OBJECTIVES

After completing this chapter, the reader should be able to

  • Define tumor markers, describe the characteristics of an ideal tumor marker, and discuss the usefulness of tumor markers in the diagnosis, staging, and treatment of malignant diseases

  • List malignant and nonmalignant conditions that may increase carcinoembryonic antigen levels and define the role of carcinoembryonic antigen in the management of colon cancer

  • Describe how cancer antigen 125 may be used to diagnose and monitor ovarian cancer

  • Describe how human chorionic gonadotropin and α-fetoprotein are used to diagnose and monitor germ cell tumors

  • Discuss the role of estrogen and progesterone receptors and human epidermal growth factor receptor 2 in determining treatment decisions for breast cancer

  • Outline the role of the BCR-ABL gene in the diagnosis and as a target for treatment in patients with chronic myelogenous leukemia

  • Describe how mutations in epidermal growth factor receptor, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, v-Raf murine sarcoma viral oncogene homolog B1, or anaplastic lymphoma kinase are used in determining treatment decisions for melanoma, lung cancer, and colorectal cancer

  • Review biomarkers for immunotherapy, including programmed death-ligand 1 and tumor mutation burden, and their role in treatment for various cancers

For most types of cancer, treatment is likely to be most successful if the diagnosis is made while the tumor mass is relatively small. Unfortunately, many common types of cancer (eg, carcinomas of the lung, breast, and colon) are frequently not diagnosed until the tumor burden is relatively large and the patient has developed symptoms related to the disease. As the search for more effective treatments for cancer has intensified, much effort and many resources have been dedicated to elucidating new methods of detecting cancers earlier while the tumor burden is low and the patient is asymptomatic. These efforts have led to improved radiologic and other diagnostic imaging and identification of biologic substances, which occur in relation to the tumor and can be detected even at low concentrations in the blood or other body fluids.

The term tumor marker is used to describe a wide range of proteins that are associated with various malignancies. Typically, these markers are either proteins that are produced by or in response to a specific type of tumor or other physiologic proteins that are produced by malignant cells in excess of the normal concentrations. In either case, the concentration of the marker usually correlates with the volume of tumor cells (eg, as the tumor grows or the number of malignant cells increases, the concentration of the marker also increases). In other cases, the presence of a biologic marker may be used to predict response to treatment (eg, the estrogen receptor [ER] or progesterone receptor [PR] in breast cancer) or monitor the effects of treatment. More recently, some tumor markers have been shown to be essential to the viability of tumor cells, and specific therapies have been developed that target these markers of disease. These tumor markers are often identified by genetic mutations, translocations, or amplification of genetic material.

This chapter describes tumor markers that are used clinically to detect cancers, monitor cancer burden, and help choose drug therapy as well as the laboratory methods used to measure them. In addition, the sensitivity, specificity, and factors that may interfere with evaluation of these tests are briefly discussed. For tumor markers that are widely used to screen for cancers, confirm a cancer diagnosis, or assess response to treatment, the clinical applications are described. More commonly, molecular markers are being discovered and driving therapy.

TUMOR MARKERS

Tumor markers may be found in the blood or other body fluids or may be measured directly in tumor tissues or lymph nodes. They can be grouped into three broad categories: (1) tumor-specific proteins, markers that are produced only by tumor cells, usually in response to genetic changes such as translocation of an oncogene, that contribute to the proliferation of the tumor, (2) protein expression typically isolated to embryonic development that can be expressed by cancer cells, and (3) proteins that are normally found in the body but are expressed or secreted at a much higher rate by malignant cells than normal cells.1 In addition to the laboratory tests described in this chapter, it also should be remembered that abnormalities in other commonly used laboratory tests may provide some evidence that a malignancy exists. However, they are not related to specific tumors. For example, suppression of blood counts may represent infiltration of the bone marrow by tumor cells. Increased uric acid and lactate dehydrogenase are frequently associated with large tumor burdens. Alkaline phosphatase is frequently elevated in patients with tumors of the biliary tract or bone. Occasionally, tumors may also produce hormones in excessive amounts, such as calcitonin or adrenocorticotropin. Common tumor markers are reviewed in this chapter, but many are not covered, including B-cell immunoglobulin gene rearrangement, bladder tumor antigen, chromogranin A, C-kit, gastrin, ROS1 gene rearrangment, thyroblobulin, and others. The intent of this chapter is to introduce common tumor markers; however, not every tumor marker is covered in detail, and progress in this field is rapidly moving forward.

Clinical Uses

Tumor markers are used for several purposes, including detection of occult cancers in asymptomatic individuals (eg, cancer screening and early detection), determining the relative extent or volume of disease (staging), estimating prognosis, predicting and assessing responsiveness to treatment, and monitoring for disease recurrence or progression.1 Table 21-1 lists many of the commonly used tumor markers that are used most often for screening and monitoring of response to a variety of treatments. Table 21-2 lists tumor markers with their clinical applications. The characteristics of an ideal tumor marker are somewhat dependent on the specific application. Normal values are provided, although laboratory reference ranges (normal values) may slightly differ, as will the interpretation of the laboratory value in individual patients. For example, rising levels of a tumor marker that are still in the normal range may indicate early tumor recurrence.

TABLE 21-1.

Serum Tumor Markers in Clinical Use

TUMOR MARKER

MALIGNANT DISEASE

SCREENING

DIAGNOSIS

STAGING OR PROGNOSIS

MONITORING TREATMENT OUTCOME OR DISEASE RECURRENCE

COMMENTS

PSA

Prostate carcinoma

X

X

X

Usually combined with digital rectal examination of the prostate for screening

Inflammatory disorders of the prostate, instrumentation of the genitourinary tract, and mechanical manipulation of the prostate by biopsy, transurethral resection of the prostate or prostatectomy may increase PSA

Certain medications may decrease PSA, including 5-α reductase inhibitors, NSAIDs, statins, and thiazide diuretics

Herbal products (eg, saw palmetto) may also decrease PSA

CEA

Colon, breast carcinoma, ovarian, pancreatic, and others

X (in colon)

X

Hepatic cirrhosis, hepatitis, pancreatitis, peptic ulcer disease, hypothyroidism, ulcerative colitis, or Crohn disease may elevate CEA

CA 15-3, CA 27.29

Breast carcinoma

X

X Metastatic disease only

Other cancers (eg, gastric, colorectal, lung), benign breast disease, and liver disease may all elevate levels

CA-125

Ovarian carcinoma

X

X

Endometriosis, ovarian cysts, liver disease, or pregnancy may elevate CA-125; in certain high-risk groups (strong family history) CA-125 in combination with ultrasound technology may be used to screen asymptomatic patients

hCG

Germ cell tumors of ovaries and testes; hydatidiform mole; trophoblastic tumors

X

X

X

Pregnancy, other types of cancer, or marijuana use may elevate hCG

CA 19-9

Pancreatic carcinoma

X

X

Pancreatitis, cirrhosis, gastric, and colon cancer may elevate CA 19-9

AFP

Hepatocellular carcinoma; testicular (nonseminomatous germ cell tumors)

X (hepatocellular carcinoma)

X

X

X

Pregnancy; hepatitis; cirrhosis; and pancreatic, gastric, lung, and colon cancers all can elevate AFP; some non-U.S. countries, which have a high incidence of hepatocellular cancer, use AFP to screen for hepatocellular cancer

B2M

Multiple (plasma cell) myeloma; chronic lymphocytic leukemia

X

X

Lymphomas, chronic lymphocytic leukemia, and renal failure may elevate

NSAIDs = nonsteroidal antiinflammatory drugs.

TABLE 21-2.

Tumor Markers Used to Personalize Treatment

TUMOR MARKER

MALIGNANT DISEASE

TEST OUTCOME OF INTEREST

IMPACT ON DRUG SELECTION

ALK

Lung

ALK rearrangement

ALK rearrangement predicts response to ALK-inhibitors (crizotinib, ceritinib, lorlatinib, alectinib, and brigatinib)

BCR-ABL translocation

CML or Ph+ ALL

BCR-ABL translocation

TKI therapy used in treatment (imatinib, bosutinib, dasatinib, nilotinib, ponatinib)

BRAF

Melanoma

Lung

Thyroid

BRAF V600E and V600K mutation

BRAF mutation predicts response to vemurafenib, dabrafenib, and trametinib

EGFR

Lung

Mutation in exon 19 or 21

Mutation predictive of response to EGFR TK inhibitors predicts response to afatinib, erlotinib, gefitinib, osimertinib, dacomitinib

ER/PR

Breast carcinoma

ER or PR positive disease

Hormonal therapy indicated in treatment (tamoxifen, fulvestrant, exemestane, letrozole)

KRas

Colorectal cancer

Mutation versus wild type (nonmutated)

EGFR monoclonal antibodies (cetuximab and panitumumab) only effective against wild-type

HER2

Breast carcinoma, gastric cancer

HER2 overexpression

HER2 monoclonal antibodies or TKI therapy may be indicated (ado-trastuzumab emtansine, fam-trastuzumab deruxtecan, lapatinib, neratininb, pertuzumab, trastuzumab, and tucatinib)

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

For a tumor marker to be clinically useful in screening for cancer, 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 result should indicate, with some certainty, that the patient does not have the cancer. Chapter 1 describes the methodology in determining sensitivity and specificity and should be reviewed before reading this chapter. Knowledge of the sensitivity, specificity, and predictive values of tumor marker tests is particularly important when they are used to screen asymptomatic patients. If a tumor marker test is positive only in a portion of patients who actually have cancer or if a test result 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 test results not only cause a high level of anxiety but also typically result in the performance of costly and sometimes invasive additional diagnostic tests. Because of 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 gives the patient, the family, and 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 actual disease is 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 (eg, 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-negative results) can be affected.

Accessibility

If a tumor marker test is to be used to screen asymptomatic individuals for cancer, both patients and 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.

Cost-Effectiveness

Widespread screening of asymptomatic individuals with a tumor marker test can be 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 a patient’s productivity.

Prostate-Specific Antigen

Standard reference range: 0 to 4 ng/mL (4 mcg/L)

Prostate-specific antigen (PSA) is a protein produced by both normal (benign) and malignant 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 25.

Carcinoembryonic Antigen

Normal range: <2.5 ng/mL nonsmokers (<2.5 mcg/L); <5 ng/mL smokers (5 mcg/L)

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 <2.5 ng/mL. CEA is a protein generally found in a fetus, and the protein diminishes at birth. 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 also is elevated in patients with lung cancer. CEA levels are usually modestly increased in individuals who smoke; the normal serum level in these individuals is usually considered to be <5 ng/mL. Nonmalignant conditions are usually not associated with CEA levels >10 ng/mL. However, many patients with malignant conditions 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; 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 previously 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 and further diagnostic workup, a baseline serum CEA level is usually measured to determine 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 after 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. After surgical removal of colon cancer, the CEA level should return to normal (<2.5 ng/mL) within 4 to 6 weeks.5 If the CEA level remains elevated beyond this point, it may indicate that either residual primary tumor or metastatic disease is present.

In early-stage colon cancer (stages II and III), CEA levels should be followed every 3 to 6 months for 5 years after the completion of all cycles of adjuvant chemotherapy.6 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 to 3 months during therapy.6 Rising levels may indicate therapy failure, although increasing levels may result from chemotherapy at the beginning of treatment and require careful evaluation.6,7 When CEA levels are monitored in conjunction with other follow-up tests, including computed tomography 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.6,8

Carcinoembryonic antigen may also be used to monitor patients with metastatic breast cancer. The American Society of Clinical Oncology 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.9 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 (Minicase 1).

CA 15-3

Normal range: <30 units/mL

Cancer antigen 15-3 (CA 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.10 This test is used in combination with imaging studies, physical examination, and medical history to monitor response to treatment in women with metastatic disease in whom no other reasonable measure of disease is feasible11,12 (Minicase 2).

CA 27.29

Normal range: <38 units/mL

Cancer antigen 27.29 (CA 27.29) is also defined by an assay using a monoclonal antibody that detects circulating mucin antigen in blood.9 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 it is not useful as a screening test or for detection of recurrence after primary therapy in early-stage disease.9

CA-125

Normal range: <35 units/mL

Cancer antigen 125 (CA-125 antigen) is a protein 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 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.11 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. Levels are lower in women who use systemic contraceptives and decline after menopause.11

A Case of Elevated Carcinoembryonic Antigen Levels

Phil L., a 64-year-old white man, 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. Patient has a history of stage I colon cancer, following resection, and no further treatment 2 years ago. 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 reveals lethargy and slight confusion but no apparent distress. Vital signs show a sitting blood pressure of 125/75 mm Hg (standing blood pressure not measured), a regular heart rate of 86 beats/min, and a rapid and shallow respiration rate of 36 breaths/min. His physical examination is pertinent for signs of dehydration (poor skin turgor). Laboratory values are drawn. They are unremarkable except for serum sodium (153 mEq/L), serum creatinine (1.7 mg/dL), and blood urea nitrogen (45 mg/dL). The decision is made to admit him based on his dehydration and worsening diarrhea. Additional laboratory values are drawn in the hospital, including a CEA level of 27 ng/mL.

QUESTION: What is the most likely cause of this patient’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 if he has a malignant tumor?

DISCUSSION: This patient most likely has a malignant tumor in his colon that has relapsed. 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 this patient.

Although other nonmalignant conditions and smoking also are associated with increased CEA levels, levels >10 ng/mL indicate a high likelihood of cancer. An elevated CEA level alone is not enough to make a diagnosis of colon cancer; a complete workup, including computed tomography scan and a tissue diagnosis, need to be obtained prior to therapy. Additional laboratory values that may be useful are CA 19-9 levels and a complete hepatic panel to assess for metastatic disease.

The CEA level may be used to monitor the success of treatment, check for evidence of tumor recurrence following primary treatment, and provide some indication of his prognosis.

A Case of Using Tumor Markers for Breast Cancer

Sarah H., a 41-year-old white woman, was recently diagnosed with breast cancer. She presents for her first scheduled routine mammogram, and a small lump is detected in her left breast. A fine-needle biopsy is performed, and the lump is 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; she only takes seasonal allergy medicine and drospirenone/ethinyl estradiol oral contraceptives.

A review of systems is noncontributory. Her physical examination is 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 this patient? Are there any other tumor markers or tests you would recommend be performed on her?

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 this patient. Her ER/PR receptors were found to be positive. There are many ways to report ER/PR status, with most being determined by IHC. Because the presence of even small amounts of ER/PR has been correlated with prognosis and the need for hormonal therapy, ER/PR status is commonly reported as either positive or negative. Because her ER/PR status is positive, she will benefit from hormonal therapy that targets the ER receptor. The use of RT-PCR in determining ER/PR status could be done to confirm her ER/PR status.

She also has her HER2 status reported. Her value is 2+ as determined by IHC. This value is in the inconclusive range. Because HER2 status is critical in determining the benefit from anti-HER2 therapies (eg, trastuzumab and lapatinib), inconclusive values require further workup. The confirmatory test that should be performed is a FISH assay. This test measures 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 >6 or a FISH ratio >2.2.

She should have this test performed. If positive, she will be offered trastuzumab as part of her adjuvant therapy. If she has a negative FISH test result for HER2, then she will not receive adjuvant therapy with trastuzumab and will instead receive a standard adjuvant chemotherapy regimen followed by hormonal therapy.

Additional markers, such as CEA, CA 15-3, and CA 27.29, would not be useful in following this patient because she does not have metastatic disease, and these markers only are useful for determining progressive disease during treatment for metastatic breast cancer.

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 >65 units/mL strongly associated with the presence of a malignancy. However, such levels should not be considered diagnostic.11,13 Several studies evaluating serial levels of CA-125 in healthy women have shown that serum levels may start to rise 1 to 5 years before the detection of ovarian cancer.11 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 <60%; thus, many cases would not be detected. Using CA-125 levels with other tests, such as transvaginal ultrasound, has been investigated to increase the use 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.11,14 However, recent data of the largest published prospective screening trial conducted to date demonstrated that using serial biomarker measurements doubled the number of screen-detected ovarian cancers.15 This study used serial CA-125 levels in conjunction with a risk algorithm and compared this to serial CA-125 levels alone. The investigators concluded that using the algorithm in combination with serial CA-125 levels increased the number of cancers detected at screening.15 The impact of this on ovarian cancer mortality is still unknown. 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; however, this approach has not proven beneficial and may result in unacceptable morbidity in women at average risk for ovarian cancer.

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.11 This indication is useful in women whose tumors expressed CA-125 prior to surgery. For women who have undergone a tumor debulking operation before chemotherapy, a level measured approximately 3 weeks after surgery correlates with the amount of residual tumor mass and is predictive of overall survival.16 Serial levels during and after 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 as determined by a second-look laparotomy to pathologically evaluate the disease.17 A more rapid decline of serum CA-125 during treatment has been associated with a more favorable prognosis.11,18,19 Nadir values <10 units/mL predict improved survival, and increases in CA-125 from the nadir (even when <35 units/mL) may be used to predict disease progression.17,20 Failure of CA-125 level to decline may also be used to identify tumors that are not responding to chemotherapy; an increase usually indicates progression.20 However, a large European trial involving >1,400 women failed to demonstrate an improvement in survival in treating women based on rising CA-125 levels alone.21 Additional trials are ongoing to confirm these results. Subsequently, rising CA-125 levels without any other evidence of disease require careful clinical interpretation to determine if patients require treatment interventions.

Human Chorionic Gonadotropin

Normal range: serum <5 milli-International Units/mL (<5 International Units/L)

Human chorionic gonadotropin (hCG) is a glycoprotein consisting of α and β subunits that is normally produced by the placenta during pregnancy.22,23 Elevations in nonpregnant women and in men requires evaluation for malignant conditions. The β subunit is most commonly used as the determinant in both serum as a tumor marker and in urine tests for pregnancy. hCG 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 (eg, 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 various automated assays, and false-positive hCG immunoreactivity also has 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.22 hCG has an important prognostic role, with levels >50,000 milli-International Units/mL indicating a poor prognosis in nonseminomatous disease.23 Most frequently, hCG is used to monitor response to therapy (ie, an elevated level is evidence of residual disease following surgery) and monitor for evidence of disease progression or recurrence during or after treatment.22,23 hCG has a half-life of only 18 to 36 hours, so serum levels decline rapidly after therapeutic interventions; failure to do so may indicate residual disease.22-24

CA 19-9

Normal range: <37 units/mL

Cancer antigen 19-9 (CA 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 for this indication.6 It is primarily used in pancreatic cancer to help discriminate benign pancreatic disease from cancer, monitor for disease recurrence, and assess response to treatment interventions.6,25 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.26 An elevated CA 19-9 level is a poor prognostic factor in patients with inoperable pancreatic cancer.27

α-Fetoprotein

Normal range: <20 ng/mL

α-Fetoprotein (AFP) is a glycoprotein made in the liver, gastrointestinal tract, and fetal yolk sac. It is found in high concentrations in serum during fetal development (~3 mg/mL); 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 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 ovarian germ cell, pancreatic, gastric, lung, and colon cancers.23 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.23 In some parts of the world, AFP is used as a screening test for hepatocellular carcinoma in patients who are positive for HBsAg, and are at increased risk for hepatocellular carcinoma. In the United States, however, AFP is used primarily to assist in the diagnosis of hepatocellular carcinoma. AFP levels >1,000 ng/mL are common in patients with hepatocellular carcinoma.28,29

These AFP levels also are used to monitor patients with hepatocellular carcinoma or ovarian and testicular germ cell tumors for disease progression or recurrence and to assess the impact of treatment interventions. The serum half-life of AFP is 5 to 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.25 After successful treatment for nonseminomatous germ cell tumors of the testis, hCG and AFP tests 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 radiographs.25 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.23

β2-Microglobulin

Normal range: <2.5 mcg/mL

β2-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 chronic lymphocytic leukemia lymphoma. B2M is renally excreted and may be elevated in nonmalignant conditions such as renal failure.30

B2M is a reflection of tumor mass in multiple myeloma and is considered standard for the measurement of tumor burden. Measurement of serum B2M is most commonly done in the evaluation of multiple myeloma and is an important part of the staging and prognosis for that disease. Additionally, B2M is used to follow patients with multiple myeloma for treatment efficacy, with increases in B2M potentially indicating progressive disease.30-32

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 biopsy. ER status is also a prognostic factor, with ER-negative tumors having a worse prognosis than ER-positive ones. For >30 years, it has been the standard of practice to evaluate breast cancer tissue for these protein receptors and use that information in directing therapeutic interventions. The relative expression of hormone receptors can be determined using small amounts of tumor tissue.

The current standard of practice is to measure each protein using immunohistochemistry (IHC) to determine if patients will benefit from endocrine therapy; this method detects protein expression through an antibody–antigen interaction.33 Although the method (eg, antibody) used can vary, the biopsy is read by pathologists with the results reported as a percent positive cells. If ≥1% of cells is positive, one is considered to have ER-positive or PR-positive disease.31 Biopsies scored 1% to 10% may be considered “weakly” positive, and risks and benefits of hormonal therapy should be discussed with patients, but the patient is still considered to have ER-positive or PR-positive disease. This category is now described as “ER Low Positive.”34 Because of the variety of methods to evaluate IHC staining and intraobserver/interobserver 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.34 This test demonstrated statistically significant superiority over IHC in predicting relapse in tamoxifen-treated, ER-positive patients in one retrospective trial.34 Further validation of the test is needed before it becomes routinely used in clinical practice.34

Positive ER/PR 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.33 In addition, ER/PR content in tumor biopsies correlates with benefit from adjuvant hormonal therapy after surgical removal of the tumor. After 15 years of follow-up in ER/PR-positive breast cancer patients, adjuvant tamoxifen decreased mortality by 9% in women who received 5 years of therapy.35

Human Epidermal Growth Factor Receptor 2

Human epidermal growth factor receptor 2 (HER2) is a transmembrane glycoprotein member of the epidermal growth factor receptor (EGFR) family with intracellular tyrosine kinase (TK) activity.36 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 B2.37 About 20% of samples from human breast cancers exhibit amplification of c-erb B2 or overexpression of HER2.35

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 (eg, anthracyclines, taxanes). (3) HER2 status may be used to predict resistance to other therapies (eg, tamoxifen). (4) HER2 status predicts benefit from anti-HER2 therapies, such as monoclonal antibodies (eg, trastuzumab, ado-trastuzumab emtansine, and pertuzumab) and TK inhibitors (eg, lapatinib).38-43

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 a woman should receive specific adjuvant therapy or whether endocrine therapy should be used.10

It is well-established that HER2 overexpression is predictive of a response to treatment with trastuzumab, ado-trastuzumab, and pertuzumab, which are all monoclonal antibodies against HER2; lapatinib, a TK inhibitor of human epidermal growth factor receptor 1 (HER1) and HER2; and neratinib and tucatinib, a TK inhibitor of HER2.39,40,44-48 Therefore, it is necessary to evaluate all invasive breast cancers for HER2 status to select appropriate patients for these anti-HER2 therapies.43,49

Although 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.43 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 women with a score of 0 or 1+ should be considered HER2 negative and do not benefit from anti-HER2 therapy, and women who have a score of 3+ are HER2 positive and benefit from therapy.39,43 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 because of decreased variability and increased ability to predict efficacy of therapies aimed at the HER2 receptor.43 The FISH assay measures both the number of gene copies of HER2 gene and provides a ratio of HER2/CEP 17 (also called FISH ratio). Tumors are measured by the ratio of HER2 signals divided by the number of signals determined by the centromeric portion of chromosome 17 (CEP 17). A positive test for HER2 gene amplification is a gene copy number >4 or a FISH ratio >2. HER2-negative tumors are defined as a gene copy number <4 or FISH ratio <2, and FISH ratios between 1.8 to 2.2 are inconclusive. Additional cells should be scored and the results compared.41 Only patients with FISH-positive tumors derive benefit from anti-HER2 therapy.43

In summary, routine testing for HER2 with either IHC or FISH is recommended in all patients with invasive breast cancer, with FISH as the preferred method.10,43 Patients who are HER2 positive benefit from HER2-targeted therapy in neoadjuvant, adjuvant, and metastatic settings.37,38,44 The use of HER2 testing to determine benefit of additional therapies (eg, tamoxifen, anthracyclines, taxanes) is inconclusive at this time10,43 (Minicase 2).

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 also can 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 and form the characteristic BCR-ABL fusion gene on the Ph chromosome, is diagnostic for CML and is present in almost all patients with the disease by definition.50,51 The BCR-ABL gene encodes a protein with deregulated, constitutively active TK 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 used 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. The quantitative PCR is typically drawn every 3 months; labs can use their own assay, but BCR-ABL1 transcripts obtained should be converted to the international scale (IS) by applying a lab-specific conversion factor. Bone marrow cytogenetics are obtained at diagnosis and if patient does not achieve the expected response or if there is a loss of response. There is a strong correlation between results obtained from peripheral blood using quantitative PCR and bone marrow cytogenetics, allowing for molecular monitoring without bone marrow aspirations. Table 21-3 lists the response criteria for CML using cytogenetic and molecular monitoring.50,51

TABLE 21-3.

Criteria for Cytogenetic and Molecular Response in Patients with Chronic Myelogenous Leukemia

CYTOGENETIC RESPONSE

MOLECULAR RESPONSE

Complete: Ph +0%

Early molecular response: BCR-ABL1 (IS) ≤10% at 3 and 6 mo

Major: Ph +0% to 35%

Partial: Ph +1% to 35%

Major molecular response: BCR-ABL1 (IS) ≤0.1% or a ≥3-long reduction in BCR-ABL1 mRNA from the standardized baseline, if qPCR (IS) is not available

Minor: Ph +36% to 65%

Complete molecular response: variably described, but best defined by the assay’s level of sensitivity

Therapies (eg, imatinib, nilotinib, dasatinib, bosutinib, and ponatinib) have been developed that target the abnormal TK activity of the BCR-ABL gene.4,52 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 resistance to the currently available TK inhibitors. Patients who present in advanced-phase disease or have an inadequate or loss of response to TK inhibitors should undergo mutational analysis, so that the appropriate therapy may be selected. Patients with threonine-to-isoleucine mutation at codon 315 (T315I) were previously referred for stem cell transplantation because of a lack activity of the available TK inhibitors to this mutation. Ponatinib is a U.S. Food and Drug Administration (FDA)-approved TK inhibitor that is active against this mutation, and patients who have the T315I mutation should be considered for this therapy. Failure with multiple therapies may lead to a referral for stem cell transplantation.53

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 TK activity (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, gefitinib, afatinib, dacomitnib, and osimertinib (EGFR TK inhibitors [TKi]) that are used in non–small cell lung (NSCL) cancer. 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 women, 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 EGFR TKi drugs except for osimertinib. 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.54 For patients with NSCL cancer, it is recommended that next-generation sequencing (NGS) or another broad, panel-based approach be performed to identify oncologic drivers, including EGFR mutational analysis and would many EGFR variants, including rare variants and variants of unknown significance. In contrast, a RT-PCR can also be used but would only detect the common EGFR variants, such as exon 18 through 21.55,56 RT-PCR detects overexpressed protein caused by the underlying fusion transcript, and target break-apart FISH probes can detect a rearrangement regardless of the fusion partner but is not able to screen for large samples for other rearrangements (such as ALK, ROS1, and RET) that occur at a low frequency.57 Some EGFR mutations show a decreased response from TKI inhibitors, such as EGFR exon 20 insertions and p.T790M. Osimertinib was FDA-approved for metastatic patients with EGFR T790M mutation positive NSCL cancer in patients who have progressed with EGFR TKIs. It was approved with a companion diagnostic test, Guardant360 CDx assay, that uses a tumor or plasma specimen. This was the first liquid biopsy companion diagnostic to use NGS technology to guide treatment decisions.58

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 many cell surface receptors, including EGFR. When activated 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 currently, no therapy has reached the market that inhibits this signal. 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, other methods, including direct gene sequencing, can be used, but this is typically part of a larger panel.59,60

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 two most common mutations are V600E mutation (glutamic acid for valine substitution at amino acid 600) and V600K mutation (valine to lysine substitution at amino acid 600).61 This mutation means that the kinase is always turned on, signaling downstream partners in the mitogen-activated protein kinase pathway, such as mitogen-activated extracellular kinase (MEK).62 Vemurafenib was the first BRAF inhibitor approved to treat melanoma in patients whose tumor contains this mutation. Concurrent with the approval of vemurafenib, the CobasC 4800 BRAF V600 mutation test was introduced, which uses real-time PCR to identify the V600E mutation in tumors; now other tests are available to detect V600E and V600K mutations, including NGS technology. An NGS companion test was approved in patients with NSCL cancer, Oncomine Dx Target Test (Thermo Fischer Scientific, Waltham, MA) for dabrafenib, a BRAF inhibitor, in combination with trametinib, a MEK 1 and 2 inhibitor; this test can also detect other markers such as ROS1 fusion positivity and EGFR L858R and exon 19 deletion positive patients, which would be indications for other therapies. The prescribing information requires that the test be performed and the result be positive for the mutation before using the drug.63 Encorafenib is another BRAF inhibitor approved to treat metastatic colorectal cancer in combination with cetuximab in patients with the V600E mutation and t iis indicated in metastatic melanoma in combination with binimetinib in patients with the V600E or V600K mutation.64 BRAF inhibitors are approved for various malignancies, including melanoma, NSCL cancer, thyroid cancer, and colorectal cancer.

Anaplastic Lymphoma Kinase

Anaplastic lymphoma kinase (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. Multiple ALK inhibitors are available that are highly effective for patients with lung cancer whose tumors contain this translocation; these agents are crizotinib, alectinib, brigatinib, ceritinib, and lorlatinib. The mutation most commonly occurs in nonsmokers with lung adenocarcinoma, and it rarely occurs in combination with KRas or EGFR mutations. Although the mutation is found only in 2% to 7% of patients with non–small cell lung cancer, it should be routinely tested for because of significant improvement in outcomes with ALK inhibitors that target this mutation. As stated in the EGFR section, NGS technology, such as FoundationOne CDx, should be performed on these patients to detect the possible tumor markers and direct therapy. ALK can also be detected by PCR, FISH, or IHC.65,66

Programmed Death-Ligand 1

Programmed death-ligand 1 (PD-L1) is a ligand of programmed death 1 (PD-1). In some cancers, PD-L1 can be overexpressed, which is an oncologic driver and inhibits the proliferation and differentiation of T cells.67 The PD-1/PD-L1, also known as a checkpoint pathway, is important for immune tolerance, and tumor cells can turn off the function of normal immune response, causing loss of T-cell activity. Expression of PD-L1 can be detected by IHC.68 Targeting this pathway has played important role in advancement of treatment for many cancers, such as lung cancer, breast cancer, bladder cancer, melanoma, ovarian cancer, gastrointestinal malignancies, and many others. PD-1 inhibitors (nivolumab, pembrolizumab, avelumab, and cemiplimab) and PD-L1 inibitors (atezolizumab and durvalumab) bind to PD-1 and PD-L1, respectively, blocking the interaction between PD-1 with PD-L1 on tumor cells and reactivating the immune system, allowing T cells to destroy the tumor. PD-L1 can be tested on tumor cells and is reported as a tumor proportion score. Tumor and inflammatory cells can also be reported and resulted as a combined positive score. PD-L1 expression has shown to be predictive for PD-1/PD-L1 inhibitors; however, some tumors show benefit with low or no expression. Companion tests have been approved for various indications but are not required for all indications.69

Genomic alterations of the mismatch repair (MMR) system can also predict efficacy PD-1 and PD-L1 inhibitors. MMR recognizes and repairs errors arising during DNA replication. It is made up of four major genes: MLH1, PMS2, MSH2, and MSH6. If a patient has a germline or somatic mutation or a deletion of any these four genes, the patient would have deficient MMR (dMMR). MMR deficiency may induce microsatellite instability (MSI). Microsatellites are small repeating segments of a consistent length. Instability is considered if the length of the microsatellite repeat in tumor DNA is different from the length in corresponding germline DNA. Five standard sites are tested, and a patient is considered MSI-high (MSI-H) if two or more sites are altered.70 MSI-H and dMMR are considered interchangeable terms because there is a high level of consistency between them in tumors. These tumors contain increased neoantigen formation, which can be a target for the immune system; also these tumors can upregulate immune checkpoints, such as PD-L1 in infiltrating lymphocytes, making them susceptible PD-L1 inhibitors. The FDA has approved a tumor agnostic indication for pembrolizumab in solid tumors that are MSI-H or dMMR that have progressed following prior treatment and have no satisfactory treatment options as well as for colon cancer. Pembrolizumab can be used in the first-line setting for unresectable or metastatic MSI-H or dMMR disease that has progressed with a fluoropyrimidine, oxaliplatin, and irinotecan.71 MSI-H can be found in endometrial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, and many more malignancies. Sensitive standardized detection of MSI status is necessary with tests such as NGS; older methods include PCR.70 NGS is a massively parallel DNA sequencing method that allows the simultaneous analysis of millions of fragments of DNA. A sample from the patient’s tumor is sequenced alongside a sample of normal tissue from the patient, which allows genetic variants to be identified. The results show somatic mutations, which are found only in the tumor, or germline mutations that are inherited and found in both the tumor sample and the normal tissue.72

Another biomarker that shows promise in predicting the use of PD-1/PD-L1 inhibitors is tumor mutation burden (TMB). Currently there is no consensus on how to measure this. Nivolumab in combination with ipilumumab has been evaluated in patients with NSCL cancer. Patients with high TMB had increased PFS with nivolumab and ipilumuab compared with chemotherapy.73

SUMMARY

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 follow-up 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 fusion gene found in CML patients, BRAF V600E and V600K mutations found in melanoma, and EGFR mutations found in lung cancer provide a specific target in which therapeutic strategies have been developed to inhibit the actual pathogenesis of the cancer. Molecular targets detected by NGS technology have become a mainstay of treatment for many patients with cancer.

LEARNING POINTS

1. If a patient with CML has a complete cytogenetic response, is the patient considered to be cured of leukemia and thus can stop therapy?

ANSWER: Obtaining a complete cytogenetic response to therapies demonstrates that the patient is responding to treatment. However, molecular responses, particularly complete molecular responses, are the most sensitive test to determine if the Ph chromosome is still present. Unfortunately, reaching undetectable levels of BCR-ABL transcripts in a patient is not common and does not indicate cure; therefore, the patient should continue therapy.

2. A patient with testicular cancer has serum AFP level drawn 2 days after his surgery and it is still elevated (250 ng/mL). Is this cause for concern?

ANSWER: Using serum tumor markers after surgery in testicular cancer is common, and the rate by which they decline has prognostic implications. However, because the serum half-life of AFP is 5 to 7 days, a level drawn so close to surgery is of little value. In contrast, hCG has a half-life of only 18 to 36 hours. If hCG does not decrease within 2 days after surgery, this should be cause for concern.

3. In a patient is diagnosed with metastatic melanoma, which pathway mutations should be checked prior to initiating therapy?

ANSWER: The two most common mutations in metastatic melanoma are the BRAF V600E and the V600K mutations. This results in the constitutive activation of the mitogen-activated protein kinase pathway. Currently, we have therapies that inhibit the components BRAF and MEK in these mutation-positive patients. Trials show that the combination of BRAF and MEK inhibitors is synergistic and may be used in combination if the patient is mutation-positive.

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QUICKVIEW | Carcinoembryonic Antigen

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<2.5 ng/mL (<2.5 mcg/L)

Children

Unknown

Critical value

Yes, levels >10 ng/mL (>10 mcg/L) generally indicate cancerous process

Inherent activity

Unknown

Location

Production

Intestine, pancreas, liver

Normally found during fetal development only; detected in serum of patients

Storage

Unknown

Secretion/excretion

Unknown

Causes of abnormal values

High

Cancer (mainly colon), smoking, hepatitis, pancreatitis, peptic ulcer disease, hypothyroidism, ulcerative colitis, Crohn disease

Usually <10 ng/mL in nonmalignant conditions

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Not reliable to screen for cancers because elevated in other conditions; can be used to monitor effectiveness of therapy in patients with cancer

QUICKVIEW | CA-125

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<35 units/mL

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Production

Detected in serum of patients

Storage

Unknown

Secretion/excretion

Unknown

Causes of abnormal values

High

Cancer (mainly ovarian, cervical, and fallopian tube carcinomas), endometriosis, ovarian cysts, liver disease, pregnancy, menstruation

Low

Luteal phase of cycle, patients on oral contraceptives, menopausal women

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Not reliable to screen for cancers because elevated in other conditions; can be used to monitor effectiveness of therapy in patients with ovarian cancer; rate of rise and fall of levels may indicate disease recurrence or residual disease

QUICKVIEW | CA 15-3

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<30 units/mL

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Serum

Production

Unknown

Antibody detects circulating mucin antigen secreted

Storage

Unknown

Secretion/excretion

Secreted from breast tissue

Causes of abnormal values

High

Breast cancer, may be elevated in other cancers of lung, colon, ovary, and pancreas origin and benign breast and liver disorders

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Mainly used in combination with other markers or in clinical trials

QUICKVIEW | CA 27.29

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<38 units/mL

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Serum

Production

Unknown

Antibody detects circulating mucin antigen secreted

Storage

Unknown

Secretion/excretion

Secreted from breast tissue

Causes of abnormal values

High

Breast carcinoma, may be elevated in benign breast disorders

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Mainly used in combination with other markers or in clinical trials

QUICKVIEW | Human Chorionic Gonadotropin

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<5 milli-International Units/mL (<5 International Units/L)

β subunit commonly measured, serum levels drawn when used as a tumor marker

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Production

Made by cells of the placenta

Detected in patient serum and urine

Storage

Unknown

Secretion/excretion

Secreted from the placenta or malignant germ cells

Causes of abnormal values

High

Pregnancy, mixed germ cell tumors, or choriocarcinoma of the testes or ovary, increased in other rare tumors

If elevated in men or in nonpregnant women, cancer is suspected

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Most commonly used in testicular cancer as a prognostic factor as well as to monitor effects of treatment; levels >50 milli-International Units/mL indicate a poor prognosis

QUICKVIEW | CA 19-9

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<37 units/mL

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Production

Pancreas, gastric cells, colon

Detected in patient serum

Storage

Unknown

Secretion/excretion

Secreted from breast tissue

Causes of abnormal values

High

Pancreatic, gastric, and colon carcinomas; also in benign pancreatic disorders

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Only recommended to evaluate treatment response and recurrence in patients with pancreatic cancer

QUICKVIEW | α-Fetoprotein

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<20 ng/mL

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Production

Protein made normally during fetal and neonatal stages by liver, gastrointestinal tract, and yolk sac cells

Detected in patient serum; levels should decline after birth

Storage

Unknown

Secretion/excretion

Unknown

Causes of abnormal values

High

Cancer (mainly liver and testicular); can be elevated in other cancers, such as pancreatic, gastric, lung, and colon carcinomas; elevated in nonmalignant conditions, including pregnancy, hepatitis, and cirrhosis

High results may be used to screen for liver cancer in parts of the world at increased risk for this malignancy

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Only recommended to evaluate treatment response and recurrence in patients with testicular cancer

QUICKVIEW | β2-Microglobulin

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

<2.5 mcg/mL

Children

Unknown

Critical value

Not applicable

Inherent activity

Unknown

Location

Protein found on surface of lymphocytes and other MHC I molecules

Also present in small amounts in urine and blood; level should decline after birth

Production

Unknown

Storage

Unknown

Secretion/excretion

Unknown

Causes of abnormal values

High

Multiple (plasma cell) myeloma, lymphoma, and in patients with renal failure

Renally excreted so elevated levels may indicate renal failure

Low

Not applicable

Signs and symptoms

High level

May see signs of renal failure

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Used in patients with multiple myeloma to assist in determining disease stage, prognosis, and response to treatment

MHC = major histocompatibility complex.

QUICKVIEW | Estrogen and Progesterone Receptors

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Not applicable

Not a normal serum laboratory value, only determined in breast biopsies; if >1% of cells are positive for the receptor, it is considered ER-positive or PR-positive

Children

Not applicable

Critical value

Not applicable

Inherent activity

Growth of breast and other hormone sensitive cells

Location

Throughout the body (eg, breast tissue, ovaries, bone)

Also present in small amounts in urine and blood; level should decline after birth

Production

Unknown

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Not applicable

It is unknown if the levels are higher in cancer, but they are checked to determine if blocking them with hormonal therapy will be useful

Low

Not applicable

Signs and symptoms

High level

May see signs of renal failure

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Antiestrogens (eg, tamoxifen) and aromatase inhibitors (eg, anastrozole) often given if these receptors are positive in women with breast cancer

QUICKVIEW | Human Epidermal Growth Factor Receptor 2

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Considered positive by IHC if 3+ cells stain for HER2 or by FISH if HER2 gene copy number >4 or FISH ratio >2

Not a normal serum laboratory value, only determined in breast biopsies; FISH preferred

Children

Not applicable

Critical value

Not applicable

Inherent activity

Protein involved in normal growth and development of cells by activating intracellular pathways that send growth signals to the nucleus

In cancer the growth signal is abnormal and amplified, leading to uncontrolled proliferation of the cancerous cells

Location

Surface of many epidermal cells

Also present in small amounts in urine and blood; level should decline after birth

Production

Not applicable

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Cancer

Either number of receptors may be higher or there may be an increase in HER2 gene copies indicating increased function of the gene

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Anti-HER2 therapies (eg, trastuzumab, ado-trastuzumab, fam-trastuzumab deruxtecan, pertuzumab, lapatinib, neratinib, and tucatinib) often given if positive

QUICKVIEW | BCR-ABL

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Not applicable

This is an abnormal fusion gene that results from a genetic translocation producing a fusion; mRNA normally not present in any significant amount unless a malignancy is present

Children

Not applicable

Critical value

Not applicable

Inherent activity

When present, causes abnormal growth of cells

Translocation results in an abnormal fusion protein with increased TK activity, which continually signals cells to grow

Location

Chromosome 22 resulting from t(9;22) translocation

Also present in small amounts in urine and blood; level should decline after birth

Production

Not applicable

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Cancer

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Rising levels of BCR-ABL mRNA correlate with increasing disease activity whereas falling levels are consistent with response to therapy

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Called the Philadelphia chromosome; levels of BCR-ABL mRNA should decrease with therapy, and failure to do so indicates treatment failure

QUICKVIEW | EGFR Mutation (Exon 19 and 21)

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Not applicable

This is a gene that codes for a transmembrane receptor; it does not normally contain any mutations

Children

Not applicable

Critical value

Not applicable

Inherent activity

When present, causes abnormal growth of cells

Mutation in lung cancer cells leads to perpetual signaling

Location

Located on chromosome 7p12; region of interest is exon 19 and 21h

Production

Not applicable

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Not applicable

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

The presence of a mutation in exon 19 or 21 in lung cancer indicates a higher likelihood of response to erlotinib, afatinib, gefitinib, dacomitnib, and osimertinib

QUICKVIEW | KRas Mutation

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Not applicable

This is a gene that codes for a GTPase that is a binary switch in cell signaling; it does not normally contain any mutations; when mutations are not present it is referred to as WT

Children

Not applicable

Critical value

Not applicable

Inherent activity

When present, causes abnormal growth of cells

Mutation in colorectal cancer cells leads to perpetual signaling and resistance to monoclonal antibodies targeting EGFR

Location

Chromosome 12p12; region of interest is exon 1 (codon 12 and 13)

Production

Not applicable

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Mutation common in lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, and colorectal carcinoma

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Cetuximab and panitumumab only should be used for patients with colorectal cancer with WT KRas tumors

WT = wild-type.

QUICKVIEW | BRAF Mutation

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Not applicable

This is a gene that codes for a kinase involved in cell signaling through the MAP kinase pathway; it does not normally contain any mutations

Children

Not applicable

Critical value

Not applicable

Inherent activity

When present, causes abnormal growth of cells

Mutation in cancer cells leads to perpetual signaling

Location

Chromosome 7q34; mutation of interest is at amino acid 600 (BRAF V600E or V600K)

Production

Not applicable

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Mutation common in non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, thyroid carcinoma, non–small cell lung carcinoma, and adenocarcinoma of lung

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Patients with malignant melanoma should receive only vemurafenib and dabrafenib if they have a tumor with the V600E mutation; or if the combination of dabrafenib and trametinib, vemurafenib and cobimetinib, or encorafenib and binimetinib is used, a V600E or V600k mutation should be present

MAP = mitogen-activated protein.

QUICKVIEW | Anaplastic Lymphoma Kinase Mutation

PARAMETER

DESCRIPTION

COMMENTS

Common reference range

Adults

Not applicable

This is a fusion gene between EML4-and ALK; the resulting protein promotes cancer growth through increased kinase signaling activity; cells do not normally contain this gene fusion

Children

Not applicable

Critical value

Not applicable

Inherent activity

When present, causes abnormal growth of cells

Mutation in cancer cells leads to increased signaling

Location

Chromosome 2 contains the genes for EML4 and ALK; mutation of interest is translocation/fusion gene EML4-ALK

Production

Not applicable

Storage

Not applicable

Secretion/excretion

Not applicable

Causes of abnormal values

High

Mutation most commonly found in adenocarcinoma of the lung in nonsmokers

Low

Not applicable

Signs and symptoms

High level

Not applicable

Low level

Not applicable

After event, time to….

Initial elevation

Not applicable

Peak values

Normalization

Causes of spurious results

Not applicable

Additional info

Patients with metastatic non–small cell lung cancer should receive only crizotinib, alectinib, brigatinib, ceritinib, or lorlatinib if they have a tumor with the ALK rearrangement