OncoDefender

Early Stage Colorectal Cancer – the Real Risks and New Opportunities for Improved Patient Outcomes:

Historically, early stage colorectal cancer (i.e. Stage I/II) was believed to be relatively easily treated with surgical removal of the tumor and that few patients would experience a recurrence of their colorectal cancer.  Furthermore, it was believed that adjuvant chemotherapy for Stage I/II colorectal patients was not required because it had little impact on recurrence or survival rates.  This legacy perspective was largely driven by the assumption that all Stage I/II tumors were essentially the same and were all “low-risk tumors”.

Clinically Important New Insights:

Recently completed studies have provided clinically important insights which reveal that all early stage colorectal tumors are not the same:

1. Recurrence and mortality rates associated with Stage I/II Colorectal Cancer are much higher than previously realized¹:
    
   • Stage I Colorectal Cancer:  ~1 in 7 patients with Stage I colorectal cancer, (that have undergone surgical resection of their tumor), will have a recurrence of their cancer
   • Mortality Rates for Recurrent Stage I Patients:  Over half of those patients (>50%) that recur will die from their disease
   • Stage II Color Cancer:  ~1 in 3 patients of Stage II colon cancer patients, (that have undergone surgical resection of their tumor), will have a recurrence of their cancer
   • Mortality Rates for Recurrent Stage II Patients:  Most of those patients that recur (>80%) will die from their disease
2. All early stage colorectal cancer tumors are not the same, some types of Stage I/II colorectal cancer tumors have a much higher risk of recurrence and mortality:
    
   • “Of the 176 patients…only 4 (low risk patients) developed tumor recurrence (2.3%), compared with 33.3% for intermediate (risk), and 68% for high-risk patients.²”
     • Patients with intermediate-risk tumors are ~14X more likely to have a recurrence than patients with low risk tumors.
     • Patients with high-risk tumors are ~29X more likely to have a recurrence than patients with low risk tumors.
3. There is a growing body of evidence that Stage I/II patients with high-risk tumors have recurrence and mortality rates that are more typically associated with Stage III tumors³:
    
   • “While the overall survival in Stage II patients is approximately 70%–80% 5 years after surgery, in high-risk stage II disease, the clinical outcome is similar to that of patients with stage III disease.”
   • “Currently, these high-risk patients are identified by tumors that not only penetrate the bowel wall but also show evidence of adhesion to or invasion of surrounding structures, free perforation, obstruction, or aneuploidy.”
   • “More importantly, recent data, using molecular markers or the presence of micro-satellite stable tumors, have helped to identify a subgroup of patients with both stage II and stage III CRC who may have much worse prognoses and in whom the administration of chemotherapy may be beneficial”
4. Importantly, recent studies have demonstrated that patients identified with high-risk tumors benefit significantly from adjuvant therapy; (earlier studies suggested that adjuvant therapy had little impact on recurrence rates precisely because they were unable to distinguish early stage low-risk tumors from early stage high-risk tumors):
    
   • “High-risk patients (n=102), but not other patients with stage II colon cancer, benefited from adjuvant therapy (3-year disease-free survival: 96.4% vs. 84.7%, p = 0.045; 5-year overall survival: 100% vs. 86.4%, p = 0.015)⁴.”
5. Patients identified with high-risk early stage colorectal cancer tumors benefit significantly from both surgical removal of their tumor followed by adjuvant therapy.

Important Implications for Early Stage Colorectal Cancer Patients:

1. Colorectal cancer patients with high-risk tumors are likely to benefit from adjuvant therapy, with the potential for lower rates of recurrence of their cancer and improved survival rates.
2. This requires distinguishing between patients that have an early stage low-risk tumor and those that have early stage high-risk tumor.
3. In order to accomplish these improved outcomes requires a testing method that will help physicians identify patients with low-risk versus high-risk tumors.

The Breakthrough - OncoDefender-CRC Distinguishes Between High-Risk and Low Risk Tumors, Predicting Risk of Recurrence: 

OncoDefender-CRC is the world’s first molecular diagnostic test capable of accurately distinguishing between high-risk and low-risk tumors, predicting the risk of recurrence of cancer in patients previously treated with surgical resection of a Stage I/II colon cancer tumor or Stage I rectal cancer tumor.  According to an independent statistical review, test results correlate well with accurately predicting colorectal cancer recurrence. In studies, no other currently available molecular diagnostic test approaches the performance capabilities of OncoDefender-CRC in this regard.

• The OncoDefender–CRC test is performed at the company’s Ann Arbor, Michigan headquartered laboratory.  The tumor analysis requires formalin-fixed, paraffin-embedded tumor tissue.
• Results Reported Rapidly:  Results of the OncoDefender-CRC assay are available within 7 to 10 days from the date the tumor sample is received by Everist Genomics.

Clinical Utility of OncoDefender-CRC: 

Patients identified by OncoDefender-CRC at high-risk of cancer recurrence are likely to benefit from adjuvant therapy or other more aggressive treatment options.

• Independent studies⁵ have demonstrated a significant improvement in overall survival rates for high-risk Stage I and Stage II colorectal cancer patients receiving adjuvant therapy within 60 days following surgical resection of their tumor.
• The OncoDefender-CRC test will help physicians make appropriate post-surgical decisions about the need for adjuvant therapy.

The OncoDefender-CRC test is a prognostic gene-based laboratory assay intended to help guide treatment decisions following tumor resection in patients with a pathologically confirmed stage I or II colorectal cancer diagnosis. Based on Everist Genomics patented Evolver™ platform, the OncoDefender-CRC assay evaluates the expression levels of 5 specific genes from colorectal cancer tissue samples with a proprietary computational rule. The results report distinguishes between patients that would be considered either high risk or low risk for cancer recurrence within 3 years of surgery.

Performance in Stage 1 Colorectal Cancer Patients: 
Because of the limitations of legacy testing methods for Stage 1 colorectal cancer, no patients are currently receiving adjuvant therapy; the Everist Genomics OncoDefender-CRC™ test represents a major advance for these patients.

Performance in Stage 2 Colon Cancer Patients:  In Stage 2 patients the Everist Genomics OncoDefender CRC™ test represent a major advance for physicians, patients and insurance companies.

 
Everist Genomics OncoDefender-CRC™ Prognostic Test Compared to Other Available Molecular Diagnostic CRC Tests:  The Everist Genomics test has major advantages over other molecular diagnostic CRC tests:

1. Currently available competing molecular diagnostic tests are typically priced at more than $3,000 per patient tested.  However a competing test fails to provide a test result in roughly 25% of all patients tested.  Competitors also refer to this as a ‘no-call result’.  This equates to more than $750,000 dollars being spent (250 patient) per 1000 patients to get no usable results.
2. The Everist Genomics OncoDefender CRC™ test produces zero no-call results and provides a test result for every patient when the appropriate sample collection guidelines are followed
3. Everist Genomics has succeeded in creating a breakthrough colorectal cancer molecular diagnostic test which overcomes many limitations of currently available competing products and exceeds performance characteristics achieved by applying standard of care guidelines alone.

Performance characteristics of OncoDefender-CRC in predicting recurrence in stage 1 and 2 colon and rectal cancer patients are summarized in the table below:

OncoDefender-CRC was developed through extensive clinical research and assay validation studies involving over 500 patients from around the world. According to independent biostatistical review, OncoDefender-CRC test results correlate well with colorectal cancer recurrence.

Our clinical data indicate that the performance characteristics of the test are equal to or superior to other methods currently in use to predict recurrence.  Furthermore, our studies demonstrate that high-risk OncoDefender-CRC results correlate with cohorts with higher risk of colorectal cancer recurrence within 3 years of surgery, while a low-risk result correlates more closely with those patients without recurrence in 3 years.

Clinicopathologic staging of colorectal cancer, most commonly using the American Joint Committee on Cancer TNM Staging System, is currently the prognostic ‘gold standard’ for clinical management of colorectal cancer (Source: Colon and Rectum. In: AJCC Cancer Staging Manual, 7th ed. Edge, S.B.; Byrd, D.R.; Compton, C.C.; Fritz, A.G.; Greene, F.L.; Trotti, A. (Eds.) 2010, New York: Springer-Verlag (www.cancerstaging.net) ISBN 978-0-387-88440-0.)

This system only considers anatomic tumor features, categorizing the size and depth of invasion of the primary tumor (T), the extent to which regional lymph nodes (N) are involved, and the presence or absence of distant metastases (M). TNM staging, however, fails to take into account a cancer’s underlying molecular and genomic heterogeneity, characteristics that are increasingly being recognized as key to fully understanding the natural history of the disease.This omission has clinical consequence for early-stage colorectal cancer is evidenced, in part, by the notable incongruent 5-yr survival rates for Stage IIB (72%) and supposedly more advanced Stage IIIA (83%) disease.

The National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology, based on the AJCC TNM Staging System, are currently used by most oncologists in the U.S. who are responsible for devising the optimal clinical management plans for their patients. Although there is consensus that, following resection with curative intent, adjuvant chemotherapy and adjuvant chemo-radiotherapy are indicated for Stage III colon and Stages II/III rectal cancers, respectively. The optimal post-op management of Stages I/II colon and Stage I rectal cancers remains ill-defined and, consequently, controversial. More information about postoperative management of pathologic Stages I/II colorectal cancer can be obtained from the NCCN Clinical Practice Guidelines in Oncology (National Comprehensive Cancer Network Practice Guidelines in Oncology – Colon Cancer V.2.2011; Rectal Cancer V.2.2011 (www.nccn.org)

The advantage of the OncoDefender-CRC in accurately assessing risk of colorectal cancer is depicted in the following diagrams which compare standard pathologist assessment procedures with the OncoDefender-CRC assessment:

OncoDefender - CRC assay protocol is summarized below:

1. Either formalin-fixed paraffin-embedded (FFPE) slides or blocks are accepted. If blocks are sent, slides are prepared using 5-micron slices from the blocks.
2. Slides are reviewed by an independent pathologist at Everist Genomics to verify tissue type, tumor quantity, and location of tumor on the slides.
3. The FFPE tumor tissue affixed to the slides is scraped into RNAse-free microfuge tubes and deparaffinized in xylene.
4. RNA is extracted and purified following the manufacturer’s standard protocol for a RecoverAll™ Total Nucleic Acid Isolation Kit (Applied Biosystems/Ambion, Austin, TX).
5. Purity and quantity of RNA is determined by measuring UV absorption ratios of 260/280 and 230/260 nM using a Nanodrop 1000 UV/Vis spectrophotometer.
6. A minimum of 100 ng of RNA is transcribed into single stranded cDNA using a High Capacity cDNA archive kit (Applied Biosystems, Inc., Foster City, CA), employing random hexamers and poly (dT) as primers. cDNA archive plates are either used immediately for RT-PCR or sealed and stored at -80o C.
7. Gene expression is assayed using TaqMan® custom array 384-well micro fluidic cards that allow 384 simultaneous real-time PCR reactions. One Hundred ng of cDNA per 48 wells are applied to the cards and all assays are performed in duplicate according to instructions of the manufacturer using an Applied Biosystems 7900HT Fast Real-Time PCR System. Output data are in number of PCR cycles (CT) needed to reach a constant threshold set at 0.2.
8. The gene expression data is processed to determine risk of cancer recurrence based on comparison to results of studies performed by Everist Genomics.

OncoDefender-CRC is a prognostic tool that is designed to be used immediately after surgery for Stage I/II colon carcinoma and Stage I rectal carcinoma patients.

OncoDefender-CRC is a prognostic tool intended to support treatment decisions and should be used in conjunction with, not in lieu of, standard medical treatment guidelines.

OncoDefender-CRC is registered and certified through Everist Genomics high-complexity clinical reference laboratory that is regulated by the federal Clinical Laboratory Improvement Amendments (CLIA) guidelines established in 1988.

Independent research and validation studies have already been performed on OncoDefender-CRC for Stage I/II colon cancer patients and Stage I rectal cancer patients. OncoDefender-CRC is currently commercially available for this indication. OncoDefender-CRC was developed by Everist Genomics.

Other colorectal cancer prognostic tests are for different applications (such as Stage II and/or III colon cancer only). By comparison, OncoDefender-CRC is useful for Stage I and II colon and Stage I rectal cancers.

While OncoDefender-CRC does measure certain gene expression levels, it is not a test for hereditary or genetic predisposition of any disease or disorder. The gene expression values are combined mathematically in a unique way to produce a relative measure of risk of recurrence; actual genetic information is not reported.

The OncoDefender-CRC technology is derived from research and commercialization development by Everist Genomics in Ann Arbor, MI. The company was formed in 2002 and is privately owned.

The OncoDefender-CRC development and validation process followed eight steps:

• Definition of specific aim
• Selection of genes for generation of a custom colorectal cancer specific gene panel
• Selection of housekeeping genes
• Specification of sample and data inclusion criteria
• Selection and design of scientific components
• Processing and normalization of data
• Data analysis and development of predictive rule

Each of these steps is summarized below:

• Definition of specific aim
  After confirming medical need, the aim of Everist Genomics was to develop a molecular diagnostic laboratory test (OncoDefender-CRC) that determines the expression level of certain genes found in excised primary tumor tissue from CRC patients and applies those values to a proprietary rule that accurately predicts whether or not surgically resected CRC in a particular patient is likely to recur within 3 years.

• Selection of genes for generation of a custom colorectal cancer specific gene panel
  An initial custom colorectal cancer (CRC) panel of genes was selected based on information from the scientific literature according to the following criteria:
  • A core set of genes that are generally well-known to be associated with tumorigenesis, tumor progression and metastasis regardless of tumor type. Many of these genes are well-known oncogenes and proto-oncogenes.
  • Genes that encode for key regulatory proteins in cellular processes known to be greatly utilized by tumors including cell cycle progression, angiogenesis, survival and apoptosis.
  • Genes reported to be specifically involved in the initiation and progression of colorectal cancer.
  • Genes that have been reported to be prognostic for patients with colorectal cancer in terms of aggressiveness, recurrence and patient survival.
  • Genes that have been shown to be predictive or influence tumor response to current chemotherapies for colorectal cancer.
  • Genes that have been shown to be differentially expressed between normal and malignant colorectal tissue.
• Selection of reference genes
  The data used for development of OncoDefender-CRC was normalized using five reference genes to correct for differences that might arise from technical variability and deviation in RNA quality and quantity in each assay. Reference genes were selected from 9 candidates that are among those well-known in the literature as constitutively expressed genes in colorectal cancer and other tissues. The five reference genes selected for the prognostic test showed the lowest levels of expression variability out of the 9 candidates among the patient specimens tested as determined by methods using the software packages GeNorm, Bestkeeper, and Normfinder.

• Specification of sample and data inclusion criteria
  The following minimal criteria were set for acceptance of tissue samples, RNA, and data for analysis.
  • Tissue samples were required to contain at least 50% tumor of the correct diagnosis.
  • The concentration of extracted RNA was required to be at least 10 µg/µl.
  • Extracted RNA was required to have UV absorption ratio at 260/280 nM of 1.8 or higher.
  • Genes that showed little or no amplification during the PCR process in one or both replicates were not included in the analysis.
  • Based on the increased variation in higher CT numbers, values above 35 were considered unreliable and not used in the analysis.
  • Out of 417 initial genes, certain transcripts were not expressed at all or had CT values greater than 35 in the majority of samples. Genes were included in the analysis only if they were expressed with CT values <35 in 80% of the samples.
  • RNA integrity has been shown to dramatically influence results from PCR analyses where CT values rise as RNA degradation increases. It follows, then, that the normalization factor used in this analysis (i.e. the average CT of the 5 house-keeping genes) would rise or fall with the RNA degradation and could be used as an indicator of sample integrity. Based on prior data for expression of the 5 house-keeping genes in colorectal cancer tissue, an upper limit of 29.087 was set for the average of these 5 genes. Thus, any sample with an average house-keeping gene CT above this threshold was eliminated from consideration.
• Selection and design of major scientific components
  The appropriate mRNA reference sequence (REFSEQ) accession number was identified for each gene and the consensus sequence accessed through the NCBI Entrez nucleotide database. RT-PCR primers and probes were designed by Applied Biosystems (Foster City, CA). Amplicon sizes were kept to a minimum with most being less than 100 bases in length.
   
  RNA was extracted and purified from FFPE tissue using the RecoverAll™ Total Nucleic Acid Isolation Kit (Applied Biosystems/Ambion, Austin, TX). FFPE tumor tissue affixed to glass slides was scraped into RNAse-free microfuge tubes using a disposable scalpel. The tissue was deparaffinized in xylene and RNA extracted as described by the manufacturer’s standard protocol. Purity and quantity of RNA solutions were determined by measuring UV absorption ratios of 260/280 and 230/260 nM using the Nanodrop 1000 UV/Vis spectrophotometer.
   
  A minimum of 100 ng of RNA was transcribed into single stranded cDNA using the High Capacity cDNA archive kit (Applied Biosystems, Inc., Foster City, CA), employing random hexamers and poly (dT) as primers. cDNA archive plates were either used immediately for RT-PCR or sealed and stored at -80oC.
   
  Gene expression was assayed using TaqMan® custom array 384-well micro fluidic cards that allow 384 simultaneous real-time PCR reactions. 100 ng of cDNA per 48 wells were applied to the cards and all assays were performed in duplicate according to instructions of the manufacturer using the Applied Biosystems 7900HT Fast Real-Time PCR System. Output data are in number of PCR cycles (CT) needed to reach a constant threshold set at 0.2.

• Processing and normalization of data
  Expression data for each gene was produced in duplicate as CT values. Each pair of individual gene replicates was inspected for congruence and a correlation coefficient was generated for each replicate set of genes. The replicates were averaged and the resulting values used in the normalization process. The data were normalized by subtracting the CT for each rule gene (RG CT) from the average of the 5 reference genes (Ave. 5HK CT). Since CT values are expressed as logarithmic numbers to the base 2, the data was linearized by taking the antilog and the result was scaled by a selected factor.

• Data analysis and development of predictive rule
  The predictive rule within OncoDefender-CRC was developed using a proprietary machine learning technique based on genetic programming (GP). Gene expression values as assayed by RT-PCR were used to classify the primary tumor tissue samples into those associated with recurrence within 3 years and those from patients that did not recur. GP uses the available data to produce a set of classifiers ("rules") that are optimized in an iterative fashion through successive retention of the better performing rules. The predictive rule and the OncoDefender-CRC assay process have been validated through clinical study which is summarized in a presentation given at the ASCO GI Symposium January 18th 2011(pdf).

The OncoDefender-CRC test process requires either 1 formalin-fixed, paraffin-embedded (FFPE) tissue block or 15 unstained slides from the patient’s resected tumor. Results of the OncoDefender-CRC test are typically available within 10 business days from the date the sample and completed Test Requisition Form are received at Everist Genomics. 

To order OncoDefender-CRC:

1. Obtain free sample collection and pre-paid mailing kits by contacting Everist Genomics. When ordering a mailer, please specify sample type (slides or block).
2. Affix a bar code label from the collection/mailing kit to the OncoDefender-CRC Test Requisition Form. Complete the Test Requisition Form and send to Everist Genomics.
3. Forward the collection/mailing kit and a copy of the Test Requisition Form to your Pathologist. (If you have included a Pathologist on the Test Requisition Form, you may also request that Everist Genomics fax a copy of the order form to them.)
4. Your Pathologist will prepare and package the sample materials and send them to Everist Genomics using the pre-paid collection/mailer kit.

We accept either a formalin-fixed, paraffin-embedded (FFPE) tissue block or 15 unstained slides. If sending a block, please select one with the greatest amount of invasive cancer and related stroma. Everist Genomics will return any unused tissue from this block to the Pathologist.

If sending tissue slides, please prepare 15 slides containing unstained FFPE tissue that, when taken together, provide a total of at least 375 sq mm of colorectal tumor foci (i.e., ≥25 sq mm tumor/slide) and that are 5 microns thick.

Affix a bar code label from the collection/mailing kit to each block or slide sample. These labels must be included and must match the Test Requisition Form label.

The OncoDefender-CRC results report includes an indication of risk for cancer recurrence within 3 years of surgery (resection) relative to results obtained in the clinical validation study. A sample report is available.

Reports are available by fax, overnight mail, or email to the ordering physician and any other physicians or health care providers identified on the Test Requisition Form.

The Test Requisition Form provides options for you to select your preferred reporting method.

Regardless of insurance coverage or financial status, the Everist Genomics Customer Care team will assist patients in obtaining the testing they need. We will directly bill insurance companies on behalf of insured patients and will accept insurance payments as payment in full with a signed Patient Information and Assignment of Benefits form (AOB). Everist Genomics also has a financial assistance program for those patients that qualify based on financial eligibility. Qualifying patients may be eligible to have 80% of the cost of the OncoDefender-CRC test covered through one of the Everist Genomic Financial Assistance programs.  Within 3 to 4 working days of receiving the patient’s application for testing, Everist Genomics will provide the patient and their doctor with written confirmation of their financial benefits.