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Plasma Testing of Gene Expression Biomarkers for Colorectal Neoplasia Discovered in Neoplastic Colorectal Tissue
AGA Abstracts
Results: In 2006, 10,054 (49% male, median age 59) persons were invited of which 4,990 (50%) returned the test. Of these, 4,697 (94%) persons tested negative. In 17 (0.4%) persons (65% male, median age 62 (range 55 to 76) CRC was diagnosed within 3 years after the negative FOBT (median interval 27 months (range 3 to 31)). In 9 persons, a guaiac test had been performed and in 8 persons a FIT. Eight cancer cases were diagnosed clinically, through regular care based on symptoms; 9 cases were screen-detected in the second round using FIT. CRC family history was negative in 16 out of 17 cases. Location was proximal in 3 cases and distal in 14. In 3 cases coincident serrated polyps and in 7 cases coincident adenomas were identified at endoscopy or at surgical resection. One patient (6%) had multiple serrated polyps, satisfying the criteria for hyperplastic polyposis syndrome. Molecular analysis could be performed in 16 of the 17 CRC cases. APC mutations were detected in 4 (25%) and KRAS mutations in 7 (44%); one CRC had both an APC and KRAS mutation. No BRAF mutations were identified. One tumour (6%) was MSI-high, the others were microsatellite stable. Conclusion: In FOBT screening, interval CRCs are observed in a minority of individuals. These CRCs represent a molecular heterogeneous group involving both APC- and KRAS mutations, in the absence of BRAF mutations.
(mean interval 4.44 yrs). 5(10%) had SA (mean interval 4.54 yrs) and no patients had AA. 21 patients with adenomas initially had a follow up colonoscopy (mean interval 3.64 yrs). 7(33.3%) had SA and 1(4.8%) had AA. Discussion: The high number of adenomas seen in this moderate risk group supports the need for screening. The predominance of left sided advanced neoplastic lesions is similar to the average risk population. However the number of adenomas was low in patients < 45. In patients of any age with no adenomas on initial colonoscopy. the incidence of adenomas on follow up colonoscopy was low, suggesting that screening intervals could be increased in this group. Su1150 Plasma Testing of Gene Expression Biomarkers for Colorectal Neoplasia Discovered in Neoplastic Colorectal Tissue Melissa L. Thomas, Susanne K. Pedersen, Aidan McEvoy, Emma Vizgoft, Snigdha Gaur, Iain Beeston, Lawrence C. LaPointe Background: There is growing emphasis on the detection and removal of precancerous colorectal adenomas as a key objective for colorectal cancer screening. We have previously described a set of genes that are sensitive and specific for colorectal neoplasia in tissue specimens. While most colorectal cancer biomarker research starts with tissue-based discovery, the question of tissue-to-blood portability for gene expression biomarkers has not been well studied. This study explores the plasma-based expression levels of validated tissuebased biomarkers. Aim: To assess whether RNA biomarkers up-regulated in neoplastic colorectal tissues are likewise differentially expressed in the plasma of patients with and without colorectal neoplasia. Method: A total of 10 phenotypic panels from colonoscopyconfirmed patients (Proteogenix, USA) consisting of 15 normal, 15 adenoma and 15 cancer samples (45 specimens per panel) were used to evaluate 64 unique TaqMAN assays, targeting 48 different genes. RNA was extracted from 2.0 mL blood plasma aliquots using the QIAamp Circulating Nucleic Acid Kit (Qiagen, USA) and armored RNA enterovirus (arRNA) (Asuragen, USA) for extraction quality control. RNA was converted to cDNA (1:1 v/v) using Supercript VILO cDNA Synthesis Kit (Invitrogen, USA). qPCR assays were run in triplicate by adding 2.5μL cDNA per patient to a 25μL reaction TaqMAN assay for each gene (Applied Biosystems). Cycle threshold (Ct) values were determined (2nd derivative maximum) and normalized to arRNA recovery. Mean Ct values for each patient were calculated and fold changes determined relative to the median Ct for normal specimens. Results: GAPDH TaqMAN assays were positive for all samples with an average Ct of 30.21 (95% CI: 27.5 to 32.9). Of the 48 different genes tested, 21 (44%) failed to yield any detectable RNA transcripts in any clinical specimen; 24 (50%) were detectable but showed no phenotypic profile; and 3 (6%) were detectable with elevated expression level in neoplastic samples. In particular, KIAA1199 was up-regulated in plasma RNA measured from 6 distinct specimen panels with an average sensitivity of 74% (CI: 58-90%) and specificity of 66% (CI: 45-87%). Conclusions: Upregulation of RNA transcripts in neoplastic colorectal tissues translates to differential expression in patient plasma for only a small percentage of RNA biomarkers. Nevertheless, detection of differentially expressed RNA biomarker transcripts in patient plasma shows promise for development of a robust and sensitive non-invasive assay for colorectal neoplasia screening.
Su1148 Fit Testing With Three Samples Discovers More Advanced Adenomas Than One Sample Alone Belinda Frost, Aaron P. Fieker, Mohammad Madhoun, Ted Bader BACKGROUND: Fecal immunochemical testing (FIT) is usually performed with 1 or 2 samples. The Veteran's Administration (VA) purchased a large order of 3 FIT test kits from Poly Medco (FIT-CHEK®) rather than the usual single sample test. No data exist concerning the performance of 1 versus 3 samples of FIT-CHEK®. METHODS: We report our retrospective local VA experience from March, 2010 to November, 2010 in table 1. Advanced adenomas (AA) were defined as adenomas ≥ 10mm. The FIT samples were labeled by our laboratory as 1, 2 or 3 when received. Fisher exact t test was used for differences. RESULTS: The presence of more positive FIT cards was associated with finding more AA: one positive (32/ 171, 18.7%) versus 2 or 3 positive (39/115, 33.9%), p=.005; and more likely to have colorectal cancer (CRC): one positive (0/171, 0%) versus 2 or 3 positive (5/115, 4.35%) p= .009. If the first FIT sample labeled as “number 1” was the only sample analyzed, there would have been 26 patients with AA or CRC referred for colonoscopy; whereas, with all 3 tests accounted for, 62 patients were determined to have AA or CRC. With regard to the 5 patients with CRC: 4 of 5 had their first card positive and would have been discovered with a single sample. Four of 5 were asymptomatic and were discovered only with FIT testing. CONCLUSIONS Using 3 FIT samples rather than a single sample in the same population dramatically increased the number of patients determined to have AA/CRC. When more samples are positive in a given patient, more advanced pathology is found. These data provide a rationale for a prospective trial to examine the optimal number FIT tests to be used. Table 1. FIT positivity versus colonoscopy findings
Su1151 Incidence of Colorectal Cancer After a Negative Colonoscopy or Flexible Sigmoidoscopy Yemi M. Fasakin, Andy Tau, Yasser H. Shaib Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United States. Studies that directly compare flexible sigmoidoscopy (FS) and colonoscopy in CRC prevention are limited. The aim of this study is to compare the incidence of rightsided colorectal neoplasia after a negative flexible sigmoidoscopy or negative colonoscopy at a large VA over a 10-year period. Method: Retrospective chart review identified 252 patients with negative FS and 83 with negative colonoscopy in the year 2000. These patients all had repeat colonoscopy within ten years. Patients' demographics, procedure indications, adenomas, and CRC classified according to location were recorded and compared statistically. Primary outcome is the incidence of CRC after a negative FS or colonoscopy over a ten year period. The secondary outcomes were incidence of all adenomas and CRC within 10 years and incidence of CRC after a negative FS within 5 years. Results: Of the 252 patients with initial negative FS, the incidence of all adenomas and CRC on follow up colonoscopy was 35.7% (90/252) and the incidence of CRC was 3.6% (9/252). All CRC in the FS group were right sided (RS). Of the 83 patients who had negative initial colonoscopies, the incidence of CRC on follow up colonoscopy was 0% (0/83) and the incidence of all adenomas was 22.9% (19/83). Patients' demographics and follow-up periods were not statistically different between the two groups. The incidence of RS-CRC over a 5-year period in pts who had an initial negative FS was 4% (1/25). The incidence of all adenomas on follow-up colonoscopy was significantly greater amongst the FS group (p=0.043), but the incidence of CRC between the two groups was not (p=0.081). Conclusions: The incidence of all adenomas and CRC in patients who had an initial negative FS was statistically greater than those who had an initial negative colonoscopy. The difference in the incidence of right-sided colorectal cancer (RS-CRC) was not statistically significant between the two groups despite a trend in that direction. However, for every 17 patients who underwent FS in lieu of colonoscopy one developed RS-CRC over a ten year period. Furthermore, to our knowledge, this is the first report that showed nil RS-CRC over a long period of time after an initial normal colonoscopy. Our data also showed that the incidence of RS-CRC over a 5-year period in patients who had an initial negative FS is 1 out of 25 (4%).
Highest degree of pathology recorded. Totals: 1/3 = 171; 2/3 = 67; 3/3 = 48. Su1149 What is the Optimum Colonoscopy Surveillance Strategy in an at Risk Group for Colorectal Cancer Who Do Not Meet the Amsterdam Criteria? Robert Moran, Ann O'Connell, Nathan Maher, Victoria Swan, Jason Trickovic, Padraic Mac Mathuna Introduction: Colorectal cancer (CRC) screening based on a family history of CRC outside the Amsterdam criteria reduces the incidence of future CRC. This group can be described as having a moderate risk for development of CRC. The reported adenoma rate in this group is variable as is the approach to screening. Our goal was to report the results of a well characterised moderate risk group based on a family history of CRC in terms of adenoma yield, location and colonoscopy follow up. Methods: Data from a high risk familial CRC screening clinic based at a large tertiary referral centre was examined from 1997 to 2010. 762 moderate risk patients were identified. Complete data was available on 260 patients currently. Ninety two were excluded leaving 168 for analysis. Family history of CRC was assessed by questionnaire, death certificates and inquiries to the national cancer registry. Initial colonoscopy was offered 10 years prior to the age of the youngest relative diagnosed with CRC. Follow up colonoscopy was 3 yearly until 2003 and 5 yearly after 2003. Advanced adenomas (AA) were defined as: high-grade dysplasia, ≥ 10mm or villous component. Simple adenomas (SA) were defined as tubular adenomas < 10mm. Results: 168 patients had an initial colonoscopy and 71 had ≥ 1 follow up colonoscopies with a total of 404 colonoscopy follow up years. 103(61.3%) were female and 65(38.7%) were male. Over the course of the study 50(29.8%) patients had an adenoma, 36(21.5%) had SA only and 14(8.3%) had AA. The distribution of SA were; 17(39.5%) left sided only, 9(21%) both left and right sided and 17(39.5%) right sided only. The distribution of AA were; 13(81.3%) left sided only, 2(12.5%) both left and right and 1(6.2%) right sided only. At initial colonoscopy: mean age was 46.53, 80 adenomas were detected in 43(25.6%) patients, 30(17.9%, mean age 51.3) patients had SA only and 13(7.7%, mean age 53.2) patients had AA. 72(42.9%) patients were < 45 at initial colonoscopy. 8(11.1%) had SA and 2(2.8%) had AA. 96(57.1%) patients were ≥ 45 at initial screening colonoscopy. 26(27.1%) had SA and 11(11.5%) had AA. 50 patients with no adenomas initially had a follow up colonoscopy
AGA Abstracts
S-416
Results: In 2006, 10,054 (49% male, median age 59) persons were invited of which 4,990 (50%) returned the test. Of these, 4,697 (94%) persons tested negative. In 17 (0.4%) persons (65% male, median age 62 (range 55 to 76) CRC was diagnosed within 3 years after the negative FOBT (median interval 27 months (range 3 to 31)). In 9 persons, a guaiac test had been performed and in 8 persons a FIT. Eight cancer cases were diagnosed clinically, through regular care based on symptoms; 9 cases were screen-detected in the second round using FIT. CRC family history was negative in 16 out of 17 cases. Location was proximal in 3 cases and distal in 14. In 3 cases coincident serrated polyps and in 7 cases coincident adenomas were identified at endoscopy or at surgical resection. One patient (6%) had multiple serrated polyps, satisfying the criteria for hyperplastic polyposis syndrome. Molecular analysis could be performed in 16 of the 17 CRC cases. APC mutations were detected in 4 (25%) and KRAS mutations in 7 (44%); one CRC had both an APC and KRAS mutation. No BRAF mutations were identified. One tumour (6%) was MSI-high, the others were microsatellite stable. Conclusion: In FOBT screening, interval CRCs are observed in a minority of individuals. These CRCs represent a molecular heterogeneous group involving both APC- and KRAS mutations, in the absence of BRAF mutations.
(mean interval 4.44 yrs). 5(10%) had SA (mean interval 4.54 yrs) and no patients had AA. 21 patients with adenomas initially had a follow up colonoscopy (mean interval 3.64 yrs). 7(33.3%) had SA and 1(4.8%) had AA. Discussion: The high number of adenomas seen in this moderate risk group supports the need for screening. The predominance of left sided advanced neoplastic lesions is similar to the average risk population. However the number of adenomas was low in patients < 45. In patients of any age with no adenomas on initial colonoscopy. the incidence of adenomas on follow up colonoscopy was low, suggesting that screening intervals could be increased in this group. Su1150 Plasma Testing of Gene Expression Biomarkers for Colorectal Neoplasia Discovered in Neoplastic Colorectal Tissue Melissa L. Thomas, Susanne K. Pedersen, Aidan McEvoy, Emma Vizgoft, Snigdha Gaur, Iain Beeston, Lawrence C. LaPointe Background: There is growing emphasis on the detection and removal of precancerous colorectal adenomas as a key objective for colorectal cancer screening. We have previously described a set of genes that are sensitive and specific for colorectal neoplasia in tissue specimens. While most colorectal cancer biomarker research starts with tissue-based discovery, the question of tissue-to-blood portability for gene expression biomarkers has not been well studied. This study explores the plasma-based expression levels of validated tissuebased biomarkers. Aim: To assess whether RNA biomarkers up-regulated in neoplastic colorectal tissues are likewise differentially expressed in the plasma of patients with and without colorectal neoplasia. Method: A total of 10 phenotypic panels from colonoscopyconfirmed patients (Proteogenix, USA) consisting of 15 normal, 15 adenoma and 15 cancer samples (45 specimens per panel) were used to evaluate 64 unique TaqMAN assays, targeting 48 different genes. RNA was extracted from 2.0 mL blood plasma aliquots using the QIAamp Circulating Nucleic Acid Kit (Qiagen, USA) and armored RNA enterovirus (arRNA) (Asuragen, USA) for extraction quality control. RNA was converted to cDNA (1:1 v/v) using Supercript VILO cDNA Synthesis Kit (Invitrogen, USA). qPCR assays were run in triplicate by adding 2.5μL cDNA per patient to a 25μL reaction TaqMAN assay for each gene (Applied Biosystems). Cycle threshold (Ct) values were determined (2nd derivative maximum) and normalized to arRNA recovery. Mean Ct values for each patient were calculated and fold changes determined relative to the median Ct for normal specimens. Results: GAPDH TaqMAN assays were positive for all samples with an average Ct of 30.21 (95% CI: 27.5 to 32.9). Of the 48 different genes tested, 21 (44%) failed to yield any detectable RNA transcripts in any clinical specimen; 24 (50%) were detectable but showed no phenotypic profile; and 3 (6%) were detectable with elevated expression level in neoplastic samples. In particular, KIAA1199 was up-regulated in plasma RNA measured from 6 distinct specimen panels with an average sensitivity of 74% (CI: 58-90%) and specificity of 66% (CI: 45-87%). Conclusions: Upregulation of RNA transcripts in neoplastic colorectal tissues translates to differential expression in patient plasma for only a small percentage of RNA biomarkers. Nevertheless, detection of differentially expressed RNA biomarker transcripts in patient plasma shows promise for development of a robust and sensitive non-invasive assay for colorectal neoplasia screening.
Su1148 Fit Testing With Three Samples Discovers More Advanced Adenomas Than One Sample Alone Belinda Frost, Aaron P. Fieker, Mohammad Madhoun, Ted Bader BACKGROUND: Fecal immunochemical testing (FIT) is usually performed with 1 or 2 samples. The Veteran's Administration (VA) purchased a large order of 3 FIT test kits from Poly Medco (FIT-CHEK®) rather than the usual single sample test. No data exist concerning the performance of 1 versus 3 samples of FIT-CHEK®. METHODS: We report our retrospective local VA experience from March, 2010 to November, 2010 in table 1. Advanced adenomas (AA) were defined as adenomas ≥ 10mm. The FIT samples were labeled by our laboratory as 1, 2 or 3 when received. Fisher exact t test was used for differences. RESULTS: The presence of more positive FIT cards was associated with finding more AA: one positive (32/ 171, 18.7%) versus 2 or 3 positive (39/115, 33.9%), p=.005; and more likely to have colorectal cancer (CRC): one positive (0/171, 0%) versus 2 or 3 positive (5/115, 4.35%) p= .009. If the first FIT sample labeled as “number 1” was the only sample analyzed, there would have been 26 patients with AA or CRC referred for colonoscopy; whereas, with all 3 tests accounted for, 62 patients were determined to have AA or CRC. With regard to the 5 patients with CRC: 4 of 5 had their first card positive and would have been discovered with a single sample. Four of 5 were asymptomatic and were discovered only with FIT testing. CONCLUSIONS Using 3 FIT samples rather than a single sample in the same population dramatically increased the number of patients determined to have AA/CRC. When more samples are positive in a given patient, more advanced pathology is found. These data provide a rationale for a prospective trial to examine the optimal number FIT tests to be used. Table 1. FIT positivity versus colonoscopy findings
Su1151 Incidence of Colorectal Cancer After a Negative Colonoscopy or Flexible Sigmoidoscopy Yemi M. Fasakin, Andy Tau, Yasser H. Shaib Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United States. Studies that directly compare flexible sigmoidoscopy (FS) and colonoscopy in CRC prevention are limited. The aim of this study is to compare the incidence of rightsided colorectal neoplasia after a negative flexible sigmoidoscopy or negative colonoscopy at a large VA over a 10-year period. Method: Retrospective chart review identified 252 patients with negative FS and 83 with negative colonoscopy in the year 2000. These patients all had repeat colonoscopy within ten years. Patients' demographics, procedure indications, adenomas, and CRC classified according to location were recorded and compared statistically. Primary outcome is the incidence of CRC after a negative FS or colonoscopy over a ten year period. The secondary outcomes were incidence of all adenomas and CRC within 10 years and incidence of CRC after a negative FS within 5 years. Results: Of the 252 patients with initial negative FS, the incidence of all adenomas and CRC on follow up colonoscopy was 35.7% (90/252) and the incidence of CRC was 3.6% (9/252). All CRC in the FS group were right sided (RS). Of the 83 patients who had negative initial colonoscopies, the incidence of CRC on follow up colonoscopy was 0% (0/83) and the incidence of all adenomas was 22.9% (19/83). Patients' demographics and follow-up periods were not statistically different between the two groups. The incidence of RS-CRC over a 5-year period in pts who had an initial negative FS was 4% (1/25). The incidence of all adenomas on follow-up colonoscopy was significantly greater amongst the FS group (p=0.043), but the incidence of CRC between the two groups was not (p=0.081). Conclusions: The incidence of all adenomas and CRC in patients who had an initial negative FS was statistically greater than those who had an initial negative colonoscopy. The difference in the incidence of right-sided colorectal cancer (RS-CRC) was not statistically significant between the two groups despite a trend in that direction. However, for every 17 patients who underwent FS in lieu of colonoscopy one developed RS-CRC over a ten year period. Furthermore, to our knowledge, this is the first report that showed nil RS-CRC over a long period of time after an initial normal colonoscopy. Our data also showed that the incidence of RS-CRC over a 5-year period in patients who had an initial negative FS is 1 out of 25 (4%).
Highest degree of pathology recorded. Totals: 1/3 = 171; 2/3 = 67; 3/3 = 48. Su1149 What is the Optimum Colonoscopy Surveillance Strategy in an at Risk Group for Colorectal Cancer Who Do Not Meet the Amsterdam Criteria? Robert Moran, Ann O'Connell, Nathan Maher, Victoria Swan, Jason Trickovic, Padraic Mac Mathuna Introduction: Colorectal cancer (CRC) screening based on a family history of CRC outside the Amsterdam criteria reduces the incidence of future CRC. This group can be described as having a moderate risk for development of CRC. The reported adenoma rate in this group is variable as is the approach to screening. Our goal was to report the results of a well characterised moderate risk group based on a family history of CRC in terms of adenoma yield, location and colonoscopy follow up. Methods: Data from a high risk familial CRC screening clinic based at a large tertiary referral centre was examined from 1997 to 2010. 762 moderate risk patients were identified. Complete data was available on 260 patients currently. Ninety two were excluded leaving 168 for analysis. Family history of CRC was assessed by questionnaire, death certificates and inquiries to the national cancer registry. Initial colonoscopy was offered 10 years prior to the age of the youngest relative diagnosed with CRC. Follow up colonoscopy was 3 yearly until 2003 and 5 yearly after 2003. Advanced adenomas (AA) were defined as: high-grade dysplasia, ≥ 10mm or villous component. Simple adenomas (SA) were defined as tubular adenomas < 10mm. Results: 168 patients had an initial colonoscopy and 71 had ≥ 1 follow up colonoscopies with a total of 404 colonoscopy follow up years. 103(61.3%) were female and 65(38.7%) were male. Over the course of the study 50(29.8%) patients had an adenoma, 36(21.5%) had SA only and 14(8.3%) had AA. The distribution of SA were; 17(39.5%) left sided only, 9(21%) both left and right sided and 17(39.5%) right sided only. The distribution of AA were; 13(81.3%) left sided only, 2(12.5%) both left and right and 1(6.2%) right sided only. At initial colonoscopy: mean age was 46.53, 80 adenomas were detected in 43(25.6%) patients, 30(17.9%, mean age 51.3) patients had SA only and 13(7.7%, mean age 53.2) patients had AA. 72(42.9%) patients were < 45 at initial colonoscopy. 8(11.1%) had SA and 2(2.8%) had AA. 96(57.1%) patients were ≥ 45 at initial screening colonoscopy. 26(27.1%) had SA and 11(11.5%) had AA. 50 patients with no adenomas initially had a follow up colonoscopy
AGA Abstracts
S-416