Exact Sciences overcame significant obstacles to bring cancer screening innovation to patients
By Graham P. Lidgard, PhD
Colorectal cancer (CRC) is the second-leading cause of cancer-related death among men and women in the United States, behind only lung cancer.
Screening guidelines of the American Cancer Society (ACS) call for men and women at average risk to begin screening for CRC at age 50.1 Screening is critical because most CRCs develop slowly over several years. In most cases, a benign polyp appears on the inner lining of the colon or rectum. These polyps can take 10 to 15 years to become malignant, so early identification and removal (through routine screening) can effectively prevent most CRCs from ever forming. Further, because most polyps and early-stage cancers cause no symptoms, the only way to detect them early is through screening. When diagnosed early, 5-year survival can be greater than 90%.
Unfortunately, according to the Centers for Disease Control and Prevention (CDC), 23 million Americans are noncompliant with screening recommendations.2 The lack of patient compliance with screening has resulted in more than 60% of all CRC cases not being detected until the late stages, making treatment difficult and 5-year survival a mere 12%.3
As a result, CRC is often considered the most preventable, yet least prevented cancer in the United States.
THE SCREENING CHALLENGE
Today, the standard procedure used to diagnose CRC is colonoscopy. What makes colonoscopy so effective is that doctors are able to visually inspect the colon for signs of cancer and, during the same procedure, to remove potentially problematic tissue or polyps, reducing the chances that they will later develop into cancer.
The challenge is that many patients are unwilling to undergo colonoscopy because the procedure is invasive; requires bowel preparation (including a clear liquid diet and laxatives); and often involves sedation, which means taking time off from work and arranging for transportation to and from the procedure. Another screening tool, called sigmoidoscopy, takes less time, but is still invasive and requires bowel preparation and sedation.
Alternative tests, such as the fecal occult blood test (FOBT) and fecal immunochemical test (FIT), were developed to help meet the need for noninvasive screening options. Both are designed to detect occult blood in the stool. However, because there are unrelated conditions that can cause blood in the stool, and not all polyps or lesions bleed, FOBT and FIT on their own may not be reliable for the detection of cancer. In addition, FOBT has a low level of sensitivity and a long list of potential causes for false positive results, while FIT delivers only a moderate level of sensitivity.4–6
IN SEARCH OF A NEW OPTION
Exact Sciences is a molecular diagnostics company with an initial focus on the early detection of CRC. The company also owns and operates a large research and development facility and 30,000 square foot, state-of-the-art laboratory. Committed to playing a role in the eradication of this deadly disease, the company set out in the mid-1990s to develop a noninvasive, patient-friendly, and accurate test processed almost entirely in its lab.
The concept underlying such a test was straightforward. Colorectal cancer arises from accumulated genetic and epigenetic alterations, which also provide a basis for the analysis of stool to identify tumor-specific changes. As stool passes through the colon, it picks up normal cells as well as abnormal cells shed by cancer or precancers. A multitarget test that detects altered DNA from abnormal cells, as well as blood from precancerous lesions or cancerous tumors, would represent a significant diagnostic breakthrough.
However, turning this simple concept into a commercially viable product was a challenge.
As far back as 1992, scientists had identified the potential for finding mutant genes associated with colorectal cancer in stool.7 For more than 20 years afterward, scientists in the diagnostics industry attempted to create a product based on the idea of using stool to detect CRC, but with little success. Others tried to use blood samples to detect the disease, but again, fell short.
Exact pioneered the idea of detecting DNA from abnormal cells in stool early on in its history, but was unable to achieve the level of sensitivity or specificity required to enable a successful commercial assay. It wasn’t until the company entered into a partnership with the Mayo Clinic, in 2009, that the project gained momentum. The partnership gave Exact access to critical clinical samples and enabled the company to overcome several technical challenges that previously had stymied the development of a reliable stool-based DNA (sDNA) screening test.
Purification and Capture. The first challenge was to purify sufficient quantities of intact DNA from stool samples to enable reliable analysis. The issue is that DNA from cancer cells is partially degraded in the digestive tract, making it difficult to purify. Moreover, quantities of these molecules are very limited, and there are excessive amounts of bacterial DNA and inhibitors present in stool (such as nonspecific DNA, RNA, protein, lipids, carbohydrates) that make isolation of “good” DNA difficult.
Previously known methods for purification included ethanol precipitation of DNA from the sample, which resulted in a gelatinous mass that was difficult to get back into solution. Another previously used method relied on capture of specific sequences with biotinylated oligonucleotides on streptavidin beads. This was problematic because of the potential for patients’ stool to contain large concentrations of free biotin from diet or vitamin supplements.
Because there were no commercially available products capable of meeting Exact’s need for the purification of consistently high quality DNA from stool samples, Exact developed this critical component of the product from scratch. The innovation evolved primarily through trial and error that ultimately led the company to a combination process that includes inhibitor removal by binding inhibitors of the polymerase chain reaction (PCR) to insoluble polyvinylpyrrolidone (PVPP), filtering out the PVPP in a proprietary filter device, and then further purifying the DNA by sequence-specific DNA target capture on magnetic particles with oligonucleotides coupled to them.
The overall process of DNA purification from stool has yielded eight US patents awarded to Exact Sciences, with several more pending.
Detection. Following the purification of stool DNA by inhibitor removal and target capture, it was necessary to further process the DNA for detection of epigenetic modifications indicative of cancer; specifically, the detection of methylated gene sequences that are part of the cancer biology. This requires detecting methylated cytosine residues in the DNA sequence by modifying those cytosine-containing sequences that are not methylated. None of the existing literature or known commercial methods were rapid or robust enough for Exact’s clinical product needs.
As a result, the team had to develop proprietary amplification and detection technologies with analytical capabilities superior to other available tools and capable of honing in on the tiniest amounts of target DNA. This involved developing an improved method for modifying the DNA, one that allowed for consistent detection of epigenetic modifications indicative of adenomas or cancer. Exact’s method utilizes higher concentrations of bisulfite ions, optimal times and temperatures, and concurrent modification and purification of the methylated or modified DNA on magnetic particles. Patents for these innovations have been applied for.
Amplification. Next, although various real-time, quantitative PCR methods were available, Exact needed better performance. As a result, the company combined existing, licensed technology with its own newly invented and optimized quantitative allele-specific real-time target and signal amplification (QuARTS) method to create its own version of real-time PCR. QuARTS employs a combination of polymerase and flap endonuclease, as well as PCR thermal cycling parameters, inventive primer designs, and improved methods of specificity for the detection of mutations and epigenetic methylation. The method can detect mutant or methylated gene copies in a milieu of 10,000-fold excess of wild-type DNA copies. The technology has resulted in six issued US patents with additional patents pending.
Biomarker Selection. Searching for and validating the proper biomarkers was a significant challenge. It took more than 2 years to collect enough samples from patients with known disease states to design studies that would enable the team to select the right biomarkers from among hundreds of possible candidates.
Exact worked extensively with the Mayo Clinic throughout the marker selection process, which involved several steps. First, a list of candidate mutation and methylation markers was generated from previous studies, and Mayo Clinic researchers used sophisticated DNA sequencing methods to screen and identify genes that were methylated in colorectal cancer and could be used as potential markers. Any markers with high background, such as normal tissue or normal stool, were eliminated. Candidate markers were then tested on colorectal cancer biopsy tissue of known pathology; those markers with good performance on disease tissue were tested in stool as individual markers. All the data were analyzed to determine which markers were complimentary and which ones did not add to the levels of sensitivity.
The result is a robust assay used to detect 11 distinct biomarkers—all known to be associated with CRC or precancers—including methylated DNA; beta-actin (DNA normalization target); blood; seven different aberrent forms of the KRAS gene; and two hypermethylated genes, NDRG4 and BMP3, the latter of which was discovered by the Mayo Clinic.
Automation. After demonstrating technical success, the team realized that automation would be key to making the assay feasible in a clinical environment. There were several reasons for this.
First, some of the steps required instrumentation that was simply not available. Needs included technology capable of ensuring homogenization of the whole stool sample in the shipping stabilization preservative solution. In response, Exact created a device for holding and mixing the stool much like a shaker that might be found at a paint store. In addition, for the target capture purification step, the company developed a customized incubator and shaker as well as a magnetic separator and aspirator.
The second reason for automation concerned the large number of steps requiring repetitive and accurate pipetting. For example, error-free dispensing of reagents and addition of samples to a 96-well microtiter plate is done far more effectively by a robotic pipettor than by a person. Full automation of the process includes integration of the steps for sample preparation, DNA modification, blood marker detection, reagent dispensing, sample addition, PCR amplification, and detection, within the laboratory information system—plus an algorithm for calling results positive or negative. Laboratory technologists play integral roles in this automated process, as they must pay meticulous attention to sample and reagent set up as well as the transfer of samples from instrument to instrument.
All of these instrument components are unique to Exact and were developed specifically to support processing and analysis of the company’s colon cancer screening test. In addition, development of the assay required discovery and optimization of:
- A preservative solution for stool samples at ambient temperature.
- A collection container for stool samples from which DNA could be obtained.
- A collection device for an immunoassay from stool.
- Controls, calibrators, and methods of quantitation.
Exact now holds 17 US patents covering all aspects of its technology, collection system, and analysis process.
BRINGING COLOGUARD TO MARKET
In 2012, Exact and Mayo Clinic published early proof-of-concept data demonstrating that the QuARTS method provided a promising approach for quantifying methylated markers. In the study, the markers assayed accurately discriminated colorectal neoplasia from healthy epithelia.8
The following year, the results of a blinded, multicenter, case-control study were published. In this study, researchers collected stool samples from 459 asymptomatic patients before screening or surveillance colonoscopies and from 544 referred patients. The results demonstrated that the automated, multitarget sDNA assay developed by the two organizations was capable of identifying individuals with colorectal cancer with 98% sensitivity at 90% specificity. The study concluded that this new, automated, high-throughput system could detect colorectal cancer and premalignant lesions with levels of accuracy previously demonstrated with a manual process and, as a result, could be a widely accessible, noninvasive approach to general colorectal cancer screening.9
Early on, Exact was approached to participate in the parallel review pilot program, in which FDA and the Centers for Medicare and Medicaid Services (CMS) review medical technologies simultaneously. The parallel review program is designed to expedite the process for bringing life-saving innovations to patients. Participation, however, was a risk—it could save time and years of regulatory review, or bring the entire project to a halt. Bolstered by strong early data, Exact decided to move forward, and joint review by FDA and CMS was agreed upon at the onset of the project.
As one of the first companies to go through the parallel review process, Exact had to navigate uncharted territory and rely heavily on a positive relationship and open communication with FDA and CMS at every turn in order to improve the chances of success. This communication began even before initiation of the company’s pivotal trial, when Exact held several meetings with FDA and CMS to determine exactly which studies needed to be completed in order to support both the regulatory and reimbursement submissions, and then to review the design of the clinical trial. Feedback from the agencies was essential in ensuring that the study was conducted properly.
The DeeP-C Study. The DeeP-C study was designed to determine the performance characteristics of the test, named Cologuard, and to compare that performance to a leading fecal immunochemical test (FIT), the Polymedco OC-CHECK, a commonly used noninvasive colorectal cancer screening test. (For more information, see the companion article, “The Cologuard Process.”)
DeeP–C enrolled more than 10,000 patients at 90 sites throughout the United States and Canada in just 18 months. Study sites included private practice and academic settings; the Mayo Clinic provided input on the study design and was one of the main sites at which patient samples were collected. The target population was asymptomatic persons between the ages of 50 and 84 years who were considered to be at average risk for colorectal cancer and who were scheduled to undergo screening colonoscopy. All participants were required to provide a stool specimen and undergo screening colonoscopy within 90 days after providing informed consent. Stool was collected before routine bowel preparation. No dietary or medication restrictions were required.
During the colonoscopy, colonoscopists were required to describe the extent of the examination, document cecal visualization, rate the quality of preparation (on a modified Aronchick scale), and record the size and location of lesions. Although colonoscopists reported the location and size of all lesions, only the most advanced colorectal epithelial lesion (the index lesion) and its location (proximal or distal) were used to categorize participants for the analysis. The biopsy and surgical specimens underwent histopathological analysis at the laboratory typically used by each study site.
The primary outcome was the ability of the DNA test to detect colorectal cancer (ie, adenocarcinoma), with disease stage determined using the staging system developed by the American Joint Committee on Cancer (AJCC). The secondary outcome was the performance of the DNA test for the detection of advanced precancerous lesions.
The study was designed to have a power of 90% to test the prespecified hypothesis that the DNA test would have a sensitivity of 65% or more for the detection of colorectal cancer (AJCC stages I through IV) under the null hypothesis, at a one-sided type I error rate of 0.05. A secondary hypothesis was to rule out a 5% noninferiority margin of sensitivity for the detection of colorectal cancer with the DNA test as compared with FIT, at a one-sided type I error rate of 0.05. Testing of the two hypotheses with a power of at least 80% required the diagnosis of 49 and 56 adjudicated colorectal cancers, respectively, which required the enrollment of 10,500 to 12,000 participants, under the assumption of a colorectal-cancer prevalence of 4.5 cases per 1000 population.
In the context of the DeeP-C study, sensitivity measured the ability of the Cologuard test to correctly identify positive results, and referred to the percentage of patients who were determined by colonoscopy to have precancerous polyps or cancer and also had a positive Cologuard test result. Specificity measured the test’s ability to correctly identify negative results, and referred to the percentage of patients who were determined by colonoscopy not to have precancerous polyps or cancer and also had a negative Cologuard test result.
In April 2013, Exact Sciences announced preliminary top-line results, which showed that Cologuard met or exceeded all primary and secondary endpoints of the DeeP-C pivotal clinical trial.10 In these top-line results, Cologuard demonstrated 92% sensitivity for the detection of colorectal cancer and 42% sensitivity for the detection of precancerous polyps, including 66% sensitivity for polyps equal to or greater than 2 cm in size. The test achieved a specificity of 87% during the trial.
In April 2014, the full results of the DeeP-C study were published in the New England Journal of Medicine.11 The results, which elaborated on the top-line data announced a year earlier, found that Cologuard was accurate and specific in identifying cancers and the most advanced precancerous polyps in average-risk patients. Specifically, the data found:
- Sensitivity of Cologuard in detecting patients with colorectal cancer was 92% versus 74% for FIT.
- Sensitivity for detecting patients with colorectal cancers in Stages I–III—those determined by AJCC to be associated with an increased rate of being cured—was 93% for Cologuard versus 73% for FIT.
- Sensitivity for patients with advanced precancerous lesions was 42% for Cologuard versus 24% for FIT.
- Cologuard detected 69% of patients with polyps with high-grade dysplasia versus 46% for FIT.
- Cologuard achieved a specificity of 87% versus specificity of 95% for FIT.
Regulatory Review. Exact’s parallel review regulatory submission was modular, meaning that each section—design control and manufacturing, analytical studies, and clinical study—was submitted separately for consideration. After each section was submitted and during each review, there were conference calls and written communications among Exact, FDA, and CMS to answer questions, clarify data, and secure guidance for moving forward. As a result of these regular and ongoing communications, Exact was able to address questions fully, provide data or information for any deficiencies, and ensure that the final submission package was comprehensive.
In August 2014, FDA approved Cologuard, making it the first noninvasive stool DNA test for colorectal cancer approved by the agency, and the first product approved under the parallel review program. On the same day, CMS provided a proposed coverage memorandum. CMS issued its final payment decision regarding Cologuard in November 2014, making the test broadly available as a new colorectal cancer screening option to the more than 50 million people covered by Medicare.
THE IMPACT OF COLOGUARD
Since approval, thousands of physicians have started prescribing Cologuard, more than 35,000 patients have completed the test, and multiple health systems and a growing number of private payors, including Aetna Medicare Advantage and Anthem Blue Cross & Blue Shield, have signed on to offer and cover it. Cologuard is now also included in the American Cancer Society’s colorectal cancer prevention and early detection national guidelines for CRC screening, marking a pivotal benchmark in providing patients and clinicians with information and access to the test.
Early results demonstrate that Cologuard is not just a scientific breakthrough; it is actually helping to motivate people who had previously avoided CRC screening. In fact, the test has achieved patient compliance rates in excess of 70%, meaning that more than 70% of people who receive a kit actually complete and return it for analysis. This exceeds the overall national screening compliance rate of 58% for CRC screening, and is far above the 38% who comply when their doctor orders a colonoscopy. Further, about half of all patients screened with Cologuard have never undergone a colonoscopy, indicating that the test is helping to reach a new population of previously unscreened patients.
Looking ahead, Exact is exploring potential applications of this unique molecular screening technology platform for the detection of other deadly cancers in the earliest stages of disease when they are most treatable and survivable. Some of the technology developed for Cologuard—along with the lessons learned from its development—is poised to transform the cancer screening marketplace and will serve as an important starting point for development of new early detection screening tests for such diseases as esophageal, pancreatic, and lung cancers—innovations with the potential to transform the cancer screening marketplace for years to come.
Graham P. Lidgard, PhD, is chief science officer at Exact Sciences. For further information, contact CLP chief editor Steve Halasey via [email protected].
- American Cancer Society guidelines for the early detection of cancer [online]. Atlanta: American Cancer Society, 2015. Available at: www.cancer.org/healthy/findcancerearly/cancerscreeningguidelines/american-cancer-society-guidelines-for-the-early-detection-of-cancer. Accessed January 13, 2016.
- Vital signs: colorectal cancer tests save lives [online]. Atlanta: Centers for Disease Control and Prevention, 2013. Available at: www.cdc.gov/vitalsigns/colorectalcancerscreening. Accessed January 13, 2016.
- Colorectal Cancer Facts & Figures 2014–2016 [online]. Atlanta: American Cancer Society, 2014. Available at: www.cancer.org/acs/groups/content/documents/document/acspc-042280.pdf. Accessed January 13, 2016.
- Ahlquist DA, Wieand HS, Moertel CG, et al. Accuracy of fecal occult blood screening for colorectal neoplasia: a prospective study using Hemoccult and HemoQuant tests. JAMA. 1993; 269(10):1262–1267.
- Lieberman D. Progress and challenges in colorectal cancer screening and surveillance. Gastroenterology. 2010;138(6):2115–2126; doi: 10.1053/j.gastro.2010.02.006.
- Nadel MR, Shapiro JA, Klabunde CN, et al. A national survey of primary care physicians’ methods for screening for fecal occult blood. Ann Intern Med. 2005; 142(2):86–94.
- Sidransky D, Tokino T, Hamilton SR, et al. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science. 1992; 256(5053):102–105.
- Zou H, Allawi H, Cao X, et al. Quantification of methylated markers with a multiplex methylation-specific technology. Clin Chem. 2012; 58(2):375–383; doi: 10.1373/clinchem.2011.171264.
- Lidgard GP, Domanico MJ, Bruinsma JJ, et al. Clinical performance of an automated stool DNA assay for detection of colorectal neoplasia. Clin Gastroenterol Hepatol. 2013; 11(10):1313–1318; doi: 10.1016/j.cgh.2013.04.023.
- Top-line data show Exact Sciences’ Cologuard test demonstrates 92% sensitivity in the detection of colorectal cancer (press release]. Madison, Wis: Exact Sciences, 2013. Available at: http://investor.exactsciences.com/investor-relations/press-releases/press-release-details/2013/Top-Line-Data-Show-Exact-Sciences-Cologuard-Test-Demonstrates-92-Percent-Sensitivity-in-the-Detection-of-Colorectal-Cancer/default.aspx. Accessed January 13, 2016.
- Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014; 370(14):1287–1297; doi: 10.1056/nejmoa1311194.