(in colon)
CA 27.29
Testicular (nonseminomatous germ cell tumors)
(hepatocellular)
Chronic lymphocytic leukemia
AFP = alpha fetoprotein; B2M = beta-2 microglobulin; CEA = carcinoembryonic antigen; HCG = human chorionic gonadotropin; PSA = prostate specific antigen.
CML = chronic myeloid leukemia; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; PR = progesterone receptor.
ALK = anaplastic lymphoma kinase; BRAF = v-Raf murine sarcoma viral oncogene homolog B1; EGFR = epidermal growth factor receptor; KRas = V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog.
Sensitivity and Specificity
In order for a tumor marker to be clinically useful, it must have a high degree of sensitivity and specificity. That is, the presence of the marker should correlate with the presence of the tumor, and a negative test should indicate, with some certainty, that the patient does not have the cancer. Chapter 1: Definitions and Concepts describes the methodology in determining sensitivity and specificity and should be reviewed prior to reading this chapter. Knowledge of the sensitivity, specificity, and predictive values of tumor marker tests are particularly important when they are used to screen asymptomatic patients. If the tumor marker test is positive only in a portion of the patients that actually have the cancer or if the test is negative in patients who do have the disease, then diagnoses would be missed. In the case of malignant diseases, delay of the diagnosis until symptoms or other clinical findings appear may mean the difference between curable and incurable disease.
Outcomes studies evaluating the usefulness of tumor marker testing in asymptomatic individuals must result in decreased mortality rates due to the disease, not just establishment of a diagnosis. On the other hand, false-positive tests not only cause a high level of anxiety, they also typically result in the performance of very costly and sometimes invasive, additional diagnostic tests. Due to these limitations, the only tumor marker routinely used to screen for malignancies is prostate specific antigen (PSA). Although this is the single example, the use of PSA alone for prostate cancer screening is declining because it does not appear to reduce mortality.2
Sensitivity and specificity are also important when tumor marker tests are used to monitor for recurrent disease in patients who have previously been treated for the cancer. A negative tumor marker test that is known to have a high degree of specificity will give the patient, their family, and their clinicians a great deal of comfort and sense of security that the disease has been eliminated. If the test has a lower degree of sensitivity, then it is likely that other screening and diagnostic tests will need to be performed at regular intervals to monitor for disease recurrence. In some cases, the presence of a positive tumor marker may be indication enough to resume cancer treatment. A decision to initiate or resume treatment should be made when there is a high degree of certainty that there is actual disease present because most cancer treatments are associated with significant toxicity and a small, but appreciable, mortality risk. When tumor markers are used to assess the extent of disease or the presence of specific tumor characteristics (e.g., HER2/neu), the quantitative sensitivity may also be important in determining prognosis, appropriate diagnostic tests, and treatment options. Genetic mutational testing is a dichotomous endpoint that is present or not present; however, depending on the quality of tissue and type of testing the sensitivity (false negatives) can be affected.
Accessibility
If a tumor marker test is to be used to screen asymptomatic individuals for cancer, both the individuals and their clinicians are more likely to include them if they do not necessitate painful, risky, or lengthy procedures to obtain the necessary fluid or tissue. Most clinicians request and patients willingly provide samples of blood, urine, or sputum in the course of regular physical examinations. However, if a test requires biopsy of other tissues or involves procedures that are associated with a significant risk of morbidity, patients and clinicians are likely only to consent to or include them in physical examinations if there is a high likelihood—or other evidence that supports the presence—of the disease. Tumor markers that are obtained from tumor tissue directly are obtained at the time of diagnosis with the original tissue.
Widespread screening of asymptomatic individuals with a tumor marker test can be quite expensive. It is not surprising that insurance companies, health plans, and health policy decision-makers are also more likely to support the inclusion of these tests during routine physical examinations or other screening programs if health economic evaluations demonstrate that they may result in lower overall treatment costs and a positive benefit to society, such as prolongation of the patient’s productivity.
Prostate Specific Antigen
Standard reference range: 0–4.0 ng/mL
Prostate specific antigen (PSA) is a protein produced by both malignant and normal (benign) prostate tissue that is secreted into the blood. The role of PSA in the screening, diagnosis, and monitoring of treatment response of patients with prostate cancer is reviewed in Chapter 22: Common Medical Disorders of Aging Males—Clinical and Laboratory Test Monitoring.
Carcinoembryonic Antigen
Normal range: <2.5 ng/mL nonsmokers; <5.0 ng/mL smokers
Carcinoembryonic antigen (CEA) is a protein that is found in fetal intestine, pancreas, and liver. In healthy adults, the level of this protein is usually less than 2.5 ng/mL. Serum CEA levels are frequently elevated in patients with colon, breast, gastric, thyroid, or pancreatic carcinomas and a variety of nonmalignant conditions including hepatic cirrhosis, hepatitis, pancreatitis, peptic ulcer disease, hypothyroidism, ulcerative colitis and Crohn disease. Occasionally, CEA is also elevated in patients with lung cancer. Carcinoembryonic antigen levels are usually modestly increased in individuals who smoke, and the normal serum level in these individuals is usually considered to be less than 5.0 ng/mL. Nonmalignant conditions are usually not associated with CEA levels greater than 10 ng/mL. However, many patients with malignant conditions will have CEA levels that greatly exceed 10 ng/mL.
Blood samples for CEA testing preferably should be obtained in a red top tube. Following separation of the serum (or plasma), the specimen can be refrigerated if it is to be assayed within 24 hours or frozen at –20°C if the specimen is to be assayed later. Immunoassays from different manufacturers may provide different values, and, therefore, the same laboratory and assay method should be used whenever possible for repeat testing in an individual patient.
Carcinoembryonic antigen is most commonly used in the assessment of colon cancer. Unfortunately, this test does not have adequate sensitivity or specificity to make it a useful screening test for asymptomatic individuals. It may be elevated in a wide variety of conditions as noted above and may be negative in patients with widely metastatic disease. It is most commonly used in monitoring patients with a known history of colon cancer.2 Following detection of early stage colon cancer by screening tests such as fecal occult blood and colonoscopy or sigmoidoscopy with biopsy confirmation of suspicious areas, a baseline serum CEA level is usually measured to assess if the tumor produces excessive amounts of CEA. If the CEA level is grossly increased, then the CEA level may be used to monitor the success of treatment or for evidence of tumor recurrence following successful treatment.
The CEA level also may provide some information on a patient’s prognosis.3,4 The elevation of CEA level may relate to the extent of disease (stage), which often correlates with overall survival. Following surgical removal of a colon cancer, the CEA level should return to normal (less than 2.5 ng/mL) within 4–6 weeks.5 If the CEA level remains elevated beyond this point, it may indicate that either residual primary tumor or metastases are still present.
In early stage colon cancer (stages II and III), CEA levels should be followed every 3 months for at least 3 years after diagnosis once the adjuvant chemotherapy regimen ends.6 The CEA level should decline to below the 5 ng/mL level within 1 month following surgery if all tumor was successfully removed.5 If the CEA remains elevated, there is a high likelihood that the tumor will recur, and many surgeons would even consider a second-look surgery at that time for identification and removal of residual disease.7 Rising CEA levels mandate evaluation of the patient for metastatic disease. In patients with metastatic disease CEA levels should be monitored at the start of therapy and then every 1–3 months during therapy.8 Rising levels may indicate therapy failure, though increasing levels may result from chemotherapy at the beginning of treatment and require careful evaluation.8,9 When CEA levels are monitored in conjunction with other followup tests including CT scans of the liver and colonoscopy, several studies have reported improved overall survival and other benefits, including cost-effectiveness, that are attributable to earlier detection of recurrent disease.8,10
Carcinoembryonic antigen may also be used to monitor breast cancer patients with metastatic disease. The American Society of Clinical Oncology (ASCO) guidelines for use of tumor markers in breast cancer state that CEA levels in combination with imaging, medical history, and physical exam may indicate treatment failure and prompt evaluation for worsening of disease.11 Rising CEA levels alone should not be used to monitor treatment efficacy. Unlike colon cancer, monitoring of CEA levels in early stage breast cancer (stages I to III) is not recommended after a patient has received primary therapy. (See Minicase 1.)
A Case of Elevated CEA Levels
PHIL L., A 64-YEAR-OLD WHITE MALE, presents to the clinic with a 6-week history of worsening diarrhea (five to six stools a day), pain in his right upper quadrant, and general gastrointestinal discomfort. Additional past medical history includes hypercholesterolemia for the past 5 years. Medications include simvastatin 20 mg daily. He drinks one to two glasses of wine a day and has a 30 pack/year history of smoking.
A review of systems revealed lethargy and slight confusion but no apparent distress. Vital signs showed a sitting BP of 125/75 mm Hg (standing BP not measured), a regular heart rate of 86 beats per minute, and a rapid and shallow respiratory rate of 36 breaths per minute. Phil L.’s physical examination was pertinent for signs of dehydration (poor skin turgor). Laboratory values are drawn. They are unremarkable, except for serum sodium 153 mEq/L (136–142 mEq/L), serum creatinine 1.7 mg/dL (0.6–1.2 mg/dL); and BUN 45 mg/dL (8–23 mg/dL). The decision is made to admit Phil L. based on his dehydration and worsening diarrhea. Additional laboratory values are drawn in the hospital and include a CEA level of 27 ng/mL.
Question: What is the most likely cause of Phil L.’s fluid status? How is the CEA level interpreted in relation to colon cancer? Should any other laboratory or imaging tests be obtained to further assess his cancer status?
Discussion: Phil L. most likely has a malignant tumor in his colon. Common signs and symptoms of colon cancer include pain and a change in bowel habits, which result from the tumor blocking part of the colonic lumen and interfering with normal colonic function. This can lead to the severe diarrhea and dehydration as seen in Phil L.
Although other nonmalignant conditions and smoking are also associated with increased CEA levels, levels greater than 10 ng/mL indicate a high likelihood of cancer. An elevated CEA level alone is not enough to make a diagnosis of colon cancer and a complete workup including CT scans and a tissue diagnosis will need to be obtained prior to therapy. Additional laboratory values that may be useful would be CA 19-9 levels and a complete hepatic panel to assess for metastatic disease.
The CEA level may be used in Phil L. to monitor the success of treatment, to check for evidence of tumor recurrence following primary treatment, and to provide some indication of his prognosis. Following successful surgery the CEA level should return to normal (less than 2.5 ng/mL) within 4–6 weeks. Depending on the stage of disease, Phil L. will have his CEA levels followed periodically to assess for disease recurrence or progression of metastatic disease.
CA 15-3 Antigen
Normal range: <30 units/mL
CA 15-3 (cancer antigen 15-3) is defined by an assay using monoclonal antibodies directed against circulating mucin antigen shed from human breast cancer. In addition to elevation in the serum of many women with breast cancer, it may also be elevated in lung cancer and other nonmalignant conditions including liver and breast disorders. Elevated CA 15-3 has been demonstrated to be a poor prognostic factor in early stage breast cancer, but the test is not sensitive enough to use as a screening test for early stage breast cancer.12 This test is used in combination with imaging studies, physical examination, and medical history to monitor response to treatment in women with metastatic disease where no other reasonable measure of disease is feasible.11 (See Minicase 2.)
A Case of Utilizing Tumor Markers for Breast Cancer
SARAH H., A 41-YEAR-OLD WHITE FEMALE, is recently diagnosed with breast cancer. She presented for her first scheduled routine mammogram, and a small lump was detected in her left breast. A fine-needle biopsy was done, and the lump was found to be positive for breast cancer. A complete workup determines that this is local disease and, she is diagnosed with stage II breast cancer. Additional medical history is unremarkable, and she only takes seasonal allergy medicine and drospirenone/ethinyl estradiol oral contraceptives.
A review of systems is noncontributory. Her physical examination was pertinent for a small lump palpable on the left breast near her nipple. Her cancer is evaluated for the presence of tumor markers, and the pathology shows ER/PR = positive and HER2 = 2+ on IHC.
Question: How will these markers be evaluated and used to make treatment decisions in Sarah H.? Are there any other tumor markers or tests you would recommend to be performed on Sarah H.?
Discussion: The two most important tumor markers in determining prognosis and treatment decisions are ER/PR status and HER2 status and both were performed on Sarah H.
Her ER/PR receptors were found to be positive. There are many ways to report ER/PR status with most being determined by IHC. Since the presence of even small amounts of ER/PR have been correlated with prognosis and the need for hormonal therapy, ER/PR status is commonly reported as either positive or negative. Since Sarah H.’s ER/PR status is positive, she will benefit from hormonal therapy that targets the ER receptor and likely be offered 5 years of tamoxifen based on her premenopausal status. The use of RT-PCR in determining ER/PR status could be done to confirm her ER/PR status.
Sarah H. also has her HER2 status reported. Her value was 2+ as determined by IHC. This value is in the inconclusive range. Since HER2 status is critical in determining the benefit from anti-HER2 therapies (e.g., trastuzumab and lapatinib), inconclusive values require further workup. The confirmatory test that should be performed is a FISH assay. This test measures both the number of HER2 gene copies and provides a ratio of HER2/CEP 17 (also called FISH ratio). A positive test for HER2 gene amplification is a gene copy number greater than 6 or a FISH ratio >2.2.
Sarah H. will need to have this test performed. If positive, she will be offered trastuzumab as part of her adjuvant therapy. A positive result may also dictate part of her chemotherapy regimen; anthracyclines generally are recommended in HER2 positive patients. If Sarah H. has a negative FISH test for HER2, then she will not receive adjuvant therapy and will instead receive a standard chemotherapy regimen followed by hormonal therapy.
Additional markers such as CEA, CA 15-3, and CA 27.29 would not be useful in following Sarah H. since she does not have metastatic disease and these markers are only useful for determining progressive disease during treatment for metastatic breast cancer.
Normal range <38 units/mL
CA 27.29 (cancer antigen 27.29) is also defined by an assay using a monoclonal antibody that detects circulating mucin antigen in blood.11 It is a newer test than CA 15-3 but has the same clinical indications. CA 27.29 is used only in combination with other clinical factors such as imaging studies, physical examination, and medical history to monitor response to treatment in patients with metastatic breast cancer but is not useful as a screening test or for the detection of recurrence after primary therapy in early stage disease.11
CA 125 Antigen
Normal range: <35 units/mL
The CA 125 antigen (cancer antigen 125) is a protein, which is usually found on cells that line the pelvic organs and peritoneum. It may also be detected in the blood of women with ovarian cancer and those with adenocarcinoma of the cervix or fallopian tubes. It may be elevated in nonmalignant conditions including endometriosis, ovarian cysts, liver disease, and pregnancy, and occasionally in many other types of cancer.13 It is not, however, elevated by mucinous epithelial carcinomas of the ovaries. Levels of CA 125 also increase during menstruation and are lower at the luteal phase of the cycle.14 Levels are lower in women who use systemic contraceptives and also decline following menopause.15
CA 125 is assessed using a blood sample collected in a red top tube. The sample should be refrigerated within 2 hours of collection. The level of CA 125 in the serum has been reported to correlate with the likelihood of malignancy, with levels greater than 65 units/mL strongly associated with the presence of a malignancy. However, they should not be considered diagnostic.16,17 Several studies evaluating serial levels of CA 125 in healthy women have shown that serum levels may start to rise 1–5 years before the detection of ovarian cancer.16,18 It does not, however, have sufficient sensitivity to be recommended as a routine screening test for ovarian cancer in asymptomatic women. The sensitivity in early stage ovarian cancer (before symptoms are usually evident) is believed to be less than 60%; thus, many cases would not be detected.19 Using CA 125 levels with other tests such as transvaginal ultrasound has been investigated to increase the utility of CA 125. However, using transvaginal ultrasound in patients with elevated CA 125 levels does not appear to increase the detection of early tumors and the routine use of the combination is not recommended.20 Some advocate that rising serial CA 125 levels could be used as a trigger to do more extensive (and often costly) screening tests in high-risk women; this approach has not proven beneficial and may result in unacceptable morbidity in women at average risk for ovarian cancer.20
Most often CA 125 is measured to monitor for evidence of disease recurrence or residual disease in women who have undergone surgical resection of ovarian cancer.13 This use is efficacious in women whose tumors expressed CA 125 prior to surgery. For women who have undergone a tumor debulking operation prior to chemotherapy, a level measured approximately 3 weeks after surgery correlates with the amount of residual tumor mass and is predictive of overall survival.21 Serial levels during and following chemotherapy are used to monitor response to treatment, disease progression, and prognosis. However, many women, with CA 125 levels that have returned to the normal reference range during treatment still have residual disease if a second-look laparotomy is done to pathologically evaluate the disease.22 A more rapid decline of serum CA 125 during treatment has been associated with a more favorable prognosis.13,23,24 Nadir values less than 10 units/mL predict improved survival and increases in CA 125 from the nadir (even when below 35 units/mL) may be used to predict disease progression.22,25 Failure of the CA 125 level to decline may also be used to identify tumors that are not responding to chemotherapy and an increase usually indicates progression.25 However, a large European trial in over 1400 women failed to demonstrate an improvement in survival in treating women based on rising CA 125 levels alone.26 Additional trials are ongoing to confirm these results. Subsequently, rising CA 125 levels, without any other evidence of disease, requires careful clinical interpretation to determine if patients require treatment interventions.
Human Chorionic Gonadotropin
Normal range: serum <5 million International Units/mL
Human chorionic gonadotropin (HCG) is a glycoprotein consisting of alpha and beta subunits that is normally produced by the placenta during pregnancy.27 Elevations in nonpregnant females and in males requires workup for malignant conditions. The beta subunit is most commonly used as the determinant in both serum as a tumor marker and in urine tests for pregnancy. Human chorionic gonadotropin is also commonly produced by tumors of germ cell origin including mixed germ cell or pure choriocarcinoma, tumors of the ovaries and testis, extragonadal tumors of germ cell origin, and gestational trophoblastic disease (e.g., hydatidiform mole). Occasionally islet cell tumors and gastric, colon, pancreas, liver, and breast carcinomas also produce HCG. Patients with trophoblastic disease often produce irregular forms of HCG that may or may not be recognized by the various automated assays and false-positive HCG immunoreactivity has also been reported. Newer highly specific and highly sensitive immunoassays have improved the reliability of this test. Radioimmunoassays and the DPC Immulite® HCG test have been reported to have the greatest accuracy.
In patients with testicular cancer, elevated levels of HCG may be present with either seminomatous (1% to 25%) or nonseminomatous disease (10% to 70%), depending on the stage of disease, so the test is not sensitive enough to be used as a screening tool for asymptomatic patients.28 Human chorionic gonadotropin has an important prognostic role with levels greater than 50,000 million International Units/mL indicating a poor prognosis in nonseminomatous disease.29 Most frequently, HCG is used to monitor response to therapy (i.e., an elevated level is evidence of residual disease following surgery) and to monitor for evidence of disease progression or recurrence during or after treatment.30 Human chorionic gonadotropin has a half-life of only 18–36 hours, so serum levels decline rapidly following therapeutic interventions and failure to do so may indicate residual disease.27,28,31
Normal range: <37 units/mL
CA 19-9 (cancer antigen 19-9) is an oncofetal antigen expressed by several cancers including pancreatic (71% to 93% of cases), gastric (21% to 42% of cases), and colon (20% to 40% of cases) carcinomas. Serum for this test is collected in a red top tube, and the sample is frozen for shipping for analysis. The sensitivity of the test is insufficient to be useful as a screening test for early stage diseases. It was originally developed for colon cancer monitoring but is no longer recommended.6 It is primarily used in pancreatic cancer to help discriminate benign pancreatic disease from cancer, to monitor for disease recurrence, and to assess the response to treatment interventions.6,31 CA 19-9 levels have been used to evaluate the effectiveness of a chemotherapy regimen with rising values indicating a shorter patient survival and the possible need to change chemotherapy regimens.32 An elevated CA 19-9 level is a poor prognostic factor in patients with inoperable pancreatic cancer.33
Alpha Fetoprotein
Normal range: <20 ng/mL
Alpha fetoprotein (AFP) is a glycoprotein made in the liver, gastrointestinal tract, and fetal yolk sac. It is found in high concentrations in the serum during fetal development (~3 mg/mL), and following birth it declines rapidly to <20 ng/mL. Serum for AFP evaluation should be collected in a red top tube and refrigerated until assayed using radioimmunoassay. It is elevated in about 70% of patients with hepatocellular carcinoma, 50% to 70% of patients with testicular nonseminomatous germ cell tumors, and occasionally in patients with other tumors such as pancreatic, gastric, lung, and colon cancers.29 Nonmalignant conditions that may be associated with increased levels of AFP include pregnancy, hepatitis, and cirrhosis. In patients with nonseminomatous germ cell tumors, the level of AFP serum concentrations seems to correlate with the stage of the disease.27,29 In some parts of the world, AFP is used as a screening test for hepatocellular carcinoma in patients who are positive for HBsAg, and, therefore, are at increased risk for hepatocellular carcinoma. In the United States, however, AFP is used primarily to assist in the diagnosis of hepatocellular carcinoma. Alpha fetoprotein levels greater than 1000 ng/mL are common in patients with hepatocellular carcinoma.34,35
Alpha fetoprotein levels are also used to monitor patients with both hepatocellular carcinoma and germ cell tumors for disease progression or recurrence and to assess the impact of treatment interventions. The serum half-life of AFP is 5–7 days, and usually an elevation of the serum level for more than 7 days following surgery is an indication that residual disease was left behind.27,31 Following successful treatment for nonseminomatous germ cell tumors of the testis, HCG, and AFP are repeated every 1 or 2 months during the first year, every 2 or 3 months during the second year, and less frequently thereafter along with physical exams and chest x-rays.27,29 Increases in these serum tests are considered an indication for further treatment such as chemotherapy. Rising levels in patients receiving chemotherapy indicate that therapy should be changed, whereas declining levels predict a more favorable outcome.27,29
Beta-2 Microglobulin
Normal range <2.5 mcg/mL
Beta-2 microglobulin (B2M) is a protein found on the surface of lymphocytes as well as in small quantities in the blood and urine. Elevations of B2M may be seen in lymphoproliferative disorders including multiple (plasma cell) myeloma and lymphoma. B2M is renally excreted and may be elevated in nonmalignant conditions such as renal failure.36
Measurement of serum B2M is most commonly done in the workup of multiple myeloma and is an important part of the staging and prognosis for that disease. Additionally, B2M will be used to follow multiple myeloma patients for treatment efficacy with increases in B2M potentially indicating progressive disease.36 Patients with serum B2M levels ≥5.5 mcg/mL are diagnosed as stage III patients and have a median survival of 29 months.37
Estrogen and Progesterone Receptor Assays
The levels of estrogen receptor (ER) and progesterone receptor (PR) in biopsy tissue from breast cancers predict both the natural history of the disease and the likelihood that the tumor will respond to hormonal manipulations. This test is not a blood test but requires tissue from the cancer obtained by a relatively noninvasive biopsy. Estrogen receptor status is also a prognostic factor with ER-negative tumors having a worse prognosis than ER-positive ones. For over 30 years, it has been the standard of practice to evaluate breast cancer tissue for these protein receptors and to use that information in directing therapeutic interventions. The relative concentration of hormone receptors can be determined using very small amounts of tumor tissue.
The current standard of practice is to measure each protein using immunohistochemistry (IHC); this method detects protein expression through an antibody-antigen interaction.38 Although the method (e.g., antibody) used can vary, the biopsy is read by pathologists with the results reported as a percent positive cells. If greater than or equal to 1% of cells are positive, one is considered to have ER- or PR-positive disease.38 Biopsies scored 1% to 10% may be considered “weakly” positive, and risks and benefits of hormonal therapy should be discussed with patients. Because of the variety of methods to evaluate IHC staining and the intra-/inter-observer variability, newer methods of measuring ER and PR status are under investigation including the use of reverse-transcriptase polymerase chain reaction (RT-PCR), which measures gene expression of ERs in tissue. Classification of ER- and PR- positive tumors are based on cutoff points of 6.5 and 5.5 units, respectively.39 This test has demonstrated statistically significant superiority over IHC in predicting relapse in tamoxifen-treated, ER-positive patients in one retrospective trial.39 Further validation of the test is needed before it becomes routinely used in clinical practice.11
Positive ER levels correlate with response to hormonal therapies including removal of the ovaries in premenopausal women or administration of an antiestrogen, such as tamoxifen, or an aromatase inhibitor such as anastrozole.38 In addition, ER content in tumor biopsies correlates with benefit from adjuvant hormonal therapy following surgical removal of the tumor. After 15 years of followup in ER-positive breast cancer patients, tamoxifen decreased mortality by 9% in women who received 5 years of therapy.40
Human Epidermal Growth Factor Receptor 2
Human epidermal growth factor receptor 2, HER2/neu, (HER2) is a transmembrane glycoprotein member of the epidermal growth factor receptor (EGFR) family with intracellular tyrosine kinase activity.41 This group of receptors functions in the growth and control of many normal cells as well as malignant cells. The gene that encodes for HER2 is c-erb B2.41 About 20% of samples from human breast cancers exhibit amplification of c-erb B2 or overexpression of HER2.42
There are many potential clinical applications based on HER2 status in breast cancer: (1) studies have described the role of HER2 in the prognosis of patients with breast cancer, with poor prognosis seen in overexpressers; (2) HER2 status may predict responsiveness to certain chemotherapy (e.g., anthracycline, taxanes); (3) HER2 status may be used to predict resistance to other therapies (e.g., tamoxifen); and (4) HER2 status will predict benefit from anti-HER2 therapies such as trastuzumab and lapatinib.43–47
However, the considerable variability in study design and the well-recognized heterogeneity of the disease itself have made interpretation difficult, and HER-status alone should not determine whether or not a woman should receive specific adjuvant therapy or whether endocrine therapy should be used. 11 The authors did conclude that the benefit of anthracycline therapy in the adjuvant setting is greatest in HER2 positive tumors and that determining the benefit of taxane-based therapy is inconclusive at this time.11
It is well-established that HER2 overexpression is predictive of a response to treatment with trastuzumab (Herceptin®), a monoclonal antibody against HER2, and lapatinib (Tykerb®), a tyrosine kinase inhibitor of human epidermal growth factor receptor 1 (HER1) and HER2.44,45 Therefore, it is necessary to evaluate all invasive breast cancers for HER2 status in order to select appropriate patients for these anti-HER2 therapies.48
Though a portion of the HER2 receptor can dissociate from the cell and be detected in the serum, biopsies of the tumor are routinely used to evaluate HER2 status. It can be measured for overexpression of the protein by IHC or by gene amplification, most commonly by using fluorescence in situ hybridization (FISH) assays.48 Several commercial assays have been recommended to aid in the selection of patients for anti-HER2 therapy. Immunohistochemistry assays assess for the overexpression of the HER2 protein and a score of 0, 1+, 2+, or 3+ is reported. Clinical trials have demonstrated that those with a score of 0 or 1+ should be considered HER2 negative and do not benefit from anti-HER2 therapy, and those that are 3+ are HER2 positive and benefit from therapy.44,48 A score of 2+ should be considered inconclusive and requires further evaluation with a FISH assay.
The FISH assay can be used as the initial test for HER2 positivity and is preferred by some groups due to decreased variability and increased ability to predict efficacy of therapies aimed at the HER2 receptor.48 The FISH assay measures both the number of gene copies of HER2 gene as well as provides a ratio of HER2/CEP 17 (also called FISH ratio). A positive test for HER2 gene amplification is a gene copy number greater than 4 or a FISH ratio greater than 2.0. HER2 negative tumors are defined as a gene copy number less than 4 or FISH ratio less than 2.0, and FISH ratios between 1.8–2.2 are inconclusive. Additional cells should be scored and the results compared.48 Only patients with FISH-positive tumors derive benefit from anti-HER2 therapy.48
In summary, the routine testing for HER2 with either IHC or FISH is recommended in all patients with invasive breast cancer, with FISH as the preferred method.11,48 Patients who are HER2 positive benefit from trastuzumab in the adjuvant setting and both trastuzumab and lapatinib in the metastatic setting.44,45,49 The use of HER2 testing to determine benefit of additional therapies (e.g., tamoxifen, anthracyclines, taxanes) is inconclusive at this time.11,48
BCR-ABL
The identification of tumor markers in the pathogenesis of malignancy has led to the development of therapeutic strategies that specifically target the cause of the malignancy. By definition patients with chronic myelogenous leukemia (CML) possess the Philadelphia (Ph) chromosome that indicates the presence of the BCR-ABL fusion gene.50,51 The BCR-ABL fusion gene can also be found in acute lymphoblastic leukemia and rarely in acute myeloid leukemia. ABL and BCR are normally found on chromosomes 9 and 22, respectively. The translocation of ABL and BCR t(9;22) in which both genes are truncated forming the characteristic BCR-ABL fusion gene on the Ph chromosome is diagnostic for CML and is present in all patients with the disease by definition.50,51 The BCR-ABL gene encodes a protein with deregulated tyrosine kinase activity that has become the primary target for treating CML.
The Ph chromosome can be tested by the following three methods50,51: (1) conventional cytogenetic testing, in which bone marrow cells are aspirated and the individual chromosomes are examined for the presence of the Ph chromosome (the term cytogenetic remission has been developed to describe the elimination of the Ph chromosome on testing by this method after treatment); (2) FISH testing, which can be done on either blood or bone marrow cells (genetic probes are utilized to look for abnormal cells that contain the BCR-ABL gene); and (3) RT-PCR testing, which is the most sensitive test for monitoring response to therapy and counts the number of cells that contain the BCR-ABL gene (it can be done on either blood or bone marrow cells). Testing with RT-PCR is referred to as molecular monitoring and responses are called molecular responses. Table 19-4 lists the response criteria for CML using cytogenetic and molecular monitoring.50,51
Therapies (e.g., imatinib, nilotinib, dasatinib) have been developed that target the abnormal tyrosine kinase activity of the BCR-ABL gene.52 As mentioned, efficacy is monitored by the elimination of the Ph chromosome (cytogenetic or molecular) and detection of increasing amounts of the BCR-ABL fusion gene often require adjustments in therapy.
Several mutations in the BCR-ABL gene have been identified that may predict response to the currently available tyrosine kinase inhibitors. All CML patients should be tested for a threonine-to-isoleucine mutation at codon 315 (T315I) and may be referred for a stem cell transplantation since all three currently approved agents are inactive against this BCR-ABL mutation.51 In patients who do not respond or relapse on initial therapy, additional BCR-ABL mutations should be tested for and may be useful in selecting the best second-line treatment options.51
Epidermal Growth Factor Receptor
Epidermal growth factor receptor (EGFR) (human epidermal growth factor receptor, HER1, c-erb B1) is a transmembrane glycoprotein member of the EGFR family with intracellular tyrosine kinase activity (TK) (same family as HER2). When EGFR receptors are activated, they support tumor growth by influencing cell motility, adhesion, invasion, survival, and angiogenesis. The gene that encodes for EGFR can have activating mutations in exon 18 through 21, but the ones of most interest influence the sensitivity or resistance to erlotinib (an EGFR tyrosine kinase inhibitor [TKi]). Class I mutations in exon 19 account for approximately 44% of all EGFR TK-activating mutations and a point mutation in exon 21 accounts for approximately 41% of EGFR TK-activating mutations. These mutations are most commonly found in adenocarcinoma of the lung from nonsmokers. They are also more common in Asians and females, which matches patient subset analysis from clinical trials with erlotinib. Approximately 15% of all U.S. patients with adenocarcinoma of the lung have one of these activating mutations. A secondary mutation in exon 20 (T790M) has been found to convey resistance to the current EGFR TKi treatments. Recent recommendations state that all patients who are being considered for first-line therapy with an EGFR TKi should have mutational analysis run on their tumor tissue.53 Epidermal growth factor receptor mutational analysis can be performed with a number of different assays; however, standard testing for patient care includes PCR amplification and genetic sequencing of exon 18 through 21.54
V-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog
V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRas) is an intracellular GTPase that plays an important role in signal transduction. Functionally, it works like an on/off switch that is downstream of a number of cell surface receptors including EGFR. When turned on, it conveys proliferative, growth, and survival signals; in the normal setting it turns off after conveying the activation signal. Mutated or oncogenic Ras performs the same function, but mutations in exon 1 (codons 12 and 13) lead to a permanently active Ras. Oncogenic Ras is found in 20% to 25% of all human tumors and in up to 90% of pancreatic cancers. This is obviously a target for drug development, but as of today no therapy has reached the market that inhibits this signal. It is, however, routinely used to select drug therapy, with patients having wild type (WT) (nonmutated) tumors more likely to respond to therapy. Mutated KRas is present in approximately 40% of colorectal tumors, where it conveys resistance to cetuximab and panitumumab. Current national guidelines and many payers require KRas mutational testing before giving either of these anti-EGFR monoclonal antibodies for colorectal cancer. Real-time PCR methods with fluorescent probes to common mutations in codon 12 and 13 are commonly used to determine if a KRas mutation exists; however, there are other methods including direct gene sequencing that can be used.55
V-Raf Murine Sarcoma Viral Oncogene Homolog B1
V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that plays an important role in signal transduction. It has activating mutations in 7% to 8% of all cancers and 40% to 60% of melanomas. The most common mutation (approximately 90%) is a glutamic acid for valine substitution at amino acid 600, which is known as the V600E mutation.56 This mutation means that the kinase is always turned, signaling downstream partners in the mitogen-activated protein (MAP) kinase pathway. Vemurafenib, a drug specifically designed to inhibit the mutated BRAF, is now available to treat melanoma in patients whose tumor contains this mutation. Concurrent with the approval of vemurafenib, the cobas® 4800 BRAF V600 mutation test was introduced, which utilizes real-time PCR to identify the V600E mutation in tumors. The prescribing information requires that the test be performed and the result be positive for the mutation before using the drug.56
Anaplastic lymphoma kinase (ALK) (EML4-ALK) is a fusion gene formed when the echinoderm microtubule-associated, protein-like 4 (EML4) is fused to ALK. The abnormal fusion protein promotes malignant cancer cell growth. This has recently become clinically relevant because a new drug, crizotinib, is highly effective for patients with lung cancer whose tumors contain this translocation. The mutation most commonly occurs in nonsmokers with lung adenocarcinoma, and it rarely occurs in combination with KRas or EGFR mutations. Though the mutation is found only in 2% to 7% of non-small-cell lung cancer patients, it should be routinely tested for due to significant improvement in outcomes with crizotinib treatment that targets this mutation. The rearrangement/fusion is usually detected with FISH; however, PCR and IHC can be used to identify the fusion gene or its protein product respectively.57,58
SUMMARY
In order to be clinically useful as a screening tool in asymptomatic individuals, tumor markers should be both sensitive and specific. Unfortunately, most of the tumor markers identified to date lack the sensitivity to be used in this capacity. In addition, many nonmalignant conditions cause elevations of these markers. Currently, only PSA is in widespread use as a screening tool when used along with the results of a digital rectal exam. Tumor markers are valuable to monitor for disease recurrence in patients who have undergone definitive surgery for cancers or to assess a patient’s response to chemotherapy or other treatment interventions. In these situations, serial measurements of tests such as PSA for prostate cancer, CEA for colon cancer, HCG and AFP for testicular cancer, and CA 125 for ovarian cancer are considered standards in the followup care of patients with these malignancies. Increasingly tumor markers are being used to choose appropriate therapeutic strategies. Some tumor markers such as HER2 and ER are used as indicators of tumor sensitivity to therapies that target those receptors. Others such as the BCR-ABL gene, found in CML patients, provide a specific target in which therapeutic strategies have been developed to inhibit the actual pathogenesis of the cancer.
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