to be performed to improve model performance, clinical utility and characterize a colon neoplasia microbiota model.
AGA Abstracts
709 THE BET-DEPENDENT EXPRESSION OF NSD1 DEFINES AN EPIGENETIC NETWORK THAT PROMOTES THE GROWTH OF PANCREATIC DUCTAL ADENOCARCINOMA Curtis E. Adams, Yinshi Huang, Kim C. Honselmann, Keith D. Lillemoe, Carlos Fernandez-Del Castillo, Andrew L. Warshaw, Andrew S. Liss Background: While the aberrant expression of epigenetic regulators has been linked to a variety of cancers, little is known about their contribution to pancreatic ductal adenocarcinoma (PDAC). Members of the BET family of chromatin adaptors contain tandem bromodomains that bind to acetylated lysines on histones, thereby enacting transcriptional changes. We have recently demonstrated that the activity of this family contributes to malignant PDAC growth. Our transcriptomic analysis revealed NSD1 to be among the top genes downregulated in PDAC cells after inhibition of BET bromodomain activity. NSD1 is a histone
S-152
observed in patients infected with clarithromycin-resistant strains of H. pylori. Vonoprazan improved the eradication rates of H. pylori with no increase in the incidence of adverse events.
701 MICROBIOME AND METABOLOME RESPONSES TO FECAL MICROBIOTA TRANSPLANTATION FOR RECURRENT CLOSTRIDIUM DIFFICILE INFECTION IN PEDIATRIC PATIENTS Richard Kellermayer, Miriam Balderas, Dorottya Nagy-Szakal, Ruth Ann Luna, Faith Ihekweazu, Karen Queliza, Claire Bocchini, Jennifer Spinler, Numan Oezguen, Robert J. Shulman, James Versalovic, Tor C. Savidge Background & Aims: Fecal microbiota transplantation (FMT) is the most effective treatment for recurrent (antibiotic refractory) Clostridium difficile (C. difficile) infection (rCDI). Special considerations for FMT must be taken with pediatric rCDI, given a higher asymptomatic carriage rate and more frequent underlying clinical conditions when compared to adults. We performed fecal microbiome and metabolome analyses in a cohort of pediatric rCDI patients who received FMT. Methods: Eighteen rCDI patients (1.5-18 years old) received filtered, frozen-thawed fecal preparation from screened, self-designated (1 case) or universal donors (2 donors, 8 and 9 cases, respectively). Eight patients had no underlying disease. Five patients had inflammatory bowel disease (IBD: 4 ulcerative colitis [UC], 1 Crohn's disease [CD]), 1 had a heart transplant, 2 had significant neurologic impairment, and 2 had intestinal dysmotility as underlying conditions. The fecal microbiome and metabolome (in 4 responder cases) was analyzed using unbiased global high throughput methods and was compared to healthy age matched controls. Results: All 8 patients without underlying disease (44.4%) had resolution of their symptoms following a single FMT. One to 3 FMTs cleared C. difficile in all 5 IBD patients, but symptomatic improvement was observed only in 1 (20%) of these cases. The 3 patients without clearance of C. difficile were deemed as carriers of the bacterium based on their post-FMT course. The 2 remaining patients (heart transplant, and mild dysmotility) responded to 2 and 1 FMT, respectively. Post-FMT recipient microbiomes in the responders clustered closer to the standardized donors than to healthy age matched controls in most cases, and up to 16 months after the treatment. Microbiome changes in the non-responders varied. Post-FMT metabolomes in 4 responders with uncomplicated rCDI clustered with that of healthy children. Conclusions: This is the largest reported pediatric FMT cohort with high throughput microbiome and metabolome analyses utilizing age matched controls. FMT is highly effective for the treatment of pediatric rCDI in patients without complicating clinical disorders. Prolonged donor derived tax onomic changes lead to metabolic recovery in recipient microbiomes. Special attention should be taken when considering FMT for rCDI in pediatric patients with underlying conditions.
677 INFLUENCE OF CYP3A4/5 GENOTYPE STATUS FOR OUTCOME OF VONOPRAZAN-CONTAINED HELICOBACTER PYLORI ERADICATION THERAPY IN JAPANESE Mitsushige Sugimoto, Hiromitsu Ban, Taketo Otsuka, Osamu Inatomi, Shigeki Bamba, Akira Andoh Introduction: Potent acid inhibition with acid inhibitory drugs is crucial to the success of eradication for Helicobacter pylori infection. In 2015, vonoprazan, which competitively inhibits the binding of potassium ion to H+/K+-ATPase in gastric parietal cells, has been clinically available in Japan. Vonoprazan inhibits H+/K+-ATPase activity at 400-fold more potent than that of lansoprazole, proton pump inhibitor (PPI). Therefore, the eradication regimen contained vonoprazan has advantage for increasing the eradication rate, more than 90%, compared with a PPI-based therapy, irrespective with infection of clarithromycin-resistant H. pylori strain. Although vonoprazan is mainly metabolized by CYP3A4/5, not CYP2C19, it is unclear whether its acid inhibitory effect and outcome of H. pylori eradication differ among CYP3A4/5 genotypes. Our aim was to clarify the influence of CYP3A4/5 genotypes on outcome of H. pylori eradication including vonoprazan in Japanese. Methods: We investigated the influence of CYP3A4/5 and CYP2C19 genotypes and susceptibility to antimicrobial agents for outcome of vonoprazan-contained eradication regimen for 7 days in 120 Japanese: (1) with amoxicillin 750mg and clarithromycin 20mg twice daily (bid) as the first-line treatment (n=76); (2) with amoxicillin 750mg and metronidazole 250mg bid as the secondline (n=29); and (3) with amoxicillin 500mg qid and sitafloxacin 100mg bid as the thirdline (n=15). Eradication status was assessed at eight weeks via a 13C-urea breath test. CYP3A4*22, CYP3A5*3 and CYP2C19*2/*3 were genotyped for all patients. Results: Eradication rate on intention-to-treat analysis was 82.9% (95% confidence interval: 72.5%-90.6%, 63/76) in the first-line treatment, 93.1% (77.2%-99.1%, 27/29) in the second-line and 80.0% (51.9%-95.7%, 12/15) in the third-line. None with CYP3A4*22 was observed. 38.3% of patients (46/120) were CYP3A5 *1/*3 type and 55.0% (66/120) were *3/*3 type. In naive patients, the prevalence of clarithromycin-resistant strain was 42.4% (14/33). Eradication rate in patients with CYP3A5 *1 carrier type (CYP3A5 *1/*1 and *1/*3 types) was 72.7% (54.5%-86.7%, 24/33), which was significantly lower than that in the *3/*3-type (90.7%, 77.9%-97.4%, 39/43, p=0.039) in the first-line treatment. However, no significant differences of clinical outcome in the second-line and third-line therapies were seen among CYP3A4/ 5 genotypes. Conclusions: Eradication rates of vonoprazan-based eradication therapy can be achieved high compared with PPI-based therapy, irrespective with eradication history and infection of clarithromycin-resistant strain. However, because CYP3A5*3 genotype may be one of determinate for outcome of eradication regimen including vonoprazan in Japan, genotyping of CYP3A5*3 will be required to be paid attention for clinical outcome before treatment.
708 PLASMA LEVELS OF PIRNAS AS BIOMARKERS FOR PROGNOSIS AND PREDICTING TUMOR RECURRENCE IN COLORECTAL CANCER PATIENTS Tsuyoshi Ozawa, Yuji Toiyama, Naoki Takahashi, Takeshi Nagasaka, Toshiyoshi Fujiwara, Yasuhide Yamada, Masato Kusunoki, Toshiaki Watanabe, Ajay Goel Background: Accumulating evidence in recent years indicates that small non-coding RNAs (sncRNAs), such as miRNAs, lncRNAs, snoRNAs and piwi-interacting RNAs (piRNAs), play a central role in many diseases, including cancer. In this regard, although the mechanistic role piRNAs play in cancer pathogenesis continues to evolve, it is believed that they may function just like miRNAs in causing transcriptional repression of gene targets or by inducing hypermethylation of specific tumor suppressor genes. Considering their small size and stability in biological fluids, and lack of any reports for their role as disease biomarkers in cancer, we undertook this study to systematically and comprehensively evaluate the piRNA expression patterns and their biomarker potential in tissue and plasma specimens from colorectal cancer (CRC) patients. Methods: We performed a discovery phase by performing statistical analysis on RNA-Seq data from TCGA, to identify differentially expressed piRNAs between early (stage I/II) vs. late (stage IV) CRCs. Thereafter we evaluated the clinical significance of these piRNAs in 405 surgically resected tissue and 145 plasma samples from two, independent patient cohorts (testing: n=202, validation: n=203) by quantitative PCR, and analyzed our results with various clinicopathological features and patient survival. Results: We identified a panel of 3 piRNAs (piR61919, piR30652, and piR31111), which were significantly up-regulated in cancer vs. matched normal tissues in CRC patients. KaplanMeier curves revealed that high piR61919 and piR30652 expression were significantly associated with poor overall survival (p < 0.001 and 0.016, respectively), and these results were subsequently validated in an independent validation cohort (p = 0.016 and 0.005, respectively). More importantly, high levels of piR61919 and piR30652 in plasma specimens significantly associated with poor recurrence free survival (p = 0.029 and < 0.001, respectively) in stage II-III CRC patients. A Cox regression model which included combined expression levels of piR61919 and piR30652 revealed a robust AUC value of 0.779 to predict 5 years' recurrence in stage II-III CRC patients. Conclusions: We for the first time demonstrate that piR61919 and piR30652 expression levels in tissue, but more intriguingly in plasma, may serve as novel and clinically useful biomarkers to predict prognosis and tumor recurrence in CRC.
700 USE OF THE MICROBIOME AS A BIOMARKER TO IDENTIFY PATIENTS WITH COLORECTAL POLYPS Patrick M. Gillevet, Ezzat Dadkhah, Masoumeh Sikaroodi, Swati Dalmet, Louis Y. Korman, Robert Hardi, Jeffrey H. Baybick, David K. Hanzel, Greg Kuehn, Thomas Kuehn Background: Alterations in the colonic microbiota are likely to play a role in colonic neoplasia. Characterization of these complex changes could be used as a non-invasive biomarker to screen patients for pre-malignant lesions. A prospective study of patients undergoing screening or surveillance colonoscopy was performed to determine if a unique microbiome pattern could be used to identify patients with colorectal polyps (hyperplastic and adenoma). Methods: Home collected stool samples (HS), rectal swabs (SS), and sigmoid biopsies (BS) were obtained from 232 subjects. The cohort consisted of 87 females and 131 males of which 193 were Caucasian, 21 African American, and 4 Asian Americans. The microbiome in these samples were interrogated using Multitag™ sequencing on an Ion Torrent PGM Next Generation Sequencing System. We analyzed 504 samples (168 HS, 211 SS, 125 BS) that met depth of coverage criteria based on rarefaction analysis with three different operational taxonomic units (OTUs) clustering techniques (UPARSE, UPGMA, and UCLUST). Nonparametric statistical methods (Metastats, LEfSe, Kruskal Wallis, Indicator) were used to identify the taxa that were significantly different between subjects with polyps and subjects without polyps. These informative OTUs were then used to build classifying predictors for the presence of polyps using a Naïve Bayes, Support Vector Machine (SVM), and Neural Network models. The prediction power of classifiers was highest when the informative UPARSE OTUs from the HS samples were used in the model. Results: Polyps were found in 59% of the colonoscopies; one or more adenomas were found in 42% and advanced adenomas in 10%. Microbiome exploratory modeling and analysis resulted in classification accuracy for a Naïve Bayes model of 79%, the SVM 68% and the Neural Network 81%. A naïve holdout analysis was performed using these models and we observed an average false positive rate (FPR) of 12% and an average false negative rate (FNR) of 11.5% over all the models. Furthermore, only 7 out of 19 false negatives were wrong with all three models and, of these, only two were advanced adenomas. Conclusion: These results indicate that microbiome analysis combined with advanced machine learning methods could be used: as a biomarker to screen patients for polyps, to optimize the use of colonoscopy, and to reduce the associated health care costs. Additional studies with larger populations need to be performed to improve model performance, clinical utility and characterize a colon neoplasia microbiota model.
AGA Abstracts
709 THE BET-DEPENDENT EXPRESSION OF NSD1 DEFINES AN EPIGENETIC NETWORK THAT PROMOTES THE GROWTH OF PANCREATIC DUCTAL ADENOCARCINOMA Curtis E. Adams, Yinshi Huang, Kim C. Honselmann, Keith D. Lillemoe, Carlos Fernandez-Del Castillo, Andrew L. Warshaw, Andrew S. Liss Background: While the aberrant expression of epigenetic regulators has been linked to a variety of cancers, little is known about their contribution to pancreatic ductal adenocarcinoma (PDAC). Members of the BET family of chromatin adaptors contain tandem bromodomains that bind to acetylated lysines on histones, thereby enacting transcriptional changes. We have recently demonstrated that the activity of this family contributes to malignant PDAC growth. Our transcriptomic analysis revealed NSD1 to be among the top genes downregulated in PDAC cells after inhibition of BET bromodomain activity. NSD1 is a histone
S-152