| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 EXACT Sciences Corporation, Marlborough, Massachusetts; 2 Division of Gastroenterology, Mayo Clinic, Rochester, Minnesota;3 Division of Gastroenterology, Kaiser Permanente Medical Center, Walnut Creek, California;4 Division of Gastroenterology, Indiana University School of Medicine, Indianapolis, Indiana;5 Howard Hughes Medical Institute, and Department of Medicine, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio
aaddress correspondence to this author at: Molecular Instincts, 6 Parker Rd., Mendon, MA 01756; fax 508-473-6832, e-mail tshuber{at}molecularinstincts.com
Abstract
Background: The genetic heterogeneity of sporadic colorectal cancer (CRC) makes the choice of genetic markers and sequence variation–detection technologies critical to the performance of screening assays. We have previously described the effectiveness of a CRC assay composed of 22 known variants in KRAS, APC, TP53, and BAT-26 (V1). We introduce a new marker formulation (V2) that includes detection of de novo variation in APC, PIK3CA, and CTNNB1, hypermethylated sequences within SMARCA3 and VIM, and a single-base variation within BRAF. We compared the abilities of the V1 and V2 markers to detect aberrant DNA in colorectal neoplasias.
Methods: V1 and V2 marker formulations were used to analyze 144 colorectal tissue samples comprising 50 precancerous adenomas, 94 carcinomas, and 11 nonpathologic tissues. V1 analysis consisted of single-base extension analysis of the 22 V1 variants. V2 analysis consisted of DNA scanning of the APC mutation cluster region, PIK3CA exons 9 and 20, CTNNB1 exon 3, analysis for the BRAF Val600Glu substitution, and methylation-specific PCR analysis of VIM and SMARCA3.
Results: The V2 marker formulation had significantly higher sensitivity than the V1 markers for carcinomas (93.6% and 72.3%, respectively; P = 0.0002) and adenomas (92.0% and 62.0%, respectively; P = 0.0006). None of the nonpathologic samples were positive for any marker.
Conclusions: We demonstrate improved sensitivity of a new marker formulation (V2) to detect aberrant DNA in CRC and precancerous adenoma tumor tissues.
Sporadic colorectal cancer (CRC) is genetically heterogeneous (1), thus creating a need for multitarget genetic assays to improve rates of detection of CRC (2)(3). We previously described a single-base variation panel (V1) that uses modified single-base extension analysis to detect substitutions, small insertions, and deletions in known targeted hot-spot variants in KRAS, APC, TP53, and a surrogate marker, BAT-26 (2)(3)(4). A need remains for methods that detect sites where variations occur only infrequently, because such sites may account for a substantial percentage of sporadic tumors in a population. We have developed a variation-scanning technology that can detect any type of substitution in any gene of interest. We apply the variation-scanning approach to detect de novo substitutions in the mutation cluster region (MCR) of APC, exon 3 of CTNNB1, and exons 9 and 20 of PIK3CA, all of which harbor multiple variation sites in CRC (5)(6)(7)(8).
Epigenetic changes have also been detected in colorectal neoplasia (9); therefore we analyzed SMARCA3 and VIM, which have shown significant promoter methylation in colon cancer and adenomas (10)(11)(12). Additionally, analysis for the BRAF Val600Glu substitution was performed (13). We refer to this combination of all new markers as V2. Several of the V2 markers were specifically targeted to detect early-stage disease (6)(7)(13)(14). In this study, we compared the detection rates of the V1 and V2 marker formulations for CRC and adenomas.
We obtained 94 colon tumor samples from individuals who underwent surgery at the Lahey Clinic, Burlington, Massachusetts, between 1997 and 1999. All participants gave informed consent, and tissue-procurement procedures were approved by the Lahey Clinic. A small piece of the surgical specimen was removed for molecular analysis, snap-frozen in liquid nitrogen, and stored at –70 °C until DNA extraction. Samples were incubated in lysis buffer [10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl2, 4.5 mL/L Tween 20, 0.5 g/L proteinase K] overnight at 37 °C, and DNA was isolated by phenol-chloroform extraction and ethanol precipitation. Acquisition of additional tissue samples from the Mayo Clinic was approved by the Mayo Clinic Institutional Review Board. Tissue samples included apparently healthy colon mucosa from 11 patients without any gross lesions and 50 specimens from adenomatous polyps 
1 cm in diameter with low-grade dysplasia. Healthy colon tissues were frozen after collection and embedded in optimal temperature cutting compound. Ten sections (20 µm thick) were cut from each sample, followed by DNAzol (Invitrogen) treatment and ethanol precipitation. Adenoma tumor tissue DNA was prepared from formalin-fixed, paraffin-embedded samples. Four sections (20 µm thick) were cut and placed on slides for analysis. Tissues were subsequently purified with a QIAmp DNA Mini Kit (Qiagen).
All tissue samples were analyzed for V1 markers according to previously published methods (15). For V2 marker analysis, the BRAF Val600Glu variant and hypermethylation of SMARCA3 and VIM were detected as previously described (9)(15)(16). Additionally, we developed a new DNA-scanning assay for analysis of APC, PIK3CA, and CTNNB1 variations. Modified sequencing reactions were used to develop an assay for detecting de novo variants at very low heterogeneity (
1% variant molecules). Briefly, the assay consists of amplification of the region of interest, purification of single-stranded template, and then 4 scanning reactions including a scanning primer, all 4 deoxynucleoside triphosphates (dNTPs), 1 labeled AcycloTerminator (PerkinElmer), ThermoSequenase (Amersham Biosciences), and AcycloPol enzyme (PerkinElmer). We analyzed each of the 4 nucleotides of a given sequence in 4 separate reactions to maximize the signal from a genetic variant while minimizing the signal from the wild-type background. Samples were then scored against the wild-type background to detect aberrant peaks (see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol53/issue1).
We optimized terminator selection, enzyme mixtures, the ratio of labeled and unlabeled nucleotides, and PCR cycle number. Single-base extension reactions that combine fluorescently labeled acyclic-modified terminators with AcycloPol enzyme improve variant-detection sensitivity because the improved specificity of incorporating variant-specific labels increases the signal-to-noise ratios (see Fig. 2 in the online Data Supplement). In the presence of both acyclic terminators and dNTPs, however, AcycloPol enzyme inefficiently incorporates the dNTPs. We determined that adding 1.6 U of ThermoSequenase was necessary, both to efficiently incorporate nucleotide species and to improve analytic sensitivity (see Fig. 3 in the online Data Supplement). A 50:1 ratio of dNTP to labeled AcycloTerminator (2.5 µmol/L to 0.05 µmol/L) gave the best variant signal (see Fig. 4 in the online Data Supplement), and use of 60 cycles eliminated most of the nonspecific peaks and improved the variant signal (see Figs. 5 and 6 in the online Data Supplement).
All tissue samples were amplified for 60 cycles with optimized scanning methods and purified according to Whitney et al. (15). Scanning primers were designed in 50- to 100-base increments along each amplicon and run in 4 separate reactions. Reactions were prepared in 10-µL volumes containing amplified DNA, 1 µL 10x buffer (1:1 mixture of AcycloPol and ThermoSequenase buffers), 0.5 µmol/L scanning primer (IDT); (see Table 1 in the online Data Supplement for primer sequences), 0.0025 mmol/L dNTP mix (Promega), 0.01 U AcycloPol, 1.6 U ThermoSequenase, and 0.05 µmol/L of R110-AcycloTerminator (A, C, G, or T). All reactions were cycled as follows: 5 min at 94 °C, followed by 30 cycles of 94 °C for 30 s, 52 °C for 10 s, and 72 °C for 10 s. Samples were subsequently treated with 0.1 µL shrimp alkaline phosphatase (Promega) and run on an ABI 3100 instrument (Applied Biosystems). Data were analyzed with Genescan SoftwareTM (Applied Biosystems) by superimposing tissue-DNA scans against those of known, nonpathologic samples. A sample was scored as positive when one or more peaks were detected against the wild-type background. Positive samples were confirmed through reamplification and repeat scan analysis (Fig. 1
). Germ-line variations were scored as negative for alteration.
|
The V2 marker formulation was significantly more sensitive than V1 for detection of aberrant DNA in cancers (93.6% vs 72.3%, respectively; P = 0.0002, 2-sided Fisher exact test) as well as in precancerous adenomas (92% vs 62%, respectively; P = 0.0006) (Table 1
). None of the 11 nonpathologic tissues tested positive for any of the markers [95% confidence interval (CI), 74.1%–100%]. This result is consistent with previous studies that have shown >90% tissue specificity for SMARCA3, VIM, and V1 markers (10)(11)(17). These markers also were previously evaluated for specificity in stool, and results were similar to the tissue data (2)(3)(11)(15)(18)(19).
|
We compared the rates of positivity in cancers and adenomas of both the V1 and V2 analyses. For variations in APC, KRAS, and CTNNB1 genes, which occur early in the tumorigenesis process, we observed no significant difference between adenomas or cancers. Similarly, we observed no significant difference in adenoma or cancer positivity for either hypermethylation gene (SMARCA3 or VIM), although both markers displayed a trend toward higher sensitivity in the cancer tissue set. Two genes thought to undergo alteration late in colorectal tumorigenesis, TP53 and PIK3CA (1)(8), showed higher detection rates in cancer vs adenoma [22% vs 4% (P = 0.0037) and 22% vs 6% (P = 0.017), respectively].
We compared the variant-detection rate of alterations in the MCR region of APC. Within the V1 marker set, 8 of the most common somatic variation sites within the MCR were analyzed, whereas the entire MCR was interrogated in the V2 analysis. APC alterations were found in 27 of the 94 cancer samples (28.7%; 95% CI, 19.9%–39.0%) by V1 analysis, whereas 61 of 94 samples (64.9%; 95% CI, 54.4%–74.5%) were found to have APC alterations in the V2 analysis. Moreover, the scan of the APC MCR revealed 41 unique variants in 94 samples, compared with the 8 single-base variants by V1 analysis. Analysis of the adenoma tissues revealed similar trends. With V1 analysis, 28% (14/50; 95% CI, 16%–42%) of the adenoma samples contained variations within the APC MCR, whereas V2 analysis detected variants in 74% (37/50; 95% CI, 60%–85%) of samples in the same region (P = 0.0001).
A high coincidence of positive markers in the V1 and V2 formulations (see Table 2 in the online Data Supplement) suggests that use of fewer markers may be adequate. In the V1 marker set, 42% (42/99) of the 99 positive tumor samples had >1 positive marker. There was no significant difference between cancers and adenomas. Only 17% (17/99) of samples had >2 positive markers, and 4 cancers (and 0 adenomas) had >3 positive markers. Of the 134 positive tumor samples found with V2 markers, 75% (101/134) contained >1 positive marker. Furthermore, 40% (53/134) contained >2 positive markers, and 8% (11/134) contained >3 positive markers. All of the samples positive for PIK3CA were also positive for the methylation-specific PCR markers, suggesting that PIK3CA may be unnecessary. Similarly, only 1 positive tissue result for CTNNB1 as well as 1 for BRAF was not coincident with either hypermethylation or APC markers.
In summary, the improved marker formulation, V2, provides high detection rates for aberrant DNA in CRC and precancerous adenomas. Further work is needed to evaluate the specificity of the new marker panel and to validate the panel for screening fecal DNA.
Acknowledgments
Jonathan Harrington, Kristen McEachern, Kara Higgins, and Pam Shaw aided in the preparation and analysis of samples. We thank Joel Skoletsky for his help in managing the study and for critical review of the manuscript. This work was conducted at and funded by EXACT Sciences Corporation. Authors Kann, Han, Whitney, and Shuber are all employees of EXACT Sciences. Dr. Ahlquist provided nonpathogenic mucosa and adenoma tissues but was not compensated in terms of cash or stock. Funding was supported in part by a grant from the National Cancer Institute (CA 89389). Drs. Rex, Levin, and Markowitz were not compensated in cash or stock by EXACT Sciences Corporation. Dr. Markowitz is a beneficiary of a technology-licensing agreement from Case Western Reserve University to EXACT Sciences.
Footnotes
1 work performed while Mr. Shuber and Drs. Kann and Whitney were employed at EXACT Sciences Corporation. Current affiliations are: A.S., Molecular Instincts, Mendon, MA; L.K., Gensyme Corporation, Westborough, MA; D.W., US Genomics, Woburn, MA. 
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  D. A. Ahlquist, D. J. Sargent, C. L. Loprinzi, T. R. Levin, D. K. Rex, D. J. Ahnen, K. Knigge, M. P. Lance, L. J. Burgart, S. R. Hamilton, et al. Stool DNA and Occult Blood Testing for Screen Detection of Colorectal Neoplasia Ann Intern Med, October 7, 2008; 149(7): 441 - 450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Oda, J. Okada, L. Timmerman, P. Rodriguez-Viciana, D. Stokoe, K. Shoji, Y. Taketani, H. Kuramoto, Z. A. Knight, K. M. Shokat, et al. PIK3CA Cooperates with Other Phosphatidylinositol 3'-Kinase Pathway Mutations to Effect Oncogenic Transformation Cancer Res., October 1, 2008; 68(19): 8127 - 8136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ito, T. Koessler, A. E.K. Ibrahim, S. Rai, S. L. Vowler, S. Abu-Amero, A.-L. Silva, A.-T. Maia, J. E. Huddleston, S. Uribe-Lewis, et al. Somatically acquired hypomethylation of IGF2 in breast and colorectal cancer Hum. Mol. Genet., September 1, 2008; 17(17): 2633 - 2643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zou, J. J. Harrington, A. M. Shire, R. L. Rego, L. Wang, M. E. Campbell, A. L. Oberg, and D. A. Ahlquist Highly Methylated Genes in Colorectal Neoplasia: Implications for Screening Cancer Epidemiol. Biomarkers Prev., December 1, 2007; 16(12): 2686 - 2696. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
