- Email: [email protected]
Statins Potently Inhibit YAP Function and Proliferation of Pancreatic Ductal Adenocarcinoma Cancer (PDAC) Cells
of metformin on PDAC cell proliferation are mediated through AMPK. Recently, AMPK has been shown to phosphorylate YAP at a site (Ser94) that inhibits its activity. We therefore hypothesized that statins and metformin synergistically inhibit PDAC cell proliferation, thereby increasing the potency of these inhibitory drugs. Results: To determine whether statin act synergistically with metformin on PDAC cell proliferation, we examined the effect of statins without and with metformin on the ability of PANC-1 and MiaPaCa-2 cells to form colonies (>50 cells/colony), using culture medium containing a physiological concentration of glucose (5 mM). Under these conditions, metformin, at 50-100 mM, dose-dependently decreased colony formation by these PDAC cells. The key point is that a combination of metformin (100 mM) with a low concentration of cerivastatin (0.01 mM) dramatically inhibited colony formation. Similarly, a low concentration of metformin (50 mM) acted synergistically with simvastatin (0.3 mM) to inhibit colony formation by either PANC-1 or MiaPaCa-2 cells. These results underpin a new combinatory approach for PDAC chemoprevention using FDA-approved drugs. In previous studies, we found that active-site mTOR inhibitors induce ERK over-activation in PDAC cells and that co-targeting these pathways enhanced inhibition of PDAC cell proliferation (Mol Cancer Ther. 14:1014-23, 2015). Accordingly, treatment of PANC-1 cells with the dual PI3K/mTOR inhibitor BEZ235 or the MEK inhibitor PD0325901 (each at a concentration as low as 5 nM) modestly inhibited colony formation while their combination resulted in a more pronounced inhibition. Strikingly, a combination of nanomolar concentrations of BEZ235, PD0325901 and cerivastatin dramatically inhibited colony formation by PANC-1 or MiaPaCa-2 cells. Collectively, our results revealed synergistic inhibition of PDAC colony formation by statins in combination with metformin or dual PI3K/mTOR inhibitors + MEK inhibitors. Conclusions: Many studies using statins in model systems in vitro have been questioned for using high concentrations of these agents (10-100 mM). The salient feature of our results is that the potentiating effects of metformin or mTOR inhibitors and MEK inhibitors on the ability of statins to inhibit long-term PDAC cell proliferation (colony forming assay), rendered statins biologically active at clinically relevant concentrations.
Mo2059 Figure 2. Network of eligible comparisons The size of circle is proportional to the number of participants received the treatment (sample size). A) Overall recurrence of colorectal adenomas; B) Recurrence of advanced adenomas; C) Serious adverse events.Every line links direct comparable treatments; The width of the lines reflect the number of trials.
STATINS POTENTLY INHIBIT YAP ACTIVITY AND PROLIFERATION OF MOUSE PANCREATIC CELLS ISOLATED FROM KC AND KPC MICE Fang Hao, Jen-Kuan Chang, James Sinnett-Smith, Guido Eibl, Hui-Hua Chang, Enrique Rozengurt Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases. The highly conserved Hippo pathway is a key regulator of development, organsize, tissue regeneration, tumorigenesis, PDAC development and a novel target for the statins. The conditional KrasG12D mouse model (LSL-KrasG12D; p-48-Cre; referred as KC) of PDAC development is characterized by the presence of an activating Kras mutation, which is conditionally expressed in pancreatic progenitor cells during embryologic development. Coexpression of a p53 mutant (p53R172H) greatly accelerates the development of invasive PDAC (model referred as KPC mice). To extend our studies on the effect of statins on PDAC (see our other Abstracts), we examined the effect of statins on pancreatic cells derived from KC and KPC mice. Results: We examined the effects of cerivastatin and simvastatin on YAP localization, YAP/TEAD-regulated gene expression and colony formation in a pancreatic cell line established from KC mice. We found that treatment of KC cells with either cerivastatin or simvastatin induced striking translocation of YAP from the nucleus to the cytoplasm, thereby blunting the ability of YAP to act as a co-activator of TEAD transcription factors. In line with this possibility, exposure to cerivastatin or simvastatin inhibited the expression of YAP/TEAD-regulated genes in a dose-dependent manner, as shown by decreased mRNA levels of Ctgf, Cyr61 and Birc5. Half-maximal effect (IC50) was produced by cerivastatin at a concentration as low as 0.03 mM. At the highest concentration tested (0.3 mM), these statins inhibited Ctgf, or Cyr61 mRNA levels by 90%. Importantly, treatment with cerivastatin or simvastatin markedly inhibited colony formation by KC cells in a dose-dependent manner. In accord with the results obtained with human PDAC cells, treatment of KC cells with metformin markedly increased the inhibitory effect of cerivastatin or simvastatin on colony formation. Additional studies showed that treatment with cerivastatin strikingly inhibited the expression of YAP/TEAD-regulated genes, including Ctgf, Cyr61 and Birc5 and the colonyforming ability of KPC cells. Cerivastatin inhibited the proliferation of KPC cells at lower concentrations than KC cells (IC50=0.01 mM), suggesting that the expression of the p53 mutant enhances the sensitivity of PDAC cells to statins. Conclusions: Our results show that statins, specific inhibitors of the 3-hydroxy-methylglutaryl CoA reductase (HMGCR), potently inhibited YAP function and proliferation in KC and KPC cells. The results, together with our accompanying Abstracts using human cells and recent epidemiological findings linking statin administration with reduced incidence of PDAC, raise the possibility of repurposing FDA-approved statins for the prevention and therapy of PDAC, a devastating disease with a dismal prognosis.
Figure 4. Median rank and SUCRA values of competing agents SUCRA=surface under the cumulative ranking curve. CrI=credibility interval. For both efficacy(recurrence colorectal adenomas) and safety(incidence of serious adverse events), SUCRA would be 1 when a treatment is certain to be the best and 0 when a treatment is certain to be the worst. The higher SUCRA, the higher probablity of the agent to be the most efficacious or safest.
Mo2060 STATINS POTENTLY INHIBIT YAP FUNCTION AND PROLIFERATION OF PANCREATIC DUCTAL ADENOCARCINOMA CANCER (PDAC) CELLS Fang Hao, Qinhong Xu, Yinglan Zhao, James Sinnett-Smith, Steven H. Young, Jan V. Stevens, Enrique Rozengurt Background: The highly conserved Hippo pathway is a key regulator of development, organsize, tissue regeneration, tumorigenesis and a novel target for the statins. Hippo signals are transduced through a kinase cascade that phosphorylates the transcriptional co-activators YAP and TAZ. Recently, YAP has been identified as a major downstream target of RAS, AMPK, growth factor signaling and cell-cell adhesion, all of which are implicated in pancreatic ductal adenocarcinoma cancer (PDAC). Here, we determined whether statins inhibit YAP function and proliferation of PDAC cells. Results: Exposure of PANC-1 and MiaPaCa-2 cells to cerivastatin, simvastatin or fluvastatin dramatically inhibited the expression of YAP/ TEAD-regulated genes, including Connective Tissue Growth Factor (CTGF) and Cysteinerich angiogenic inducer 61 (Cyr61) in a dose-dependent manner, as determined by RTqPCR. At the highest concentration tested, these statins inhibited CTGF or Cyr61 mRNA levels by 90%. Treatment with cerivastatin also blocked the increase in CTGF mRNA levels
Mo2058 STATINS INHIBIT PROLIFERATION OF PANCREATIC DUCTAL ADENOCARCINOMA CANCER (PDAC) CELLS ACTING SYNERGISTICALLY WITH METFORMIN AND INHIBITORS OF MEK AND MTOR Qinhong Xu, Fang Hao, Yinglan Zhao, James Sinnett-Smith, Enrique Rozengurt Background: The transcriptional co-activators YAP and TAZ are key regulators of development, organ-size, tissue regeneration, tumorigenesis and a novel target for the statins. Recently, YAP has been identified as a major downstream target of RAS, growth factor signaling and cell-cell adhesion, all of which are implicated in pancreatic ductal adenocarcinoma cancer (PDAC). Previously, we demonstrated that the inhibitory effects of low concentrations
S-837
AGA Abstracts
AGA Abstracts
with presumed increased risk of CRC, moderate-to-low dose aspirin and sulindac-associated combination therapy are both effective for recurrent colorectal adenomas. Future studies are required to provide more precise estimates of the optimal NSAIDs with an effective dose and low adverse events.We also suggest the further evaluation of NSAIDs-associated combination regimens and other novel agents (e.g.metformin)in the chemoprevention of CRC.
AGA Abstracts
in serum-deprived PDAC cells stimulated by crosstalk between insulin and neurotensin. Exposure to increasing concentrations of cerivastatin markedly inhibited the proliferation (increase in cell number) of PANC-1 and MiaPaCa-2 cells. Cerivastatin also potently inhibited the formation of colonies (>50 cells/colony) by these PDAC cells in a dose-dependent manner. Half-maximal effect (IC50) was produced by cerivastatin at a concentration as low as 0.03 mM. Maximal inhibition of colony formation was obtained at 0.3 mM. We extended these results to other lipophilic statins. Treatment of PANC-1 and MiaPaCa-2 cells with the FDA-approved simvastatin (Zocor) also inhibited colony formation. IC50 was produced by simvastatin at 0.3 mM. Atorvastatin (Lipitor) and fluvastatin also inhibited colony formation by PDAC cells in a dose-dependent manner. These results demonstrate that statins potently inhibit colony formation by PDAC cells. Exogenously supplied mevalonic acid (250 mM) to the culture medium reversed completely (PANC-1) or partially (MiaPaCa-2) the inhibitory effect of cerivastatin on colony formation. These results indicate that the statins block colony formation by PDAC cells, at least in part, through inhibition of HMG-CoA reductase, the rate-limiting enzyme in the generation of mevalonic acid. Conclusions: Our results show that statins, specific inhibitors of the 3-hydroxy-methylglutaryl CoA reductase (HMGCR), potently inhibited YAP function and proliferation in PDAC cells. Our results, together with recent epidemiological findings linking statin administration with reduced incidence of PDAC, raise the possibility of repurposing FDA-approved statins for the prevention and therapy of PDAC, a devastating disease with a dismal prognosis.
Demographics and Findings
Mo2061 A 3-YEAR OBSERVATIONAL STUDY OF PERSONS WITH A NEGATIVE COLONOSCOPY AND POSITIVE MULTI-TARGET STOOL DNA TEST Daniel J. Geenen, Thomas F. Imperiale, Susan L. Caskey, Katherine A. Anderson, Barry Berger Introduction: Multi-target stool DNA (mt-sDNA) is FDA approved for colorectal cancer (CRC) screening of average-risk persons age ≥50 years and is recommended by the U. S. Preventive Services Task Force. Here, we report the 3-4 year follow-up of DeeP-C pivotal study participants who were mt-sDNA positive but colonoscopy negative (false positive). The study objective is to determine whether these discordant results were followed by a later diagnosis of CRC or non-colorectal aero-digestive cancer. Methods: We reviewed our site's (WI, USA) DeeP-C study subject medical records with negative colonoscopies (187) of whom 150 had negative and 37 had positive mt-sDNA tests. Living patients with less than three years of follow-up were contacted by telephone for data on demographics, diagnosis of subsequent malignancy, subsequent endoscopy or fecal occult blood tests, and date of death/cause or date of last follow. Follow-up interval was the time between DeePC informed consent (IC) and medical records review date or interview. Deep-C colonoscopy was performed < 90 days of IC. Patients with negative colonoscopy/negative mt-sDNA were controls. Negative colonoscopies included those with only non-advanced adenomas and/or hyperplastic polyps or with no polyps at all. Results: Demographics of subjects with positive and negative mt-sDNA results were similar (Table). Of 150 subjects with negative mt-sDNA, 142 were alive and cancer free with 3.6-4.8 yrs. of follow up (mean 4.1, median 3.9, yrs.), 3 died (gangrenous gall bladder, complications of lung transplant for interstitial lung disease, and mesothelioma at 0.8, 2.3 and 3.1 years, respectively) and 5 were lost to follow-up (3 immediately after the DeeP-C colonoscopy, one each at 0.6 and 1.7 years). Ten subjects repeated colonoscopy 2-4 years later, 8 of whom had negative findings while 2 had nonadvanced colorectal neoplasia (single small non advanced adenoma). All 37 subjects with positive mt-sDNA / negative colonoscopy results were alive with 3.1-5.1 years of follow-up (mean 4.3, median 4.2). One subject had a low grade parotid mucoepidermoid cancer diagnosed 2.9 years post Deep-C. Three subjects had colonoscopy 2, 3, and 4 years post Deep-C IC which were: negative, had non-advanced adenomas (3), and negative, respectively. Summary: After 3.1-5.1 years follow-up, none of 37 subjects who were mt-sDNA positive/ colonoscopy negative had CRC or another digestive cancer (upper 95% CI 0.9%). While the sample size of test-positive, colonoscopy negative subjects is small, no mt-sDNA positive patients were lost to follow-up. These findings require validation with a greater sample size but are consistent with findings in the mt-sDNA Statement of Safety and Effectiveness projecting a very low rate of false positives due to aero-digestive cancers (1.6/1000 false positives).
AGA Abstracts
Mo2062 VITAMIN D SUPPLEMENTATION EFFECTS ON GLOBAL GENE EXPRESSION IN BARRETT'S ESOPHAGUS Linda C. Cummings, Jill Barnholtz-Sloan, Yanwen Chen, Sanford D. Markowitz, Amitabh Chak Background: Vitamin D deficiency is associated with increased risk for esophageal cancer. Vitamin D directly or indirectly controls genes that regulate proliferation, apoptosis, and differentiation. Hypothesizing that vitamin D might be of benefit, the goals of this study were to assess the effects of vitamin D supplementation on global gene expression in Barrett's esophagus (BE) patients with low-grade or no dysplasia. Methods: Patients with longsegment or short-segment BE with no or low grade dysplasia were treated with vitamin D3 (cholecalciferol) 50,000 international units weekly for 12 weeks. Blood samples and biopsies from the BE segment and from normal squamous tissue ≥2 cm proximal to the BE segment were obtained before and after vitamin D3 supplementation. Vitamin D status was assessed with serum 25-hydroxyvitamin D levels. Global gene expression was evaluated with Affymetrix GeneChip Human Gene 1.0 ST arrays using RNA isolated from the esophageal biopsy samples. Gene expression was assessed in 2 sets of paired samples: intestinal metaplasia and normal squamous esophagus samples. Gene expression after vitamin D supplementation was compared to pre-supplementation levels using paired t-tests; a paired t-test raw p value <0.001 was considered significant. Results: Eighteen BE patients with no or low grade dysplasia completed the study. Vitamin D supplementation was well tolerated. The mean age was 64 years (standard deviation, 10 years); 4 women and 14 men were enrolled. Baseline serum 25-hydroxyvitamin D levels ranged from 14 ng/mL (deficient) to 44 ng/mL (sufficient) with a median level of 27 ng/mL (insufficient). After vitamin D supplementation, 25-hydroxyvitamin D levels rose significantly (p< 0.001) with a median increase of 32 ng/ mL. After review of RNA quality control parameters, microarray data from 17 paired intestinal metaplasia samples and 15 paired normal squamous samples were evaluated. Only 1 gene within the intestinal metaplasia samples and 9 genes within the normal squamous samples were associated with a paired t-test raw p value <0.001. However, examination of patientlevel changes in expression for these genes revealed that none were associated with a unidirectional change in expression, i.e., expression increased in some subjects but decreased in others. Moreover, the associated fold changes were <1.5-fold, which is smaller than microarray technology has the ability to reliably capture. Conclusion: Baseline Vitamin D status of BE patients was insufficient. Despite significant increases in serum 25-hydroxyvitamin D levels with supplementation, no significant change in gene expression was noted in intestinal metaplasia or normal squamous tissue. If vitamin D supplementation affects gene expression in BE, a longer duration of supplementation may be needed.
S-838
Mo2059 Figure 2. Network of eligible comparisons The size of circle is proportional to the number of participants received the treatment (sample size). A) Overall recurrence of colorectal adenomas; B) Recurrence of advanced adenomas; C) Serious adverse events.Every line links direct comparable treatments; The width of the lines reflect the number of trials.
STATINS POTENTLY INHIBIT YAP ACTIVITY AND PROLIFERATION OF MOUSE PANCREATIC CELLS ISOLATED FROM KC AND KPC MICE Fang Hao, Jen-Kuan Chang, James Sinnett-Smith, Guido Eibl, Hui-Hua Chang, Enrique Rozengurt Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases. The highly conserved Hippo pathway is a key regulator of development, organsize, tissue regeneration, tumorigenesis, PDAC development and a novel target for the statins. The conditional KrasG12D mouse model (LSL-KrasG12D; p-48-Cre; referred as KC) of PDAC development is characterized by the presence of an activating Kras mutation, which is conditionally expressed in pancreatic progenitor cells during embryologic development. Coexpression of a p53 mutant (p53R172H) greatly accelerates the development of invasive PDAC (model referred as KPC mice). To extend our studies on the effect of statins on PDAC (see our other Abstracts), we examined the effect of statins on pancreatic cells derived from KC and KPC mice. Results: We examined the effects of cerivastatin and simvastatin on YAP localization, YAP/TEAD-regulated gene expression and colony formation in a pancreatic cell line established from KC mice. We found that treatment of KC cells with either cerivastatin or simvastatin induced striking translocation of YAP from the nucleus to the cytoplasm, thereby blunting the ability of YAP to act as a co-activator of TEAD transcription factors. In line with this possibility, exposure to cerivastatin or simvastatin inhibited the expression of YAP/TEAD-regulated genes in a dose-dependent manner, as shown by decreased mRNA levels of Ctgf, Cyr61 and Birc5. Half-maximal effect (IC50) was produced by cerivastatin at a concentration as low as 0.03 mM. At the highest concentration tested (0.3 mM), these statins inhibited Ctgf, or Cyr61 mRNA levels by 90%. Importantly, treatment with cerivastatin or simvastatin markedly inhibited colony formation by KC cells in a dose-dependent manner. In accord with the results obtained with human PDAC cells, treatment of KC cells with metformin markedly increased the inhibitory effect of cerivastatin or simvastatin on colony formation. Additional studies showed that treatment with cerivastatin strikingly inhibited the expression of YAP/TEAD-regulated genes, including Ctgf, Cyr61 and Birc5 and the colonyforming ability of KPC cells. Cerivastatin inhibited the proliferation of KPC cells at lower concentrations than KC cells (IC50=0.01 mM), suggesting that the expression of the p53 mutant enhances the sensitivity of PDAC cells to statins. Conclusions: Our results show that statins, specific inhibitors of the 3-hydroxy-methylglutaryl CoA reductase (HMGCR), potently inhibited YAP function and proliferation in KC and KPC cells. The results, together with our accompanying Abstracts using human cells and recent epidemiological findings linking statin administration with reduced incidence of PDAC, raise the possibility of repurposing FDA-approved statins for the prevention and therapy of PDAC, a devastating disease with a dismal prognosis.
Figure 4. Median rank and SUCRA values of competing agents SUCRA=surface under the cumulative ranking curve. CrI=credibility interval. For both efficacy(recurrence colorectal adenomas) and safety(incidence of serious adverse events), SUCRA would be 1 when a treatment is certain to be the best and 0 when a treatment is certain to be the worst. The higher SUCRA, the higher probablity of the agent to be the most efficacious or safest.
Mo2060 STATINS POTENTLY INHIBIT YAP FUNCTION AND PROLIFERATION OF PANCREATIC DUCTAL ADENOCARCINOMA CANCER (PDAC) CELLS Fang Hao, Qinhong Xu, Yinglan Zhao, James Sinnett-Smith, Steven H. Young, Jan V. Stevens, Enrique Rozengurt Background: The highly conserved Hippo pathway is a key regulator of development, organsize, tissue regeneration, tumorigenesis and a novel target for the statins. Hippo signals are transduced through a kinase cascade that phosphorylates the transcriptional co-activators YAP and TAZ. Recently, YAP has been identified as a major downstream target of RAS, AMPK, growth factor signaling and cell-cell adhesion, all of which are implicated in pancreatic ductal adenocarcinoma cancer (PDAC). Here, we determined whether statins inhibit YAP function and proliferation of PDAC cells. Results: Exposure of PANC-1 and MiaPaCa-2 cells to cerivastatin, simvastatin or fluvastatin dramatically inhibited the expression of YAP/ TEAD-regulated genes, including Connective Tissue Growth Factor (CTGF) and Cysteinerich angiogenic inducer 61 (Cyr61) in a dose-dependent manner, as determined by RTqPCR. At the highest concentration tested, these statins inhibited CTGF or Cyr61 mRNA levels by 90%. Treatment with cerivastatin also blocked the increase in CTGF mRNA levels
Mo2058 STATINS INHIBIT PROLIFERATION OF PANCREATIC DUCTAL ADENOCARCINOMA CANCER (PDAC) CELLS ACTING SYNERGISTICALLY WITH METFORMIN AND INHIBITORS OF MEK AND MTOR Qinhong Xu, Fang Hao, Yinglan Zhao, James Sinnett-Smith, Enrique Rozengurt Background: The transcriptional co-activators YAP and TAZ are key regulators of development, organ-size, tissue regeneration, tumorigenesis and a novel target for the statins. Recently, YAP has been identified as a major downstream target of RAS, growth factor signaling and cell-cell adhesion, all of which are implicated in pancreatic ductal adenocarcinoma cancer (PDAC). Previously, we demonstrated that the inhibitory effects of low concentrations
S-837
AGA Abstracts
AGA Abstracts
with presumed increased risk of CRC, moderate-to-low dose aspirin and sulindac-associated combination therapy are both effective for recurrent colorectal adenomas. Future studies are required to provide more precise estimates of the optimal NSAIDs with an effective dose and low adverse events.We also suggest the further evaluation of NSAIDs-associated combination regimens and other novel agents (e.g.metformin)in the chemoprevention of CRC.
AGA Abstracts
in serum-deprived PDAC cells stimulated by crosstalk between insulin and neurotensin. Exposure to increasing concentrations of cerivastatin markedly inhibited the proliferation (increase in cell number) of PANC-1 and MiaPaCa-2 cells. Cerivastatin also potently inhibited the formation of colonies (>50 cells/colony) by these PDAC cells in a dose-dependent manner. Half-maximal effect (IC50) was produced by cerivastatin at a concentration as low as 0.03 mM. Maximal inhibition of colony formation was obtained at 0.3 mM. We extended these results to other lipophilic statins. Treatment of PANC-1 and MiaPaCa-2 cells with the FDA-approved simvastatin (Zocor) also inhibited colony formation. IC50 was produced by simvastatin at 0.3 mM. Atorvastatin (Lipitor) and fluvastatin also inhibited colony formation by PDAC cells in a dose-dependent manner. These results demonstrate that statins potently inhibit colony formation by PDAC cells. Exogenously supplied mevalonic acid (250 mM) to the culture medium reversed completely (PANC-1) or partially (MiaPaCa-2) the inhibitory effect of cerivastatin on colony formation. These results indicate that the statins block colony formation by PDAC cells, at least in part, through inhibition of HMG-CoA reductase, the rate-limiting enzyme in the generation of mevalonic acid. Conclusions: Our results show that statins, specific inhibitors of the 3-hydroxy-methylglutaryl CoA reductase (HMGCR), potently inhibited YAP function and proliferation in PDAC cells. Our results, together with recent epidemiological findings linking statin administration with reduced incidence of PDAC, raise the possibility of repurposing FDA-approved statins for the prevention and therapy of PDAC, a devastating disease with a dismal prognosis.
Demographics and Findings
Mo2061 A 3-YEAR OBSERVATIONAL STUDY OF PERSONS WITH A NEGATIVE COLONOSCOPY AND POSITIVE MULTI-TARGET STOOL DNA TEST Daniel J. Geenen, Thomas F. Imperiale, Susan L. Caskey, Katherine A. Anderson, Barry Berger Introduction: Multi-target stool DNA (mt-sDNA) is FDA approved for colorectal cancer (CRC) screening of average-risk persons age ≥50 years and is recommended by the U. S. Preventive Services Task Force. Here, we report the 3-4 year follow-up of DeeP-C pivotal study participants who were mt-sDNA positive but colonoscopy negative (false positive). The study objective is to determine whether these discordant results were followed by a later diagnosis of CRC or non-colorectal aero-digestive cancer. Methods: We reviewed our site's (WI, USA) DeeP-C study subject medical records with negative colonoscopies (187) of whom 150 had negative and 37 had positive mt-sDNA tests. Living patients with less than three years of follow-up were contacted by telephone for data on demographics, diagnosis of subsequent malignancy, subsequent endoscopy or fecal occult blood tests, and date of death/cause or date of last follow. Follow-up interval was the time between DeePC informed consent (IC) and medical records review date or interview. Deep-C colonoscopy was performed < 90 days of IC. Patients with negative colonoscopy/negative mt-sDNA were controls. Negative colonoscopies included those with only non-advanced adenomas and/or hyperplastic polyps or with no polyps at all. Results: Demographics of subjects with positive and negative mt-sDNA results were similar (Table). Of 150 subjects with negative mt-sDNA, 142 were alive and cancer free with 3.6-4.8 yrs. of follow up (mean 4.1, median 3.9, yrs.), 3 died (gangrenous gall bladder, complications of lung transplant for interstitial lung disease, and mesothelioma at 0.8, 2.3 and 3.1 years, respectively) and 5 were lost to follow-up (3 immediately after the DeeP-C colonoscopy, one each at 0.6 and 1.7 years). Ten subjects repeated colonoscopy 2-4 years later, 8 of whom had negative findings while 2 had nonadvanced colorectal neoplasia (single small non advanced adenoma). All 37 subjects with positive mt-sDNA / negative colonoscopy results were alive with 3.1-5.1 years of follow-up (mean 4.3, median 4.2). One subject had a low grade parotid mucoepidermoid cancer diagnosed 2.9 years post Deep-C. Three subjects had colonoscopy 2, 3, and 4 years post Deep-C IC which were: negative, had non-advanced adenomas (3), and negative, respectively. Summary: After 3.1-5.1 years follow-up, none of 37 subjects who were mt-sDNA positive/ colonoscopy negative had CRC or another digestive cancer (upper 95% CI 0.9%). While the sample size of test-positive, colonoscopy negative subjects is small, no mt-sDNA positive patients were lost to follow-up. These findings require validation with a greater sample size but are consistent with findings in the mt-sDNA Statement of Safety and Effectiveness projecting a very low rate of false positives due to aero-digestive cancers (1.6/1000 false positives).
AGA Abstracts
Mo2062 VITAMIN D SUPPLEMENTATION EFFECTS ON GLOBAL GENE EXPRESSION IN BARRETT'S ESOPHAGUS Linda C. Cummings, Jill Barnholtz-Sloan, Yanwen Chen, Sanford D. Markowitz, Amitabh Chak Background: Vitamin D deficiency is associated with increased risk for esophageal cancer. Vitamin D directly or indirectly controls genes that regulate proliferation, apoptosis, and differentiation. Hypothesizing that vitamin D might be of benefit, the goals of this study were to assess the effects of vitamin D supplementation on global gene expression in Barrett's esophagus (BE) patients with low-grade or no dysplasia. Methods: Patients with longsegment or short-segment BE with no or low grade dysplasia were treated with vitamin D3 (cholecalciferol) 50,000 international units weekly for 12 weeks. Blood samples and biopsies from the BE segment and from normal squamous tissue ≥2 cm proximal to the BE segment were obtained before and after vitamin D3 supplementation. Vitamin D status was assessed with serum 25-hydroxyvitamin D levels. Global gene expression was evaluated with Affymetrix GeneChip Human Gene 1.0 ST arrays using RNA isolated from the esophageal biopsy samples. Gene expression was assessed in 2 sets of paired samples: intestinal metaplasia and normal squamous esophagus samples. Gene expression after vitamin D supplementation was compared to pre-supplementation levels using paired t-tests; a paired t-test raw p value <0.001 was considered significant. Results: Eighteen BE patients with no or low grade dysplasia completed the study. Vitamin D supplementation was well tolerated. The mean age was 64 years (standard deviation, 10 years); 4 women and 14 men were enrolled. Baseline serum 25-hydroxyvitamin D levels ranged from 14 ng/mL (deficient) to 44 ng/mL (sufficient) with a median level of 27 ng/mL (insufficient). After vitamin D supplementation, 25-hydroxyvitamin D levels rose significantly (p< 0.001) with a median increase of 32 ng/ mL. After review of RNA quality control parameters, microarray data from 17 paired intestinal metaplasia samples and 15 paired normal squamous samples were evaluated. Only 1 gene within the intestinal metaplasia samples and 9 genes within the normal squamous samples were associated with a paired t-test raw p value <0.001. However, examination of patientlevel changes in expression for these genes revealed that none were associated with a unidirectional change in expression, i.e., expression increased in some subjects but decreased in others. Moreover, the associated fold changes were <1.5-fold, which is smaller than microarray technology has the ability to reliably capture. Conclusion: Baseline Vitamin D status of BE patients was insufficient. Despite significant increases in serum 25-hydroxyvitamin D levels with supplementation, no significant change in gene expression was noted in intestinal metaplasia or normal squamous tissue. If vitamin D supplementation affects gene expression in BE, a longer duration of supplementation may be needed.
S-838