Treatments & Side Effects > About Biomarkers
BIOMARKERSPART II: UNDERSTANDING COLORECTAL CANCER BIOMARKERS
PART I: WHAT ARE TUMOR BIOMARKERS
Tumour biomarkers are chemicals that are made by tumour cells or other cells of the body, in response to cancer. Different types of cancers or tumours may be associated with different tumour biomarkers. Tumour biomarkers may be used in diagnosis, staging and prognosis of cancer, provide an estimation of tumour burden, and serve for monitoring effects of therapy, detecting recurrence, localization of tumours, and screening in general population. An abnormal level of tumour biomarker is usually not enough for a complete diagnosis of cancer and is usually combined with other tests such as a biopsy. The type of biomarker detected and its levels can give an indication to what type of cancer may be present, whether or not it is malignant and what the best treatment may be.
Tumour markers are used during the treatment of cancer in order to monitor the effectiveness of a therapy and how the patient may be responding to the treatment. If levels of a tumour biomarker decrease it may mean that the cancer is responding to treatment. If levels remain the same or increase after treatment it may be an indication that the therapy is not working. Continued monitoring of tumour biomarker levels following treatment can be used to check for recurrence of the cancer.
Different tumour biomarkers are measured in different ways and from different sources. Some markers are found in blood or urine, so these would require the patient to provide a small amount of blood or a urine sample. Other tumour biomarkers, such as those contained in the tumour tissue itself, may involve a tissue biopsy. This is a more invasive procedure than urine or stool sampling.
Tissue-based markers have been studied as possible prognostic or predictive markers of disease, while colorectal biomarkers obtained from serum (blood) are primarily used for the postoperative surveillance of patients.
- Prognostic vs. Predictive Biomarkers
Examples of each type of biomarker are provided in the next section of this document. Please see PART II.
- Serum-based Markers
- Tissue-based Markers
Integrating biomarkers into routine clinical practice will help physicians select the right treatment, for the right patient. Doing this could have many positive outcomes including sparing patients unnecessary toxicity from an ineffective treatment or being able to select a treatment that may be more effective in battling their disease. This is what is commonly referred to as personalized medicine. Specifically, personalized medicine refers to the use of new methods of molecular analysis to better manage a patient’s disease or predisposition towards a disease. It aims to achieve optimal medical outcomes by helping physicians and patients choose the disease management approaches likely to work best in the context of a patient’s genetic and environmental profile. Traditionally, all colorectal cancers have been lumped together and given similar treatments. The novelty associated with the use of biomarkers is that physicians can, in a very minimally invasive way, start to treat the disease based on its unique protein makeup through the use of therapies that target the individual’s disease state – the patient’s molecular portrait would be considered as the rationale for choice of therapy rather than based on the site or the kind of cancer alone. Truly, the prospects are very exciting.
PART II: UNDERSTANDING COLORECTAL CANCER BIOMARKERS
In recent years, significant advances in our understanding of human biology have yielded novel drug targets that may impact disease. Typically, early clinical trials test a drug target’s safety and tolerability. The efficacy of a drug is typically not tested until later stages in development. Researchers may now be able to use pharmacogenomics (the study of the relationship between a specific person’s genetic makeup and his or her response to drug treatment) to improve the efficiency of drug development. Using biomarkers, researchers can explore how a drug works in the body, allowing earlier decisions on whether to advance molecule sin clinical trials. Biomarkers may also be used to diagnose disease and for patient selection. As research continues, our understanding of the role biomarkers can play in the management of disease areas such as cancer has evolved.
A series of colorectal cancer biomarkers are addressed and described below according to the following parameters:
- Tissue or Serum-based
- Method & Timing of Measurement
Of noteworthy importance is the fact that references to genetic material or “genes” will be appearing throughout the content. Each of us has a large amount of genetic information that is organized into smaller segments known as “genes.” Genes provide the instructions the cells of the body need to perform their different functions. And depending upon how those genes are expressed (either normally or in a mutated fashion), it will dictate how biomarkers impact the management of the patient’s disease.
Figure 1: Location of the kras gene.
Utility: In order to understand the relevance of the kras gene in the treatment of colorectal cancer, it is important to understand the Epidermal Growth Factor Receptor (EGFR). The EGFR is a protein which promotes cell growth in normal epithelial tissues, such as skin and hair follicles, and is often over-expressed on a variety of tumour cells, including colorectal cancer. When growth factors (other body proteins) attach to the EGFR, the cancer cell is then stimulated to grow and divide. In order to help prevent the growth and division of cancer cells, new anti-EGFR therapies (such as erbitux and vectibix) have been developed to block the activation of the EGFR. Although considered a breakthrough in the treatment of colorectal cancer, not all cancer will respond to anti-EGFR therapies because of a kras gene mutation present in approximately 40% of the colorectal cancer patient population. Tumours that have a mutation in the kras gene have their cells continuously receiving messages to grow and divide despite anti-EGFR therapies. Because these patients do not respond to anti-EGFR therapies, detecting the mutated gene is important in determining and prescribing an alternate treatment. Patients with the non-mutant kras gene, also known as wild type, may respond to anti-EGFR therapies and a conversation about patient eligibility should take place with the treating physician.
Figure 2: Relationship between biomarkers and response to epidermal growth factor receptor (EGFR) therapies in chemorefractory metastatic colorectal cancer. (wt = wild type; MT = mutation)
Tissue or Serum-based: The kras biomarker is tissue-based which requires a sample biopsy from the primary tumour (colon or rectum) or from a metastatic lesion found in distant organ sites such as the liver or lungs.
Method & Timing of Measurement: Kras status is determined via PCR analysis of formalin-fixed, paraffin-embedded block, unstained slides, or fresh snap frozen biopsy tissue for the presence of a mutation in codons 12, 13, or 61 of the kras gene on chromosome 12. In Canada, the sample biopsy is usually performed as soon as a patient exhausts second line therapy in anticipation of third line therapy so that anti-egfr therapy eligibility can be determined.
Figure 3: Location of the BRAF gene on chromosome 7
Utility: The presence of a mutation in the BRAF gene is associated with nonresponse to anti-EGFR therapies such as erbitux and vectibix despite the fact that there is no mutation in the KRAS gene. Studies have shown that colorectal cancer patients with wild-type KRAS failed to respond when treated with either erbitux (cetuximab) or vectibix (panitumumab) when the BRAF mutation was present, compared to patients with wild type BRAF tumours. As stipulated previously, kras mutations are found in approximately 40% of colorectal cancer patients who fail to respond to standard anti-EGFR therapies. An additional 15% of patients may also be nonresponsive due to a mutation of the BRAF gene (see Figure 4 below). Screening for BRAF and KRAS mutation status in advanced stage colorectal cancer can provide important information to the treating physician regarding potential candidates for erbitux and vectibix.
Figure 4: BRAF Mutations and Anti-EGFR Therapy Response
Tissue or Serum-based: The braf biomarker is tissue-based which requires a sample biopsy from the primary tumour (colon or rectum) or from a metastatic lesion found in distant organ sites such as the liver or lungs.
Method & Timing of Measurement: BRAF status can be performed on paraffin-embedded tumor tissue or slides. The test is not routinely performed in Canada but can be requested through a treating oncologist provided payment is supplied after second line therapy is exhausted. The treating oncologist should simply complete an Oncotech/Exiqon Diagnostics Requisition (PDF download) and request BRAF Mutation Analysis (Test Code 221). Test results will be available in approximately 10 business days from time of specimen receipt. Additional information on the BRAF Mutation Analysis, including an informational sheet and sample report, is available here:
- BRAF Information Sheet
- BRAF Sample Report
iii CEA (Carcinoembryonic Antigen)
Definition/Description: CEA is a protein normally found in the tissue of a developing baby in the womb. Blood levels of this protein disappear or become very low after birth. In adults, an abnormal amount of CEA may be a sign of cancer. A blood test can be performed to measure the amount of CEA in a patient’s blood. The normal range is 0 – 2.5 micrograms per liter (mcg/L). In smokers, however, the normal range is 1 – 5 mcg/L because smoking has a tendency to increase CEA levels. Something to take note of is the fact that abnormal CEA levels can be found in people who do not have cancer.
Utility: The doctor may order this test if they suspect certain types of cancer, such as colorectal cancer. However, this test is not an accurate way to diagnose any type of cancer, and high levels can be found in people without cancer. CEA measurement is mainly used as a tumour marker to identify recurrences after surgical resection or localized cancer spread. By comparing the levels before and after surgery, the test can be used to detect recurrence of tumour and monitor for the development of distant organ metastases. The CEA level can also be used to assess the response to chemotherapy. Elevated CEA levels should return to normal after successful surgical resection or within 6 weeks of starting treatment if cancer treatment is successful. Of noteworthy significance is the fact that CEA levels may actually be normal in patients who do experience recurrence or in patients who do not respond to therapies, which is why additional testing methods are required to properly assess the management of colorectal cancer. Also, CEA is not an effective screening test for hidden (occult) cancer since early tumours do not cause significant blood elevations. Also, many tumors never cause an abnormal blood level, even in advanced disease. Because there is variability between results obtained between laboratories, the same laboratory should do repeat testing when monitoring a patient with colorectal cancer.
Tissue or Serum-based: The CEA test is serum-based (determined by drawing blood from a vein).
Figure 5: CEA testing through the drawing of blood from a vein.
Method & Timing of Measurement: CEA is most frequently tested in blood by drawing blood from a vein, usually from the inside of the elbow or the back of the hand. It can also be tested in body fluids and in biopsy tissue. The test is prescribed as soon as a diagnosis of colorectal cancer is delivered, after surgery, for monitoring purposes during treatment and through every 6-12 months for five years after the disease has been eradicated.
- MSI (Microsatellite Instability)
Microsatellites do not cause a cancer to develop, but fluctuations in the length of microsatellites (termed instability) can mean that mismatch repair genes are not functioning correctly. And it is these defects in the genes involved in mismatch repair that lead to an accumulation of mutations in a cell, which may result in the cell becoming malignant.
Figure 6: Microsatellites are short, repetitive DNA sequences. Microsatellite instability (MSI), is a marked difference in the number of repeated sequences between tumor and normal tissue. MSI is a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) tumors, and it is caused by errors in DNA replication due to mutations in DNA repair genes.
Utility: MSI testing can be performed to determine if a tumor exhibits microsatellite instability by comparing the microsatellites in the tumor specimen to a normal tissue of the individual. If the tumor specimen exhibits alterations within the microsatellite regions, it is indicative of a probable defect in the mismatch repair genes. MSI testing for demonstrating instability in the tumor specimen is helpful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC) and sporadic cancers with defective DNA mismatch repair. MSI status determination in sporadic cancers is useful in establishing prognosis and may be predictive of tumor response to certain chemotherapeutic agents such as 5FU. MSI status is determined to fall into one of three categories based on how many markers are affected with MSI:
- MSI-High: MSI is detected in at least two of the five markers (more than 30% of the markers show instability)
- MSI-Low: MSI is detected at only one marker (fewer than 30% of the markers show instability)
- MSI-Stable: None of the 5 markers shows MSI (0% of the markers show instability)
MS-Stable disease, however, may benefit from 5FU based treatment. Hence, some experts recommend testing for MSI testing as another tool to determine the need for treatment in stage II disease.
Tissue or Serum-Based: Microsatellite instability testing can be performed to determine if a tumor exhibits microsatellite instability by comparing the microsatellites in the tumor specimen to a normal tissue of the same individual. The test is, therefore, tissue-based. It is important to remember that MSI testing demonstrating instability in the tumor specimen is suggestive of HNPCC, a hereditary form of colorectal cancer, although not diagnostic since 10-15% of sporadic colon cancers (colon cancers that are not hereditary) will also exhibit MSI.
Method & Timing of Measurement: The MSI test is a fragment analysis based test. DNA, genetic material, is extracted from unstained sections of the tissue. The test is performed to detect fluctuations in the length of six microsatellite markers. The markers are
The microsatellites in the tumour specimen are compared to the microsatellites in a normal tissue of the individual. If the tumour specimen exhibits alterations within the microsatellite regions, it is indicative of a probable defect in the mismatch repair genes. MSI testing for demonstrating instability in the tumor specimen is helpful in identifying patients with HNPCC and sporadic cancers with defective DNA mismatch repair. This implies, that for HNPCC determination, testing take place as the first step in genetic testing for families with medical histories suggestive of HNPCC. And MSI status determination in sporadic cancers is useful in establishing prognosis and since it may be predictive of tumor response to certain chemotherapeutic agents, testing should take place once a diagnosis is delivered through tumour biopsy confirmation, before accessing adjuvant therapy (post-operative therapy). The sample materials are tumor and normal tissue from colon and the best material of tumor tissue is pre-treatment biopsy, while the best material of normal tissue is post-treatment resection or 2 - 4 ml of blood.
The application of MSI testing of colorectal cancers with the purpose of evaluating prognosis and selection of treatment is not currently part of routine practice in Canada but may receive support in the future, as more studies continue to confirm the findings reported in the past few years (DeDosso, S, et al., Cancer Treat Rev, 2009; Glas, Am, et al., J of Clin Oncol 2009).
v. PTEN (Phosphatase and tensin homolog)
Definition/Description: The PTEN gene provides instructions for making a protein (called the PTEN protein) that is found in almost all tissues in the body. This protein acts as a tumour suppressor, which means that it helps regulate the cycle of cell division by keeping cells from growing and dividing too quickly or in an uncontrolled manner, as is the case with cancer. This protein is commonly referred to as phosphatase. The PTEN protein acts as part of a chemical pathway that signals cells to stop dividing and triggers cells to undergo a form of programmed cell death called apoptosis. These functions prevent uncontrolled cell growth that can lead to the formation of tumours. Mutations in the PTEN gene lead to the production of a protein that does not function properly or does not work at all. The defective protein is unable to restrain cell division or signal abnormal cells to die, which can, as previously mentioned, contribute to the development of tumours.
Some gene mutations are acquired during a person’s lifetime and are present only in certain cells. These changes, which are called somatic mutations, are not inherited. Somatic mutations in the PTEN gene are among the most common genetic changes found in human cancers and they occur only in tumour cells. Mutations in the PTEN gene result in an altered protein that has lost its ability to suppress the development of tumours. This loss likely permits certain cells to divided uncontrollably, thereby allowing the growth of cancers. The PTEN gene is located on chromosome 10, as depicted in the image below.
Figure 7: The PTEN gene is located on the long (q) arm of chromosome 10 at position 23.3. More precisely, the PTEN gene is located from base pair 89,623,194 to base pair 89,728,531 on chromosome 10.
Utility: PTEN is one of the most commonly lost tumor suppressors in human cancer. During tumour development, mutations and deletions of PTEN occur that inactivate the activity of the protein made by the gene, which leads to increased growth and reduced cell death. While initial studies reported that PTEN gene mutations were rare in colorectal cancer, more recent reports have shown an approximate 18% incidence of PTEN mutations in somatic cells of colorectal tumours. More specifically, recent research (Loupakis, F, et al., Clin Oncol 2009) is speaking to the loss of PTEN expression in metastatic colorectal cancer as a predictor to resistance to treatment with anti-EGFR therapies such as cetuximab (erbitux) and panitumumab (vectibix). Identifying the genetic mutation in PTEN may be helpful in identifying patients with metastatic colorectal cancer who have a greater chance of benefiting from anti-egfr therapies. Metastatic tumours are identified as being either PTEN-positive (which means no genetic mutation in the PTEN gene) or PTEN-negative (which means there is a genetic mutation in PTEN). Patients who are PTEN-positive appear to have a greater response rate to anti-egfr therapies when compared to those patients who are PTEN-negative. This is, of course, assuming that the patients were also identified as being KRAS wild type, meaning no mutation in the KRAS gene. Researchers are, therefore, highlighting the importance of PTEN status in primary sporadic colorectal cancer, as a means of predicting anti-egfr response.
Tissue or Serum-Based: PTEN status is ascertained by performing testing on the tumour tissue.
Method & Timing of Measurement: DNA is isolated from the sample and the two copies of the PTEN gene are evaluated using a variety of methods and compared to the normal reference sequence for PTEN. Ideally, PTEN determination should be considered prior to receiving anti-egfr therapies but more research is required before its utility becomes part of treatment management protocol. PTEN determination is not currently part of routine practice in Canada but may receive support in the future to confirm anti-egfr candidacy.
vi. SEPTIN 9
Description/Definition: The Septin 9 gene provides instructions for making a protein called septin-9, which is part of a group of proteins called septins. Septins are involved in a process called cytokinesis, which is the step in cell division when the fluid inside the cell (cytoplasm) divides to form two separate cells. More importantly, the septin-9 protein acts as a tumor suppressor, which means that it regulates cell growth and keeps cells from dividing too fast or in an uncontrolled way. Methylation is a process in which a chemical group, called a methyl group, gets added to the DNA of the Septin-9 gene. When enough methyl groups are added to the DNA, the gene gets turned off and unable to control dividing cells, which may lead to cancer.
The septin-9 gene seems to be turned on (referred to as active) in cells throughout the body. Approximately 15 slightly different versions of the septin-9 protein may be produced from this gene. Alterations in the activity (or expression) of the Septin-9 gene are associated with certain types of cancers, such as colorectal cancer. The altered gene expression may enhance several cancer-related events such as cell division, cell movement and the development of new blood vessels that nourish a growing tumour. The SEPT9 gene is located on the long (q) arm of chromosome 17 at position 25. More precisely, the SEPT9 gene is located from base pair 72,827,196 to base pair 73,008,272 on chromosome 17, as demonstrated in the figure below.
Figure 8: The SEPT9 gene is located on the long (q) arm of chromosome 17 at position 25. More precisely, the SEPT9 gene is located from base pair 72,827,196 to base pair 73,008,272 on chromosome 17.
Utility: Changes in the Septin 9 gene can be detected through testing and these changes are present in an overwhelming percentage of colorectal cancer patients. Epigenomics has developed a blood-based test and is being marketed as a sanitary and non-invasive alternative to the fecal occult blood test (or FOBT) used in population-based screening programs across Canada (for patients at average risk of developing the disease). The test examines blood plasma for changes in the Septin-9 gene. Specifically, the test detects the presence of methylated Septin-9 DNA in blood, which has been strongly correlated with an increased risk of colorectal cancer (Weiss, G, et al., European Onc 2010). Although, it is not covered by provincial health-care plans, doctors may order the $445 blood test from Warnex, a private lab based in Laval, Quebec. Research performed by Epigenomics (manufacturer of the test) revealed that the blood test detected 67% of colon tumours found in 8,000 patients being screened with colonoscopies and the test’s sensitivity rate was 50% for stage I colon tumours, 80% for stages 2 and 3 and 90-100% for stage 4 tumours (PRESEPT Study). Patients who experience a positive Septin 9 test are then referred to colonoscopy for confirmation.
Tissue or Serum-Based: The test is performed on blood plasma so it is, therefore, serum-based and it appears to correlate with the presence of colorectal cancer. It can, as such, be used as an aid in the detection of this common cancer. Since lack of patient adherence to screening recommendations is the biggest hurdle to effective screening for colorectal cancer, experts believe that a blood test that is more convenient for the patients than stool tests and colonoscopy could help to get more people screened and thus be of medical and health economic benefit (Devos, t et al., Clinical Chemistry 2009).
Method & Timing of Measurement: The test could be administered as a first step in early detection of colorectal cancer. The assay is able to detect a marker (methylated Septin-9 DNA) in blood plasma that is specific to colorectal cancer. For this purpose the doctor takes blood drawn which is sent to a diagnostic laboratory for analysis. After approximately one week, the doctor receives the result. If the marker is detected in the sample, there is an increased likelihood of having colorectal cancer. In this case the patient should undergo a colonoscopy to confirm the diagnosis or as a first step in therapeutic treatment. The test would ideally be administered as part of a population-based screening program for people who are at average risk of developing colorectal cancer, starting at the age of 50 years or for those patients who are not willing to undergo colonoscopy despite being at increased risk of developing the disease. Regardless, a positive septin-9 test would ultimately urge patients to proceed directly to colonoscopy regardless of why or when they accessed the septin-9 test.
- TS (Thymidylate Synthase)
- DNA Topoisomerase I
Figure 1: http://ghr.nlm.nih.gov/gene/KRAS
Figure 3: http://ghr.nlm.nih.gov/gene/BRAF
Figure 7: http://ghr.nlm.nih.gov/gene/PTEN