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  • 1. 833© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00067-1 Introduction During normal growth and development, tissues and organs develop originally from a single cell by the process of differentiation. Although the processes involved are poorly understood, they are clearly complex and highly regulated. In cancer, cell division goes out of control, often as a result of the gradual accumulation of multiple mutations over a prolonged period. Small growths some- times occur that are harmless and not cancerous; these are said to be benign and are mostly well differentiated. Their close similarity to normal tissues makes detection difficult. In cancer, the dangerous growths are described as malignant and are predominantly less differentiated. The resulting growth and eventual systematic spread or metastasisofthediseaseoftenkillsthepatient.Carcinoma refers to tumors of skin or mucous membranes, sarcoma to tumors of connective tissue. Abnormal cancerous growth produces abnormal types and levels of substances we now recognize as cancer markers or tumor markers. These markers appear in blood and other body fluids due to the loss of polarity and/or anatomic damage from the tumor. The properties of an ideal tumor marker are sum- marized in Table 1, although no tumor marker exhibits all of these properties. Specific Bence-Jones proteins were discovered in the urine of some cancer patients as long ago as 1846. During the next 100 years, ectopic hormones and isoenzymes were identified as cancer markers. The last few decades of research have resulted in a virtual explosion in the discov- ery, validation, and clinical application of these analytes in cancer patient management, largely due to the develop- ment of monoclonal antibody (mAb) and recombinant DNA technology (Oppenheimer, 1985; Virji et al., 1988; Rittenhouse et al., 1985; Sell, 1990). This chapter attempts to provide an overview of cancer marker immunoassays with special reference to those analytes now available for routine clinical use. It is pertinent to mention, at this junc- ture, that various countries have different modes of regu- lating the use of tumor marker assays. It ranges from minimal to moderate regulatory constraints in Canada and W. Europe to substantive requirements in the USA and Japan. Until 1996, a new tumor marker assay to be intro- duced in the USA was regulated as a Class III device requiring retrospective and prospective clinical trial as part of the “premarket application” or PMA. Now, tumor markers are classified as Class II medical devices in the USA requiring a 510(k) application, a simplified process that generally requires 90–120 days for approval. A definition of tumor markers was adopted at the fifth International Conference on Human Tumor Markers held in Stockholm, Sweden, in 1988. It states that: “Biochemical tumor markers are substances developed in tumor cells and secreted into body fluids in which they can be quantitated by non-invasive analyses. Because of a correlation between marker concentration and active tumor mass, tumor markers are useful in the management of cancer patients. Markers, which are available for most cancer cases, are additional, valuable tools in patient prognosis, surveillance, and therapy monitoring, whereas they are presently not applicable for screening. Serodiagnostic measurements of markers should emphasize relative trends instead of absolute values and cut-off levels.” The potential clinical applications of tumor marker assays are listed below. SCREENING Some tumor markers have been utilized in mass screening programs of asymptomatic individuals, with limited suc- cess, in high-risk sectors of the population. However, it is to be emphasized that no biochemical tumor marker is yet specific and sensitive enough to be recommended as a definitive screening test for cancer. In some countries, screening programs have been conducted for certain can- cers highly prevalent in those regions. In China, alphafeto- protein (AFP) measurements have been used for hepatocellular carcinoma screening. Individuals with a previous history of hepatitis infection or liver cirrhosis are at a higher risk for developing liver cancer. In Japan, screening for neuroblastoma in children <1 year has been conducted by measuring urinary vanillylmandelic acid and homovanillic acid. Other screening markers tested include Cancer Markers Hoon H. Sunwoo1 (hsunwoo@ualberta.ca) Mavanur R. Suresh2 C H A P T E R 9.13 TABLE 1 Properties of an Ideal Tumor Marker 1. High clinical sensitivity 2. High clinical specificity 3. Tumor marker levels proportional to tumor volume 4. Short half-life to rapidly mirror treatment schedules 5. Reflect tumor heterogeneity 6. Low levels in healthy population 7. Low levels in benign diseases 8. Discriminatory to identify tumor and metastasis from benign to healthy states 9. Provide adequate lead times for early diagnosis and early treatment 10. Assay sensitivity to detect stage I cancers1 This Edition. 2 Previous Editions.
  • 2. 834 The Immunoassay Handbook fecal occult blood/hemoglobin for colorectal cancer, CA 125 for ovarian cancer, prostate-specific antigen (PSA) for prostate cancer, and p21 ras oncoprotein. Prostate cancer screening using PSA and digital rectal examination was recommended by the American Cancer Society in Novem- ber 1992 for men over 50 years of age. However, it remains controversial, and in spite of this strong endorsement, a significant number of oncologists question the merits of screening as related to treatment options and survival ben- efit. In 2012, the US Preventive Services Task Force (USPSTF) recommended against PSA-based screening for prostate cancer. Another prominent marker worth mentioning is the identification of mutated forms of hereditary cancer susceptibility BRCA1 and BRCA2 genes or gene products, which identifies high-risk individuals. In 2003, a new two-in-one test was approved by the FDA for the primary screening of cervical cancer. It includes a sandwich immunoassay test with the traditional 50-year-old Pap smear for women over the age of 30. The Pap test is widely used as a screening test that is done annually to detect the presence of abnormal cellular mark- ers indicative of cancerous or precancerous conditions. This was developed by George Papanicolaou and involves the collection of vaginal fluid or cells scraped from the cer- vix to predict or detect cervical cancer. The new compo- nent of the combined test (Digene, QIAGEN, USA) is the genetic test for 13 strains of the human papilloma viruses (HPVs), which are largely sexually transmitted and the likely cause of 99% of cervical cancers. While millions of women are infected with HPV, only those above the age of 30 and having persistent infections are at high risk. The Hybrid Capture® 2 DNA test uses a two-RNA probe mix- ture to distinguish between the carcinogenic and low-risk HPV types. The company offers a specific specimen col- lection device to accompany the combined test procedure, although it is similar to the traditional sample collection method. The principle of the hybrid capture method is essentially a sandwich immunoassay that detects the spe- cific DNA–RNA hybrid as the antigen. The specimen is dissolved in a base solution to dissociate the various com- ponents including the target viral DNA. The specific HPV RNA probe is added to form a DNA–RNA hybrid, which is selectively captured by a solid phase coated with an anti- body with specificity to the hybrid nucleic acids. The cap- tured DNA–RNA hybrid is detected by an alkaline phosphatase (ALP)-labeled antibody and detected by che- miluminescent dioxetane substrate. The combination test has been found to have better sensitivity than the use of either test alone. In some countries outside the USA, the HPV test is also used as a primary screening test alone or in conjunction with the Pap test. DIAGNOSIS Almost every cancer marker has been investigated for its suitability as a primary diagnostic test for cancer in symp- tomatic individuals. However, sufficient false positives and false negatives have been encountered with every marker so far discovered to preclude their use in distin- guishing malignant and nonmalignant conditions. The ultimate goal of identifying tumor-specific antigens has so far eluded oncologists because most tumor markers have been found in some normal tissues and the serum of some noncancerous individuals and in many benign diseases. For this reason, these antigens are often referred to as tumor-associated antigens. Nevertheless, a number of cancer markers have proved to be useful in confirming diagnosis, often in conjunction with a battery of other clinical methods. Another approach attempts to use mul- tiple tumor markers to diagnose tumors and to identify the primary origin of metastic disease (Wu and Naka- mura, 1997; Hanausek and Walaszek, 1998). This is in parallel with the more frequent use of drug combinations to treat tumors. DIFFERENTIAL DIAGNOSIS AND CLASSIFICATION Immunoassays for some cancer markers are used in clinics to distinguish between clinical conditions with similar symptoms, where one or both could be cancerous. For example, the measurement of neuron-specific enolase (NSE) levels allows differentiation between neuroblas- toma and Wilm’s tumor when a child presents with a pal- pable abdominal mass. Similarly, PSA and prostatic acid phosphatase (PAP) can distinguish prostate cancer metas- tasis from other secondary tumors whose primary origin is not the prostate gland. Antibody probes specific for B or T cells can establish the lineage and classify leukemias and lymphomas in an immunohistochemical assay (see HEMA- TOLOGY). Antibodies specific to lymphoid malignancies can distinguish between non-Hodgkin’s lymphoma and undifferentiated cancer of nonhematopoietic origin. RECENT DEVELOPMENTS IN CANCER DIAGNOSIS Highly sensitive diagnosis and accurate analysis of bio- markers in human samples are important for the early detection, treatment, and management of cancer. For a traditional immunometric (sandwich) immunoassay that is routinely used for protein biomarker identification, a capture antibody against a specific biomarker is first immobilized on a 96-well plate. After the binding of anti- gen, a labeled detection antibody is allowed to bind with the immobilized antigen. The concentration of the anti- gen can then be determined by indirectly measuring the concentration of the probe attached to the detector anti- body, which may include enzymes, fluorescence tags, DNA barcodes, etc. A heterogeneous immunoassay involves antibody immobilization, multiple steps of incu- bation and washing cycles, followed by signal amplifica- tion and reading. From the initial antibody immobilization to the final reading of the assay results, the immunoassay may take from hours to days to complete. A traditional immunoassay is rather time and labor intensive. To over- come these problems, the development of single-step, washing-free homogeneous immunoassays has gener- ated a lot of interest and value to the scientific commu- nity. The magnitude of light scattering by a gold nanoparticle can be significantly higher than light emis- sion from strongly fluorescing dyes. This unique charac- teristic has led to many promising applications of metal nanoparticles in the biomedical field including molecular
  • 3. 835CHAPTER 9.13 Cancer Markers and cell imaging, biosensing, bioassays, and photothermal therapy. By taking advantage of the large scattering cross- section of gold nanoparticles and the high sensitivity of “dynamic light scattering measurement,” biomarker pro- teins or other biomolecular targets can be detected at very low concentrations using gold nanoparticle probes. Uti- lizing this methodology, biomarker proteins were detected by Liu et al. (2008) at very low concentration using gold nanoparticle probes. The development of thyroglobulin (Tg) assays and recombinant human TSH (rhTSH) has provided the means for physicians to identify residual (or recurrent) thyroid cancer much earlier than few decades ago, when diagnostic technologies were limited to physical examina- tion, 131I scans after T4 withdrawal, and chest X-rays. The combined use of serum Tg and whole-body scan after rhTSH stimulation is particularly effective in the detec- tion of small residual foci of thyroid tissue. A general assumption is that earlier detection and treatment of recur- rent disease should lead to better outcomes, but proper evidence is absent in that 131I therapy of microscopic dis- ease helps the low-risk patient whose survival approaches 100%. Smallridge et al. (2007) reported that using a Tg- immunoassay with a cutoff 5- to 10-fold lower than reported by others, it is possible to follow patients with differentiated thyroid cancer and a T4-suppressed Tg below 0.1ng/mL without the need to perform rhTSH stimulation, which also has significant economic benefits. Gong et al. (2007) developed a simple, sensitive, and specific immunoassay, based on surface-enhanced Raman scattering for human AFP, a tumor marker for the diagno- sis of hepatocellular carcinoma. This methodology com- bined Ag/SiO2 core–shell nanoparticles with rhodamine B isothiocyanate dye molecules as Raman tags and used amino group-modified silica-coated magnetic nanoparti- cles as the solid-phase immobilization matrix and separa- tion tool. The system involved an immunometric (sandwich)-type immunoassay between polyclonal anti- body-functionalized Ag/SiO2 nanoparticle-based Raman tags and monoclonal antibody-modified silica-coated magnetic nanoparticles. The presence of the analyte and the antibody–antigen reaction can be monitored by the Raman spectra of the Ag/SiO2 tags. The advantages of this novel strategy include the high stability of Raman tags obtained from the silica shell-coated silver core–shell nanostructure and the use of silica-coated magnetic nanoparticles as immobilization matrix and separation tool, avoiding pretreatment and washing steps. This assay is able to measure human AFP concentrations up to 0.12µg/mL with a detection limit of 11.5pg/mL. A rapid and reproducible surface-enhanced Raman scat- tering (SERS)-based immunoassay technique, using hol- low gold nanospheres (HGNs) and magnetic beads, has also been developed by Chon et al. (2009) for the detection of lung cancer marker carcinoembryonic antigen (CEA). Gold et al. (2006) developed a new MUC1 serum immuno- assay that is able to differentiate cancer from pancreatitis. An enzyme immunoassay was established with mAB PAM4 as the capture antibody and a polyclonal anti-MUC1 anti- body as the detection probe. Patient sera were obtained from healthy, adult patients with acute and chronic pan- creatitis, and those with pancreatic and other forms of cancer, and were measured for PAM4-reactive MUC1. The sensitivity and specificity observed suggest that the PAM4-based immunoassay of circulating MUC1 may be useful in the diagnosis of pancreatic cancer. STAGING AND GRADING The degree of elevation in the concentration of several tumor markers can help to stage tumors. In general, the mean circulating levels of these tumor markers increase with the stage of the cancer. In contrast, placental ALP (PLAP) is a tumor marker related to the grade of cancer, and serum levels of this analyte are higher in Grade 1 and 2 tumors than in Grade 3 ovarian carcinomas. PROGNOSIS Prognosis is the probability of cure of a cancer patient. Positive lymph node detection is a classical method of determining prognosis invasively. The magnitude of tumor marker levels in several cancers corresponds to the mass of tumor. Moderate elevations are suggestive of better prog- nosis than persistent high levels. An important prognostic factor in ovarian and breast cancers is the amplification of the c-erbB-2 gene (HER-2/neu) and protein. Tumor aggressiveness resulting in widespread metastasis precipi- tates very high serum tumor marker levels, indicating poor prognosis. Generally, well-differentiated tumors tend to be less aggressive than undifferentiated or anaplastic tumors. While most tumor marker overexpressions indicate poor prognosis, the increased levels of progesterone and estro- gen receptors in breast cancers determine the type of treat- ment (hormone) as well as good prognosis. MONITORING AND RECURRENCE The profile of tumor marker concentration against time can mirror the condition of patients diagnosed to have cancer, for example indicating whether therapy has been successful or if remission has occurred (see Fig. 1). This is one area where tumor markers are most useful (Pannall and Kotasek, 1997; Suresh, 1996). Tumor marker profiles usually reflect one of the follow- ing classical patterns: G A rapid decline in tumor marker level to normal concen- trations following surgery or other forms of first-line therapy suggests that treatment has been successful. G The lack of a decline to basal levels following first-line therapy may indicate that treatment has only been par- tially successful. G Continued low levels of the tumor marker indicate that remission has been maintained as a result of treatment. G A subsequent rise in the concentration of the tumor marker (from the basal level) suggests a recurrence of the disease. Tumor markers can warn of renewed tumor growth or recurrence 3–12 months before other methods provide confirmation. G Decline of the marker levels after an increase has been associated with a recurrence, suggestive of the respon- siveness of a tumor to second line or subsequent treatment.
  • 4. 836 The Immunoassay Handbook G If tumor marker concentrations remain elevated after treatment, the tumor may be resistant to the therapeutic method employed and the prognosis of the patient is poor unless alternative therapeutic modalities are available. These characteristic profiles can be observed for many tumor markers, e.g., CEA in colorectal cancers, cancer antigen 125 (CA 125) in ovarian cancers, or PSA in pros- tatic cancer. Although these classical patterns in tumor marker pro- files are seen in the majority of patients, they do not reflect the clinical status of every patient. Hence, some oncolo- gists recommend the estimation of more than one marker (Wu and Nakamura, 1997). For example, in pancreatic cancer, carbohydrate antigens 19-9 (CA 19-9), 50 (CA 50), and CEA may all be elevated. However, CA 19-9 is posi- tive in 75% of the patients, whereas CEA is positive in less than 50%. In certain germ cell tumors, the combined mea- surement of human chorionic gonadotropin (hCG) and AFP is desirable to confirm diagnosis and manage patients. The association of multiple tumor markers with a range of cancers is shown in Fig. 2. FIGURE 2 The association of tumor markers with different cancer sites. FIGURE 1 Classical trends in tumor marker profiles: (a) successful first-line therapy with reduction to normal levels; (b) unsuccessful first-line therapy or partial response; (c) continued clinical remission; (d) recurrence of cancer; (e) response to second line of therapy; (f) failure of or resistance to therapy with poor prognosis.
  • 5. 837CHAPTER 9.13 Cancer Markers With the advent of automated multi-analyte analyzers for tumor markers, the assay of more than one marker is desirable and straightforward to carry out. This provides greater confidence in establishing clinical status and may become a feature of the use of tumor markers in oncology in the future. A welcome adjunct is the availability of tumor marker reference controls for several analytes like CA 19-9, CA 125, CA 15-3, and CEA from Bioref in Germany and Bio- Rad in North America. Randox has a liquid tumor marker control containing 15 cancer-related markers providing a comprehensive coverage of well-known markers. History and Classification The history of the discovery of biochemical tumor mark- ers starts with the description of a urinary substance in 1846, which is now known to be excessive secretion of the immunoglobin light chain in multiple myeloma. The next hundred years saw sporadic description of hormones (hCG, ACTH), enzymes and isoenzymes (AP, PLAP), and cytokeratins (TPA) as potential tumor markers. The devel- opment of key techniques in the latter half of the twentieth century was important for the rapid discovery and devel- opment of new tumor markers and their immunoassays. Central to the theme of this entire book is the develop- ment of the immunoassay concept in the 1950s by Yalow and Berson, involving the application of antibodies as reagents to measure specific substances in complex mix- tures. Numerous immunoassays emerged using polyclonal antibodies subsequent to this period, although most were to measure noncancerous analytes. It was in the early 1970s that the CEA immunoassay was introduced as a commercial test for cancer. MAb techniques introduced in 1975, and the development of the immunometric (sand- wich) immunoassay format in 1982, revolutionized the field of tumor markers. This resulted in a virtual explosion in the discovery of new tumor antigens and the introduc- tion of several among them as immunoassays for routine clinical use. Recombinant antibody techniques also pro- vided invaluable insights into the understanding of the structure and putative functions of tumor markers. Molec- ular biology techniques were key to the recent discovery of several emerging tumor markers belonging to the classes of oncogenes, tumor suppresser genes, and a host of other molecules including angiogenic factors, cyclins, nuclear matrix proteins (NMPs), cell adhesion factors, heat shock proteins, growth factors and their receptors, and telomer- ase (Wu and Nakamura, 1997; Suresh, 1996). Almost every new major, and many minor, molecule dis- covered in the last 10 years has been investigated as a potential new tumor marker. Hence, it is beyond the scope of this chapter to describe all of these new tumor markers, and the reader is directed to an excellent book devoted to this topic (Wu and Nakamura, 1997). A classification of tumor markers is provided in Fig. 3. Almost all tumor markers are now considered tumor- associated antigens, due to their expression to some extent in some noncancerous tissues. However, rare examples of highly specific tumor antigens can be recognized as a separate subgroup. These include the B-cell tumor immonoglobulin idiotype (the unique paratope or variable region of the specific immunoglobulin expressed on the sur- face or as a secreted myeloma protein), T-cell receptor of T-cell leukemia, mutated forms of oncogenes and tumor supressor genes, and several virus-induced antigens found predominantly in nonhuman cancers. Tumor-associated markers can be classified into two subcategories based on the size of the molecules. The macromolecular markers have several categories including proteins, genes, chromo- somes, and histologically identifiable cellular markers. NOMENCLATURE AND IDIOSYNCRASIES OF GLYCOPROTEIN TUMOR ANTIGENS The discovery of several new large glycoprotein tumor markers has been fueled by mAB technology. However, a word of caution is relevant at this juncture to illustrate the misuse of mAb technology. Claims of discovery of a new tumor marker are abundant, based mainly on the develop- ment of a new monoclonal and its putative recognition of a new tumor antigen. Several monoclonals have been devel- oped measuring complex and large glycoproteins elevated in breast, ovarian, pancreatic, gastric, lung, and colorectal cancers. The nomenclature of many of these cancer antigens (CAs) has been derived from the arbitrary clonal designa- tions of the various mAbs. For example, the mAb OC125 was developed by immunizing mice with human ovarian cancer cells (Bast et al., 1981). The antigen identified by this mAb is now recognized as CA 125. Similarly, CA 19-9 antigen was originally identified by the NS19-9 mAb. Some confusion has been introduced in the tumor marker FIGURE 3 Classification of tumor markers.
  • 6. 838 The Immunoassay Handbook literature, due to the development of several different mAbs to overlapping or distinct epitopes on the large gly- coprotein antigens with claims of identifying new markers, with increased clinical sensitivity/specificity of one over the other. Adding to this confusion is the question of what is measured by these glycoprotein tumor marker assays— epitope, antigenic determinant, domain, or the antigen. It is sufficient to state that every tumor marker assay mea- sures antigens, albeit via unique mAbs binding to unique epitopes. New tumor marker antigens/epitopes have been described purely based on the development of a new mAb without making serious attempts to compare and contrast it with preexisting mAbs and antigens. This important issue is not easy to resolve due to the idiosyncrasies of the large cancer glycoproteins. Unlike traditional analytes, these large cancer glycoproteins and mucins have special features (Suresh, 1991). The precise estimation of these glycoproteins is influenced by pH, valency, and distribu- tion or density of the epitope and serum anti-carbohydrate antibodies. An anomalous feature unique to these analytes is that many cancer serum samples exhibit increased recov- ery/estimation of the antigen upon dilution. It is not often appreciated that human serum has a substantive amount of anti-carbohydrate IgM and lgG antibodies that can inter- fere with glycoprotein immunoassays. It appears that in serum, these large cancer glycoproteins can exist as supramacromolecular complexes promoted partly due to glycan–glycan interactions and partly as a result of weak cross-linking by anti-carbohydrate antibodies. This hypothesis (Suresh, 1991) explains why upon dilution or lowering the pH of serum during assay (e.g., CA 19-9 kit, Fujirebio—previously Centocor), one can recover higher amounts of the antigen in an immunoassay, presumably due to disassociation of the large complexes. This concept also can explain the often observed phenomenon of co- expressions of CA 125, CA 19-9, CA 15-3, sialyl LewisX, LewisX, LewisY and other antigens. An attempt to critically study some of these complex issues has been initiated by the International Society For Oncodevelopmental Biology and Medicine (ISOBM). They initiated tissue differentiation (TD) workshops a few years ago, analogous to the CD workshops to classify leu- kocyte antigens. These workshops have been conducted for several tumor markers such as CEA (Hammarstrom et al., 1989; Nap et al., 1992), CA 125 (Nustad et al., 1996; Nap et al., 1996), AFP (Alpert and Abelev, 1998), PSA (Stenman et al., 1999), MUC1 (Price et al., 1998), cyto- keratins (Stigbrand et al., 1998), and sialyl Lewis A (Rye et al., 1998), primarily to standardize and compare the numerous mAbs described by different groups putatively identifying the same antigen. For example, the TD-4 workshop compared the 56 different mAbs against the MUC1 mucin (the international antigen designation for CA 15-3 or CA 27.29). The majority of the antibodies (34/56) apparently react with the 20 amino acid tandem repeat sequence of the core peptide of MUC1 mucin (TAPPAHGVTSAPDTRPAPGS). Many of the remain- ing antibodies react with carbohydrate epitopes. This type of analysis at least takes the first step toward a blind com- parison of the various antibodies, all of which are puta- tively measuring the same antigen with various affinities and overlapping epitope specificities. It is not surprising that the clinical sensitivities and specificities of breast immunoassays constructed using the various antibodies are essentially similar, but some may exhibit unique or subtle abilities to be clinically more useful than others. In the remainder of this chapter, the analytes generally regarded as being the most clinically useful are discussed in detail. Unfortunately, an exhaustive survey of all the can- cer markers utilized in various continents is not possible in this review, as also the numerous tumor markers and their assays described in the research literature (see references). A host of new analytes has appeared on the commercial scene in the last few years, and the clinical utility of many of these needs to be further explored. NEW DEVELOPMENTS Two new developments that are likely to open new vistas in cancer research need to be addressed. One is the emerg- ing fields of genomics and proteomics that have ushered in a new era of diagnostic possibilities. Several new cancer- associated/specific genes and corresponding assays have been developed. A new paradigm of understanding the total proteome fingerprint patterns is underway, and serum/plasma is the largest repository of the low and high abundant proteins. Human serum is estimated to contain 30,000 proteins, and six proteins—albumin, immunoglob- ulin G, alpha-1-antitrypsin, transferrin, immunoglobulin A, and haptoglobulin—account for ~85% of the bulk. New techniques are being developed including gene and pro- tein array technology coupled with sophisticated mass spectrometry procedures to detect the disease-specific/ associated markers. A new ovarian cancer test based on an algorithm of reading proteomic fragmentation patterns has been described with the claim of 100% accuracy. In 2003, the FDA approved the first commercially available bladder cancer-detection blood test, based on proteomics technology, called the BladderChek®. Several new groups and companies are developing multiplexed immunoassays based on microarray and nanoarray platforms. It is likely that such assays for multiple analytes may be approved for routine use in the future. Several new cancer therapeutic biotechnology medicines have been approved based on the understanding of cancer markers. These include monoclonals as cancer therapeu- tics such as Herceptin® for breast cancer-associated marker HER-2/neu, Rituximab® for B-cell cancers, and the intense research and development on CA-based thera- peutics, often referred to as cancer vaccines. Cancer mark- ers have come a long way since their first discovery nearly 150 years ago and could be the basis of dominant treatment strategies in the future. Analytes CARCINOEMBRYONIC ANTIGEN The discovery of CEA and AFP, half a century ago, ush- ered in a renewed interest in human tumor markers. The development of radioimmunoassay technology for insulin a few years earlier had set the stage for the emergence of new noninvasive techniques to aid the cancer patient. Thus, CEA and AFP are considered classical tumor
  • 7. 839CHAPTER 9.13 Cancer Markers markers. Gold and Freedman (1965), in their landmark experiment, immunized rabbits with extracts of human colon cancer tissue. The resulting rabbit antiserum was absorbed with extracts from normal human gut tissue, and the enriched polyclonal antibody obtained reacted specifi- cally to cancer tissues and their extracts. Because the anti- gen identified was also found in embryonic tissues, the term CEA was introduced. Both CEA and AFP are mem- bers of the family of oncofetal antigens that are normally expressed only in any quantity in embryonic development but are also found in adult neoplastic tissues. CEA is one of the most widely used tumor marker immunoassays with sales of $50–100 million worldwide. CEA is a heavily glycosylated cell-surface glycoprotein and one of a large family of related molecules belonging to what is now fashionably called a superfamily, which also includes immunoglobulins. This general classification is based on the degree of similarity between the domains of different proteins. Nearly 36 different glycoproteins have been identified in the CEA family, and they appear to be derived from 10 genes localized on chromosome 19 in two clusters. CEA is a non-mucinous, 180kDa glycoprotein secreted by the epithelial cells of the digestive tract in the normal fetus and in adult cancers. It exhibits β-electrophoretic mobility and contains 60% carbohy- drate by weight, constituting N-acetylglucosamine, man- nose, fucose, galactose, and sialic acid. The oligosaccharide chains are approximately 80 in number, linked to the poly- peptide core by asparagine-N-acetyl glucosamine linkages (this is termed an N-linked oligosaccharide core in con- trast to the serine or threonine-O-linked core typical of mucins) with a high proportion of branched oligosaccha- ride chains. Although CEA has a high carbohydrate con- tent, due to the composition of the sugars and the predominant N-linked oligosaccharide chains, it is not considered a typical mucin like CA 19-9 or CA 15-3. Con- siderable heterogeneity exists in CEA preparations from various sources, and this is probably due to variation in the oligosaccharide chains. In contrast, the polypeptide chain is fairly consistent between different preparations of CEA. The single protein chain consists of approximately 829 amino acids with sev- eral intra-chain disulfide bonds. Monoclonal antibodies utilized in CEA assays bind primarily to the protein chain rather than to the oligosaccharides. A number of molecules with structures similar to CEA have been discovered. These include normal cross-reactive antigens (NCA 1 and NCA 2), tumor-extracted related antigen (TEX), normal fecal antigens (NFA 1 and 2), meconium antigen (MA), and biliary glycoprotein 1 (BGP- 1). Higher molecular mass forms of CEA have been reported from some colon tumor extracts. The concept of organ-specific CEA has also been proposed. Function As with many tumor markers, the function of CEA, and the reason for its appearance in the serum of patients with cancer, is largely a mystery. As with mucins, the associa- tion of CEA with the epithelial cells of the digestive tract may suggest a protective role in the turnover of the diges- tive epithelium. It is estimated that 70mg/day of CEA is normally secreted into the digestive lumen to eventually end in feces, and its appearance in blood is presumed to be due to a reversal or loss of the normal polar secretory func- tion of the epithelial cells (Pannall and Kotasek, 1997). Reference Interval Typically, 2.5ng/mL is used as an upper limit for normal nonsmokers and 5ng/mL for smokers. The World Health Organization (WHO) made avail- able the first International Reference Preparation for CEA (73/601). One International Unit of this standard is equiv- alent to 100ng of CEA glycoprotein. Clinical Applications CEA is one of the most widely used tumor markers in oncology today, now surpassed only by PSA. Despite its widespread use, it is not suitable as a screening test for asymptomatic people, nor is it a reliable diagnostic test in patients with symptoms that may be due to cancer. This is because of the considerable incidence of false positives and false negatives. However, the presence of carcinoma is strongly indicated in patients with elevated values, and CEA is often a very useful test as part of the multiparamet- ric diagnosis of cancer. The most significant use of CEA assays is in the management of cancer patients by serial monitoring to determine the following: G the recurrence or metastatic spread of cancer after first- line therapy; G the presence of residual or occult metastatic cancer; G the effectiveness of therapy; and G the prognosis and staging of patients, when used with other additional information in colorectal and lung cancer. Most colorectal patients with preopera- tive CEA in excess of 20 ng/mL would manifest recurrence within 14 months after surgery (Wu and Nakamura, 1997). Although CEA is primarily associated with colorectal can- cers, other malignancies that can cause elevated CEA con- centrations are those arising from the lung, breast, stomach, ovary, pancreas, and other organs. A number of benign conditions may also be responsible for CEA levels significantly higher than normal. These include inflamma- tory diseases of the lung and gastrointestinal (GI) tract and benign liver disease. Heavy smokers, as a group, also have an elevated range of CEA values. However, the most useful clinical application of CEA analysis is as a noninvasive test for the recurrence of colorectal cancer. This is particularly diagnostic in patients whose postoperative levels initially decrease to a normal level within 6 weeks. CEA concentrations are significantly elevated when the liver is the metastatic site for a primary colorectal cancer. Patients with elevated preoperative lev- els of CEA that fail to reduce to normal after the first-line therapy are suspected of having residual disease or occult cancer. In all these patients, the rise or fall of CEA values generally reflects progression or regression of disease as a function of the therapeutic treatment. CEA can be used to stage disease and estimate the prog- nosis. A good correlation exists between preoperative CEA values and increased risk of recurrence of disease,
  • 8. 840 The Immunoassay Handbook particularly in Dukes’ C stage of colorectal cancer. Fully differentiated colorectal cancer tends to secrete CEA copi- ously compared to undifferentiated tumors, which are associated with low levels or do not express the antigen. Elevated CEA levels are also common in breast and lung cancer patients with disseminated disease. Increases in CEA values are usually not apparent in localized or pri- mary disease. The profiles of CEA values in patients being treated for metastatic breast cancer appear to correlate well with the therapeutic effectiveness. Limitations G Most of the immunoassay tests for CEA are reasonably well correlated. However, it is important to note that the different antibodies utilized have subtle differences in their affinity for CEA and in their cross-reactivities to CEA-like material. Hence the switching of immuno- assay kits during the course of monitoring a single patient is usually not recommended. G CEA has a low clinical sensitivity and specificity as a tumor marker and is hence not recommended for screening. Clinically, an elevated CEA value is in itself not of diagnostic value as a test for cancer and this parameter should only be used in conjunction with other clinical observations and diagnostic parameters. Some patients with colorectal cancer do not exhibit elevated CEA values and elevated CEA levels in some patients do not change in accordance with progression or regression of disease. CEA values can be elevated in a number of benign conditions. G Smokers constitute a distinct group with a higher range of baseline values. Assay Technology Assays for CEA employ the sandwich (immunometric) assay principle with either an enzyme or non-enzyme label as the signal generation method. Most immunoassays for CEA utilize a pair of monoclonal antibodies or a combina- tion of a polyclonal capture antibody with a monoclonal- labeled antibody. Types of Sample Serum or EDTA plasma. Frequency of Use Very common. ALPHAFETOPROTEIN The Russian scientific group led by Abelev in 1963 dis- covered the presence of AFP in adult mice with hepato- mas (liver cancer). The protein is abundantly present in the fetus, and levels decline rapidly after birth. This important milestone in the history of tumor marker oncology resulted in the emergence of the concept of oncodevelopmental or oncofetal antigens as possible tumor markers. The notion of regarding a tumor or can- cerous state as simulating the fetal or ontogenic pheno- type subsequently emerged. Many other oncofetal tumor markers have since been described including CEA and other cell surface glycoconjugates. The dedifferentiation of the adult cells and tissues expressing these early embry- onic antigens suggests that they may be involved in cell division and the regulation of growth. AFP is a 70kDa glycoprotein with a single polypeptide chain. It is similar to serum albumin in size, structure, and amino acid composition but has distinct immunological properties. Unlike AFP, however, albumin is not a glyco- protein. A fucosylated form of AFP has been identified as being associated with liver cancer but not in benign liver diseases. AFP is synthesized by the liver, yolk sac, and GI tract of the fetus, reaching a peak serum concentration of up to 10mg/mL at 12 weeks of gestation. This peak level gradually decreases and, 1 year after the birth of the new- born, the serum levels decrease to less than 25ng/mL. Albumin becomes the major serum component in adult serum with concentrations up to 60mg/mL. Functions AFP is one of the major components of fetal serum and is replaced by albumin postpartum. Both of these proteins are known to be responsible for the maintenance of serum osmotic pressure and to have various transport functions. Reference Interval One hundred per cent of healthy males and 97% of healthy nonpregnant females have AFP values less than 15ng/mL (Abbott AFP EIA). Clinical Applications AFP determinations are used primarily in two areas. The application of this test in the detection of open neural tube defects in the fetus is described elsewhere (see PREGNANCY). In Asia, due to the high prevalence of liver cancer, particu- larly in the hepatitis and cirrhosis groups, AFP has been successfully used in screening applications. The other common application in the field of cancer is in the management of germ cell tumors and hepatomas. The most common testicular malignancies derive from seminiferous tubules and germ cells. They are classified into two groups, namely seminomas and non-seminomas. The non-seminomatous group includes the embryonal carcinomas, teratocarcinomas, and choriocarcinomas. The magnitude of AFP elevation has been found to cor- relate with the stage of non-seminomatous testicular can- cers, particularly in the embryonal carcinoma group. For example, continued AFP elevations following orchidec- tomy (removal of a testis) suggest that the disease is at stage II or beyond. When sequential monitoring gives rise to a profile of continued elevations or a rise in AFP levels, residual disease or a recurrence is strongly suggested. Decreases in AFP levels in patients are associated with clinical remission. hCG is often used in conjunction with AFP to monitor testicular cancers, particularly non-semi- nomatous tumors of the choriocarcinoma type (see HUMAN CHORIONIC GONADOTROPIN). AFP levels are elevated in more than 60% of liver can- cers. Hepatomas are not common in the western world but are more prevalent in Africa and Asia. A strong etiological association between hepatomas and viral hepatitis, other
  • 9. 841CHAPTER 9.13 Cancer Markers infections, and aflatoxin poison ingestion, has been observed. Clinically, the response to treatment and hence prognosis of patients with liver cancer has been poor to date. Nevertheless, AFP determinations have been useful in monitoring these patients during the course of their treatment. A few AFP screening programs for hepatomas have been conducted in high-risk populations with consid- erable success. Limitations G AFP levels are known to be elevated in a number of benign diseases and conditions including pregnancy and nonmalignant liver diseases such as hepatitis and cirrhosis. G Although limited screening studies have been con- ducted with promising results, AFP is neither recom- mended as a screening test or as a diagnostic test. Assay Technology Immunometric assays are used for AFP determinations. These normally use a pair of monoclonal antibodies or a polyclonal capture antibody with a labeled monoclonal antibody. Types of Sample Serum or plasma (and amniotic fluid for pregnancy applications). Frequency of Use Common. CARBOHYDRATE ANTIGEN 19-9 (SIALYL LEWISA) Carbohydrate antigen 19-9 (CA 19-9) or sialyl Lewisa is a tumor marker predominantly associated with pancreatic, gall bladder, gastric, and colorectal cancers, which are col- lectively classified as GI malignancies. The term GI can- cer-associated antigen (GICA) has also been used, though less frequently, to identify the same antigen. The antigen was originally described as a cell surface monosialogangli- oside isolated from the SW1116 human colorectal carci- noma cell line, grown in vitro. The antigen has the chemical structure shown in Fig. 4 along with other related tumor antigens. The original hybridoma secreting the mAb 1116 NS-19.9 used to characterize the ganglioside antigen was developed by immunizing mice with the SW1116 human cancer cells. The minimal structure recognized by this antibody, and several other antibodies developed subse- quently, appears to be the terminal tetrasaccharide of the CA 19-9 antigen. Deletion of the sialic acid moiety or the fucose residue abolishes or greatly reduces the antigen– antibody interaction. The first comparative study (TD-6 Workshop) of 20 monoclonal antibodies against sialyl Lewisa and related antigens was completed by Rye et al. (1998). Cross-reactivities to closely-related oligosaccha- rides such as sialyl Lewisx, Lewisa, Lewisx, LSTa (CA 50) and others (Fig. 4) were studied. Most antibodies reacted to the sialyl Lewisa antigen and exhibited varying degrees of cross-reactivities to related structures. The subtle dif- ferences in their cross-reactivities and affinities, together with the class of the antibody (bivalent IgG versus decava- lent IgM measuring polyepitopic mucinous antigens), could explain the spectrum of clinical results obtained. Claims of superior sensitivities and specificities have been made for the various immunoassays even though these are recommended only for monitoring and not for screening or diagnosis. The CA 19-9 antigen was initially found to be present in serum from patients with GI malignancies but not in normal sera. Based upon these findings, it was hypothe- sized that the ganglioside antigen was shed into the serum. However, a more detailed investigation revealed that the circulating antigen was a high molecular mass mucinous antigen. Virtually no ganglioside antigen was found in the serum of cancer patients. Several other forms of sialyl Lewisa antigen have been described from seminal plasma, normal saliva, and human milk. A sialyl Lewisa hexasaccharide with a reducing end (i.e., without the ceramide of the CA 19-9 ganglioside) has been purified from human milk. Thus, it is now established that multi- ple species of CA 19-9 antigens exist with univalent (monosialoganglioside, hexasaccharide), oligovalent, or polyvalent (glycoproteins and mucins) expression of sialyl Lewisa residues. The mucinous form of the antigen has been further characterized following purification. The subunit struc- ture of the mucin appears to be a 210kDa glycoprotein which, in the absence of detergents or other dissociating conditions, aggregates to form higher relative molecular mass species in the range of 600–2000kDa. More than 85% of the glycoprotein is carbohydrate by weight. About 35% of the core protein is composed of serine, threonine, and proline, a feature typical of epithelial tumor-associated mucin antigens. FIGURE 4 Carbohydrate tumor and related antigens.
  • 10. 842 The Immunoassay Handbook Function The CA 19-9 antigen is a sialated derivative of the Lewisa blood group antigen. The specific function of such anti- gens is largely unknown although a number of theories have been put forward. Gangliosides are thought to be involved in interactions between cells. Mucins derived from epithelial cells may have a protective role. Milk oli- gosaccharides appear to have a bacteriostatic effect. Reference Interval Typically a reference interval of 0–37U/mL is used (this may vary according to the method). The arbitrary unit of antigen has been given a gravimetric value of 0.89ng. Clinical Applications In a significant number of GI malignancies, CA 19-9 levels are elevated above the 37U/mL level. This is particularly pronounced in pancreatic and gall bladder cancer patients, followed by gastric and colorectal cancer patients. Like all tumor marker assays currently available commercially, the clinical sensitivity of the CA 19-9 marker is moderate in early-stage disease. The important feature of CA 19-9 assays is the high specificity of the test. Less than 1% of apparently healthy blood donors exhibit elevated values. A number of benign conditions related to GI disease have been tested for the presence of the antigen, and although levels are higher than the healthy blood donor group, they tend to be much less elevated than those samples from can- cer patients that give a positive result in the test. CA 19-9 levels are found to be remarkably high in symp- tomatic pancreatic and gall bladder cancers. The mean serum level for these cancers is 10–100 times higher than those for gastric and colorectal cancers. Because benign gall bladder disease, pancreatitis, and benign hepatobiliary conditions can frequently cause CA 19-9 levels above the range for healthy individuals, it is helpful to use an elevated cutoff level for cancers of the pancreas and gall bladder. The main clinical application of CA 19-9 determina- tions is in the monitoring of pancreatic cancer patients. CEA is usually preferred to watch colorectal cancer. CA 19-9 can also be elevated in other forms of digestive tract cancer, especially cancers of the stomach and bile ducts and in some noncancerous conditions such as thyroid dis- ease, inflammatory bowel disease, and pancreatitis (inflam- mation of the pancreas). Limitations G One of the most important limitations of CA 19-9 determinations is the particular sensitivity of the assay and the tumor marker to viral and bacterial neuramini- dases resulting in false negatives. Serum samples should be carefully prepared to avoid bacterial contamination. G The Lewis blood group antigens are classified into Lewisa (approximately 40%), Lewisb (40%), Lewisab (15%), and Lewisa−b− (5%). CA 19-9 antigen is not syn- thesized in individuals who are genotypically Lewisa−b− because of the lack of the enzyme fucosyl transferase. G The distribution of antigen levels in normal donors may vary because of the population distribution of Lewis genotypes in a given geographical area. Most manufacturers recommend that establishment of cutoff values is determined by the clinical laboratory. G Nonlinear dilution, with increased recovery of the anti- gen, is common in immunoassays for mucins. This is probably due to a variety of factors such as the presence of high levels of anti-carbohydrate antibodies in serum, which generate complexes, the inherent property of mucins to aggregate and disaggregate into a range of molecular species, and other matrix-related effects (Suresh, 1991). G Elevated levels of CA 19-9 can be found in some benign conditions such as cirrhosis and other liver diseases, gall bladder disease, pancreatitis, and cystic fibrosis, thus limiting the diagnostic utility of the marker. Assay Technology Most of the kits developed for CA 19-9 utilize immuno- metric (sandwich) assay methodology, although one kit (TRUQUANT® GI™ RIA) is based on competitive inhi- bition assay. The sandwich assay format utilizes the same antibody for capture and signal generation. Thus, the CA 19-9 species measured by this homo-sandwich technology needs to be oligovalent or polyvalent for sialyl Lewisa. The competitive assay for CA 19-9 uses a solid phase coated with CA 19-9 and has the potential to measure all species of CA 19-9 irrespective of the valency for sialyl Lewisa. Types of Sample Serum or plasma. Some assays are validated only for serum samples. Frequency of Use Common. CANCER ANTIGEN 125 (MUC16) CA 125 is the most important cancer-associated marker for the management of ovarian cancer. It was discovered using a monoclonal antibody, OC125, generated by immu- nizing a mouse with a human ovarian cystadenocarcinoma cell line. This antibody exhibits specificity for staining epi- thelial ovarian carcinoma cell lines and tumor tissues. The CA 125 antigen is also expressed in a number of gyneco- logical, non-ovarian, and normal tissues of Müllerian ori- gin. Several other monoclonal antibodies have been developed subsequently to measure CA 125 antigen, and a comparative blind evaluation was the subject of the TD 1 workshop. These CA 125 antibodies appear to cluster into two major epitope groups, namely “OC125” like and “M-11” like (Nustad et al., 1996). Two newer antigens CA 130 and CA 602 were described that appeared to be CA 125-like. The CA 130 antigen employs 130-22 as the solid-phase antibody and OC125 as the tracer, while the CA 602 antigen is measured using the two anti-clear cell ovarian cancer Mabs MA602-1 and MA602-6. CA 125 is now known as mucin 16 or MUC16 as it is encoded for by the MUC16 gene. MUC16 is a large, membrane-associated glycoprotein containing about 22,000 amino acids (3–5 million Da on average, in epi- thelial cells).
  • 11. 843CHAPTER 9.13 Cancer Markers Function The CA 125 antigen is expressed in copious amounts in the tissues and serum of epithelial ovarian carcinoma patients. The antigen is minimally present in normal serum, or in adult or fetal ovaries. Using immunohisto- chemical techniques, CA 125 reactive material has been detected in some normal tissues such as adult pleura, peri- cardium, peritoneum, Fallopian tubes, endometrium, and endocervix. The antigen is also found in the chorionic membrane, extracts of maternal decidua, and, abundantly, in the amniotic fluid. Amniotic fluid CA 125 has two dis- similar subunits and is not derived from the fetus. It is present in the cornea, conjunctiva in the eye, and the respi- ratory tract and female reproductive tract epithelial cells. The high level of glycosylation creates a hydrophobic environment that acts as a lubricating barrier against for- eign particles and infectious agents. Reference Interval Tests for CA 125 typically adopt a 35U/mL discrimina- tion value that encompasses 99% of healthy donors. Clinical Applications The measurement of CA 125 antigen is very helpful in the management of serous ovarian carcinomas. Epithelial ovarian carcinomas frequently metastasize into the perito- neal cavity on the serosal surfaces, often producing ascites. Primary ovarian cancer is usually treated by surgically removing the ovaries and giving the patient chemotherapy to ablate any residual disease. CA 125 antigen measure- ment is used to monitor residual tumor burden in patients who have undergone such therapy. Antigen levels above the normal range are usually predictive of residual or recurrent ovarian carcinoma if other causes of CA 125 elevations can be eliminated (see LIMITATIONS). This intended clinical use of the CA 125 antigen assay was approved by the US FDA in 1986, and its routine use has had a strong positive impact in the management of epithe- lial ovarian cancers. Subsequently, numerous clinical reports have appeared in the literature suggesting the extension of the use of CA 125 immunoassays for a variety of other oncological applications such as limited diagnosis of ovarian cancer, monitoring of ovarian, lung and breast cancer patients, and in applications involving endometrial and fallopian tube cancer. Some studies have attempted to establish that CA 125 could be used to screen for ovarian cancer, using a higher cutoff value than the usual 99% confidence interval for normals. But because ovarian can- cer is relatively rare, and increased CA 125 levels due to a range of causes are not uncommon, this test is considered to be unsuitable for screening. Limitations G CA 125 antigen levels are elevated above the recom- mended cutoff value in 1% of normals, 5% of benign diseases, and 28% of non-gynecological cancers. The benign conditions associated with increased CA 125 in serum include ovarian cysts, severe endometriosis, menstruation, first trimester of pregnancy, cirrhosis, and pericarditis. G Higher CA 125 antigen levels are also found in non- ovarian tumors such as those originating in the breast, lung, uterus, endometrium, pancreas, and liver. Some early attempts were made to use CA 125 to classify unknown cancers as being from an ovarian primary. However, the presence of elevated CA 125 antigen lev- els in non-ovarian cancers limits the potential of such an application. G Increased CA 125 antigen levels are found in a number of ascites fluids and pleural effusions in both malignant and benign conditions. In our experience, we have found CA 125 levels in these fluids in the range of 2000–500,000U/mL. G Radiolabeled OC125 antibody has been used to identify cancer sites in vivo often missed by other diagnostic meth- ods. The injection of the mouse antibody into humans elicits a human anti-mouse antibody (anti-isotypic and anti-idotypic HAMA) response, capable of increasing the apparent CA 125 concentration in subsequent serum samples tested in immunometric assays. Assays employ- ing alternative antibodies may be used in these situations. G As with CA 19-9, increased recovery of antigen can occur in dilution experiments. Considerable caution should be exercised when carrying out comparisons between meth- ods or changing from one method to another. Assay Technology CA 125 assays are based on immunometric (sandwich) assay methodology. Types of Sample Serum or plasma. Some assays are validated only for serum samples. Ascites and pleural effusions should not be used as they have higher antigen levels than are found in serum. Frequency of Use Common. CANCER ANTIGENS 15-3 AND 27.29 (MUC1) The breast cancer-associated antigen CA 15-3 is a large mucinous glycoprotein with a native molecular mass in excess of 400kDa. The antigen is identified using a sand- wich (immunometric) assay employing two monoclonal antibodies. The solid-phase mAb 115D8 was generated by immunizing mice with defatted human milk fat globule (HMFG) antigens. The tracer mAb DF3 was developed against enriched antigens from the membrane of human breast carcinoma metastasis. mAb DF3 is more specific for cancers than 115D8. The sandwich assay that uses these two antibodies detects antigens that have been variously described as MAM6, milk mucin, human mammary epithelial anti- gen, HMFG antigen, and polymorphic epithelial mucin. The first international workshop on cancer-associated mucins assigned the name MUC1 to the breast cancer- associated mucin. It is expressed by the MUC1 gene. The antigen identified by the DF3 antibody in human milk con- sists of a single high-molecular-mass species, whereas in breast carcinomas, the antibody binds to two glycoproteins
  • 12. 844 The Immunoassay Handbook with molecular masses of 330 and 450kDa. Approximately 50% of the composition by mass is carbohydrate. The anti- genic site identified by DF3 appears to be sensitive to neur- aminidase, alkaline borohydride treatment, and proteases, suggesting that it is a combined sialyl oligosaccharide and peptide on the CA 15-3 antigen. Microheterogeneity and genetic polymorphism are observed in these epithelial anti- gens causing considerable variation in the size of native oligomers, subunits, and core proteins from different sources. Recently, a 309 base pair cDNA, encoding the sequence for the DF3 antigen, has been isolated, and using this probe, it was demonstrated that the polymorphism of these mucins is a reflection of the variations in the size of the alleles. The conserved sequence is rich in guanine and cytosine with a 60 base pair tandem repeat encoding a ser- ine-, threonine-, and proline-rich polypeptide. The num- ber of tandem repeats of this 20 amino acid sequence (PDTRPAPGSTAPPAHGVTSA) is thought to be the basis for the polymorphism in these mucins. This peptide has been synthesized without any oligosaccharide chains, and at high concentrations, it can block DF3 binding to solid-phase mucin antigen. A number of other antibodies have been prepared that apparently react with the same family of polymorphic epi- thelial mucins (e.g., CA 27.29, CA 549, MCA, etc.). These 56 mAbs were investigated in the TD-4 workshop by 16 international groups (Price et al., 1998). Most of the mAbs (34/56) were mapped within the immunodominant 20 amino acid tandem repeat domain. The bulk of the remaining antibodies appear to recognize carbohydrate- incorporating epitopes. CA 27.29 is routinely used although it does not appear to be any better than CA 15-3 in terms of clinical sensitiv- ity, but it may be more specific to cancers, i.e., it is less likely to be positive in patients without cancer. Function MUC1 is found in normal and cancerous epithelial cells and, as a mucin, it is often assumed to play a protective role. The antigen constitutes approximately 15% of the total membrane protein of HMFGs. The quantity of DF3 antigen expressed appears to correlate with the degree of breast cancer differentiation. Because human milk also contains the antigen, the DF3 antigen is considered a marker of differentiation of mammary epithelial cells. Reference Interval Typically, the normal range for CA 15-3 is considered to be less than 30U/mL in the Centocor CA 15-3 RIA kit. But women without cancer may have levels as high as 100U/mL. The normal level for CA 27.29 is usually less than 40U/mL. Clinical Applications The CA 15-3 antigen is an epithelial membrane antigen expressed on normal cells and found in serum. Elevated levels of this antigen are found in about 60% of preopera- tive breast cancer and 80% of advanced metastatic breast cancer. Breast cancer is one of the most common cancers in women in the western world, and the CA 15-3 assay has proved to be helpful in patient monitoring, with better clinical sensitivity than CEA assays. An advantage over CEA is that the antigen levels are not abnormally elevated in smokers. The CA 15-3 assay is not suitable as a diagnos- tic test because of its low sensitivity in stage I and II dis- ease, but in advanced mammary carcinomas, trends in the antigen levels provide a useful noninvasive indicator of early recurrence, presence of residual disease, and contin- ued remission or poor prognosis. Combined use of CA 15-3 and CEA does not appear to give any improved clini- cal information. Elevated levels are found in less than 10% of patients with early disease and in about 70% of patients with advanced disease. Levels usually fall if treatment is success- ful, but initially the levels can rise as dead cells release their contents into the blood. CA 27.29 is no better at detection of cancer at any stage than CA 15-3 but may be less likely to be positive in patients without cancer. It can be detected in other cancers and some noncancerous conditions. Limitations G CA 15-3 is only elevated in 10% of patients with early- stage breast cancer. G CA 15-3 is sensitive to proteases and neuraminidases, and hence it is important to prepare and store samples with great care to avoid microbial contamination. G Levels of CA 15-3 can also be elevated due to lung and ovarian cancers. G Elevated values are seen in less than 10% of benign dis- eases of liver, breast, ovary, GI tract, and lung. G The polymorphic, glycoprotein structure of MUC1, detected by CA 15-3 and CA 27.29, presents similar assay problems (e.g., dilution nonlinearity) to those described for other mucins such as CA 19-9. G CA 27.29 is not elevated in all patients with breast cancer. G CA 27.29 may be elevated in some noncancerous and cancerous conditions other than breast cancer. Assay Technology The TRUQUANT BR RIA is a competitive assay with a mucin-coated solid phase and was the first breast cancer MUC1 marker test approved by the FDA in 1995. Subsequently, the reclassification of tumor markers as class II devices by FDA allowed other similar immunoassays to be approved as well. CA 15-3 assays are usually immunometric, utilizing two different monoclonal anti- bodies. For example, mAb 115D8 is used as the solid-phase capture antibody, and labeled DF3 is employed as the sig- nal generation mAb. The tracer appears to have a more restricted antigen specificity than the capture mAb. An FDA-approved, automated, 15min, luminescence-based assay for CA 27.29 has also been developed (Siemens Healthcare Diagnostics, USA). Types of Sample Serum or plasma. Some assays may only be suitable for serum sample.
  • 13. 845CHAPTER 9.13 Cancer Markers Frequency of Use Common in Japan, Europe, and the USA. ESTROGEN RECEPTOR AND PROGESTERONE RECEPTOR Estrogens are female sex hormones synthesized by the ovary and adrenal cortex. The hormonal action of estro- gens is mediated by an estrogen receptor (ER) protein called estrophilin, which is present in the nuclei of target cells. It was originally believed that β-estradiol, the major estrogen, was bound by a cytosolic ER that subsequently underwent macromolecular size alterations prior to trans- location into the nucleus to regulate gene expression as a transcription factor. It is now understood that most, if not all, of the ER is a nuclear protein with a high affinity for estradiol. The dissociation constant (kd) is in the range of 10−10–10−9 M. This 66kDa protein has a steroid-binding site as well as a DNA-binding site. Upon binding of the steroid, the complex binds to DNA and regulates gene expression. The estrogen receptors in human breast tissue generally decrease during the development and onset of mammary carcinoma. The estimation of ER in breast can- cer tissues is an important aid in deciding the course of treatment. Progesterone is a steroid hormone that influences the endometrium to allow implantation of the fertilized ovum and its gestation. It is biochemically also a precur- sor of adrenal corticosteroids, estrogens, and androgens. The cellular progesterone receptor (PR) has two molec- ular components with molecular masses of 120 and 95 kDa. Estrogen modulates the appearance of the PR and its analysis complements the information derived from ER assays. Function The function of ER is to act as the second messenger of estrogen action by regulating gene expression in the nucleus. The estrogen–ER complex is capable of stimulat- ing gene expression by acting on nuclear DNA. Progester- one upon binding to PR promotes binding and activates the hormone-specific genes. Reference Interval A cutoff of 10fmol ER per mg cytosol protein is recom- mended for the Abbott ER enzyme immunoassay. The PR enzyme immunoassay has a cutoff level of 15fmol/mg cytosol protein. Clinical Applications Approximately two-thirds of endometrial and breast can- cers are positive for ER. At least 50% of ER-positive breast cancer patients respond favorably to endocrine therapy, while less than 10% of ER-negative patients show such a good clinical response. Hence, estimation of ER levels has become fairly routine in determining the choice of therapy for breast cancer patients. The level of ER also has a prog- nostic value as there is a good correlation between breast cancer patients who benefit from endocrine therapy and the amount of the receptor present in the sample. In addition to quantitative analysis of ER in breast can- cer tissues, several oncologists promote the use of direct visualization of the receptor in tissues by immunohistology or immunocytology. This method involves sectioning of tissue or smearing a fine-needle aspirate on a slide fol- lowed by staining ER-containing cells with a specific probe such as an anti-ER antibody. Immunocytochemical meth- ods reveal the heterogeneity in the tumor with regard to ER status and make obvious the contribution, if any, from normal tissue. With this method, it is possible to distin- guish, for example, between one patient exhibiting ER positivity in homogenates due to high receptor content in a small proportion of the tumor and another with moder- ate amounts of ER in most of the tumor cells. The latter patient is likely to respond better to endocrine therapy. Combination of ER and PR estimations appears to increase the predictive value of those patients likely to respond to endocrine therapy. Limitations G A biopsy sample is required for the assay. G Immunoassays for ER (and PR) analysis measure both the unbound and hormone-bound protein forms, unlike steroid-binding assays, which use radiolabeled estrogen or progesterone as tracers. Hence, some dis- crepancies may be observed between the two types of assays. Assay Technology The first assay method used to identify and measure recep- tors depended on the binding of 3H-labeled steroids. Such assays are affected by endogenous steroids, which block the binding sites, causing an underestimate of the receptor content. The immunometric enzyme immunoassays, using monoclonal antibodies to measure ER and PR, introduced by Abbott Laboratories, appear to be unaffected by endog- enous hormones. Types of Sample Homogenate of a biopsy tissue prepared carefully to avoid heat stress, which destroys the receptors. For immunocytochemical analysis, fresh or frozen spec- imens or fine-needle aspirates are required. Paraffin sec- tions are less desirable for the Abbott ER assay. Frequency of Use Common for ER, but uncommon for PR assays. FECAL OCCULT BLOOD A number of colorectal disorders, benign and malignant, precipitate the rupture of tissues and blood vessels, result- ing in the presence of blood in the lumen of the colon and the rectum. Some of these blood components are found in the feces, and chemical and immunochemical methods have been developed to detect their presence. All the com- mercial tests available are designed to detect hemoglobin. The chemical tests for fecal occult blood detection are popularly known as guaiac tests and are based on the
  • 14. 846 The Immunoassay Handbook pseudoperoxidase activity of heme in hemoglobin. In the presence of suitable substrates such as gum guaiac (a natural resin from the wood of Guaiacum officinale containing α-guaiaconic acid) and hydrogen peroxide, the heme cata- lyzes a peroxidation reaction generating a blue quinone product. Exploiting this principle, a variety of tests are available, based on guaiac-impregnated paper or tape, which can be used to detect fecal occult blood in a labora- tory, physician’s office, or as a home test. The presence of blood in the stool is a diagnostic aid in the detection of a number of colorectal disorders including colorectal cancer. The immunochemical test for fecal occult blood detec- tion utilizes an mAb that is highly specific for human hemoglobin and has a low cross-reactivity with hemoglo- bins from common dietary meat products. This assay appears to possess better sensitivity and specificity than chemical tests. Reference Interval Fecal occult blood tests are qualitative tests with a positive or negative end point. Clinical Applications The detection of occult blood in human feces gives a gen- eral indication of disorders in the colon and rectum and is not specific for colorectal cancer. Noncancerous condi- tions, showing a positive fecal blood test, include peptic ulcer, ulcerative colitis, and iron-deficiency anemia. Despite these limitations, the qualitative assay for detect- ing fecal blood was the first test used in the western world as a cancer screening test, with limited success. Generally, the fecal blood test is recommended as a diagnostic aid during routine physical examinations of people above the age of 50. The American Cancer Society recommends serial testing for three consecutive days to minimize false-negative results. A special diet is recom- mended for at least 2 days prior to the chemical test to avoid false positivity due to any consumption of red meat or peroxidase-rich vegetables and fruits. Large amounts of vitamin C in the diet can cause false-negative results. The immunochemical fecal blood test does not require patient compliance to the special diet. In an asymptomatic group, about 2–3% of individuals score positive for fecal occult blood, of which the incidences of adenomatous pol- yps and colorectal cancer are 1% and 0.2%, respectively. The polyps are often considered a precancerous condition. When used in conjunction with sigmoidoscopy, colonos- copy, and barium enema, the fecal blood test is a useful and simple initial test for the detection of colorectal diseases, including cancer. Limitations G The chemical tests that detect the pseudoperoxidase activity of hemoglobin are plagued by a variety of dietary factors. Red meat and peroxidase-rich vegeta- bles and fruits generate false positives while vitamin C can cause false negatives. Patient compliance to a restricted diet is essential to increase the utility of the test. The immunochemical test does not appear to be sensitive to the above factors. G Intermittent bleeding and a lack of homogeneity in the distribution of blood in the feces can cause a wide varia- tion in results. Serial testing is therefore often recommended. Assay Technology The chemical test (Hemeoccult®) is based on the genera- tion of a blue product when hydrogen peroxide is added to guaiac-impregnated paper and when the fecal smear has traces of hemoglobin. The development of a mAb reactive only to human hemoglobin has resulted in an immuno- chemical assay (Hemeselect™), from Beckman Coulter, with increased sensitivity and specificity. Type of Sample Feces. Frequency of Use Common. PROSTATE-SPECIFIC ANTIGEN PSA, also known as gamma seminoprotein or kallikrein-3 (KLK3), is a glycoprotein with a molecular mass of 34kDa with a single polypeptide chain, encoded by the KLK3 gene. Immunologically and biochemically, PSA is distinct from PAP. PSA is a serine protease (the active site of the enzyme has a serine residue), and its labile nature could be partially attributable to its autocatalytic activity. Human seminal plasma is a rich source of PSA, and histologically, it is restricted to the cytoplasm of the acinar cells and duc- tal epithelium of the prostate gland. PSA derives its name from the observation that it is a normal antigen of the prostate but is not found in other normal or malignant tis- sues, although PSA-like material has been recently described in the breast tissue. The antigen is present in benign, malignant, and metastatic prostate cancer, and immunohistochemical analysis of distant metastasis for PSA can usually identify whether the primary origin of the cancer is from the prostate. In serum, at least three forms of complexed PSA have been identified in addition to free PSA. One is bound to alpha2-macroglobulin, and it appears that the PSA epit- opes are covered and inaccessible for measurement by cur- rent assays. The second major species is the PSA–ACT (alpha-1 anti-chymotrypsin) complex. The third complex is with alpha-1 protease inhibitor (PSA–API). The dis- crepancy between the various immunoassays for PSA could be due to their epitope specificity and the relative ability of measuring the various species by the different mAbs employed. Recently, immunoassays measuring total PSA (often referred to as equimolar assays measuring PSA and PSA–ACT complex equally well) and free PSA have been introduced by several diagnostic companies. The research literature documents numerous novel immunoassays for the measurement of PSA (see Kreutz and Suresh, 1997) and the development of two ultrasensitive PSA immunoas- says deserves special mention (Yu et al., 1997; Ellis et al., 1997). Both of these novel immunoassays demonstrated utility by monitoring very low PSA antigen <0.1ng/mL,
  • 15. 847CHAPTER 9.13 Cancer Markers which was the limit of most clinical assays. In the serum of patients who have undergone radical prostatectomy, theo- retically, PSA levels should be zero or very close to it after a few weeks of surgery. The use of ultrasensitive immuno- assays for monitoring very early recurrence of metastatic disease, and potential early second-line treatment, is an exciting possibility not only for prostate cancer but for the whole range of clinically relevant tumor markers. An international workshop (TD-3) on the comparative properties of 82 PSA antibodies was organized (Stenman et al., 1999). A significant finding was that nearly 17 of these cross-reacted with human glandular kallikrein (KLK2), which shares considerable homology with PSA (see below). In the light of these observations, studies on the estimation of PSA in serum, or claims of identifying PSA in non-prostate tissues, are only credible if the assays employ monoclonal antibodies specific to unique PSA epi- topes not shared by hK2 and other kallikreins. For a com- prehensive summary of the biology and the clinical applications of PSA and kallikreins, the reader is directed to a critical review (Rittenhouse et al., 1998). PSA is now designated as KLK3. Function PSA is a protease whose role is to liquefy semen in ejacu- late, allowing sperm to swim freely. It is also believed to play a role in dissolving cervical mucous, allowing sperm to enter the uterus. It is a member of the Kallikrein family (KLK3). Reference Interval Ninety-nine percent of apparently healthy donors have total PSA levels of <4ng/mL (may vary between methods). Values in benign prostate hypertrophy (BPH) are gener- ally in the range of 4–10ng/mL, which overlaps with the levels also seen in malignancy. Total PSA values >10mg/ mL are however more likely due to malignancy. Some authors have suggested that the ratio of free and bound PSA be used to discriminate between BPH and prostate cancer. The PSA–ACT fraction is higher in cancer than in BPH. Clinical Applications Prostate cancer is the second most prevalent form of male malignancy and early diagnosis is the key to a potential cure. The diagnosis of prostatic carcinoma, like all other cancers, is done by carrying out a number of procedures in combination, such as rectal examination, fine-needle biopsy, chest X-ray, bone scan, and serum PAP tests. The development of immunoassays to measure serum PSA has provided a valuable adjunct to the diagnosis and manage- ment of patients with prostatic cancer. The American Cancer Society in 1992 recommended the use of annual PSA tests for screening prostate cancer in conjunction with digital rectal examination in males above the age of 50. While this has lead to an enormous interest in the develop- ment of PSA immunoassays for screening applications, the oncology community is split on the value of such mass applications in asymptomatic people. The US Preventive Services Task Force (USPSTF) does not recommend screening because most prostate cancer is asymptomatic, and the treatments involve considerable risks of adverse consequences for the patient. Serum PSA has been found to be more useful than PAP because of increased clinical sensitivity. However, about 5% of patients have increased PAP but normal PSA levels. For this reason, some experts recommend that a combined PSA and PAP measurement is more useful than either one in isolation. Elevations of serum PSA concentrations above 4ng/mL are found not only in prostate cancer but also in BPH. The magnitude of the serum PSA elevation is progressive with the stage of the disease, and the highest levels are seen in stage D prostatic cancer with metastatic involvement. PSA is not useful as a specific diagnostic test for prostate cancer because of the elevated values in BPH and attempts have been made to achieve discrimination based on the mea- surement of free and total PSA. The percent free PSA, as a proportion of the total, may be useful. If the free PSA is less than 10% and the total PSA is above the cutoff, the risk of cancer is much higher. Nevertheless, PSA is now a routine test in the manage- ment of patients who have been confirmed to have prostate cancer. In this clinical application for monitoring prostate cancer, PSA is superior to PAP as a reliable tumor marker. Changes in tumor marker levels correspond to classical trends (see CANCER MARKERS - INTRODUCTION) in most cases of prostate cancer. PSA is a good marker for estab- lishing prognosis in prostate cancer. Limitations G Elevation of PSA above 4ng/mL is not diagnostic of prostate cancer because benign prostatic hypertrophy and some benign genitourinary diseases also result in elevated values. G Massaging the prostate prior to blood sample collection can result in transient PSA increases. G PSA levels may be elevated for up to 2 days after ejaculation. G About 5% of patients with prostate cancer have elevated PAP but normal PSA concentrations. Assay Technology Usually, immunometric assays use monoclonal antibodies. For example, total and free PSA tests are available from Siemens for the Centaur®. Types of Sample Serum or plasma depending on the assay used. Frequency of Use Common. β2-MICROGLOBULIN β2-Microglobulin (β2M) is a single-chain aglycosyl protein composed of 100 amino acids. Its molecular mass is 11.8kDa, and it is now known to be the light-chain com- ponent of the histocompatibility antigens (HLAs). It is therefore found on all nucleated cells and is present in high
  • 16. 848 The Immunoassay Handbook concentrations on the lymphocyte cell surface. This small protein bears sequence homology with immunoglobulins and is hence classified as belonging to the superfamily of immunoglobulins. β2M, being a small protein, escapes the glomerular fil- tration network of the kidneys. Most of what passes the glomeruli is reabsorbed and catabolized by the cells of the proximal tubules. A small amount of the protein is detected in normal urine with elevated levels in patients with proxi- mal tubular dysfunction. The normal serum levels of β2M are primarily a reflec- tion of HLA metabolism and turnover. It is estimated that, on a daily basis, about 150mg of free β2M protein is secreted into the body fluids. The serum levels are altered in various benign and malignant conditions, and hence, β2M is a nonspecific tumor marker. Function β2M is an integral component of the HLA antigen system and is similar in structure to immunoglobulins. The specific role of the protein is not yet defined, but as part of the histo- compatibility complex, it is thought to be involved in molec- ular recognition, particularly in distinguishing between self and nonself. The molecule also appears to stabilize the heavy- chain conformation of the HLA class I molecule, which may be important in immune recognition and restriction. Reference Interval Typically, the normal level of β2M is less than 2.5µg/mL for normal serum and 0.16µg/mL for urine (may vary between methods). Clinical Applications Serum β2M levels are elevated in the presence of a number of solid tumors and lymphomas. However, a variety of nonma- lignant conditions such as rheumatoid arthritis, AIDS, lupus, Crohn’s disease, and renal tubular dysfunction cause elevated levels of the marker. The level of serum β2M also appears to be an indicator of acute renal transplant rejection. The role of β2M levels is less certain for solid tumors, either in monitoring the disease or as an indicator of prog- nosis. There appears to be a use for this marker in the lym- phoid malignancies such as Hodgkin’s and non-Hodgkin’s lymphoma, multiple myeloma, and chronic lymphocytic leukemia. A high initial level of β2M is an indicator of poor prognosis and an advanced stage of the disease. It is also useful for monitoring the course of the disease in these cancers, particularly in multiple myelomas. Limitations G β2M elevations are not diagnostic of cancer, as a num- ber of nonmalignant conditions also give rise to ele- vated concentrations. The changes in β2M levels found in noncancer conditions include inflammatory disor- ders such as rheumatoid arthritis, Crohn’s disease, lupus, AIDS, renal tubular dysfunction, and renal transplant rejection. G Although β2M levels are elevated in some solid tumors, the marker is not useful in prognosis or in monitoring the disease state in these situations. Assay Technology Most kits are competitive in design because of the rela- tively small size of the antigen. Solid-phase polyclonal or monoclonal antibody-based enzyme immunoassays are available. Types of Sample Serum, plasma, and urine. Frequency of Use Not very common, except in Japan. NEURON-SPECIFIC ENOLASE Enolase is a ubiquitous glycolytic enzyme, which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyr- uvate (see Fig. 5). The enzyme enolase is also referred to as 2-phospho-D- glycerate hydrolase or phosphopyruvate hydratase. It is a dimer that can be composed of three different types of subunit, namely α, β, or γ. The αα isoenzyme dimer is syn- thesized by most of the cells in the body and by glial cells in the brain. This form is sometimes referred to as non- neuronal enolase (NNE). The β enolase appears to be spe- cific to muscle tissue. The γγ and αγ isoenzymes are collectively referred to as NSE. NSE is produced by nerve cells or neurons, and neuroendocrine cells, particularly the cells of the amine precursor uptake and decarboxylation lineage. NSE is an acidic protein with a native molecular mass of 78kDa and a subunit molecular mass of 39kDa. NSE and NNE are immunologically distinct and have dif- ferent sensitivities to chloride ions and temperature. Function NSE is a glycolytic enzyme involved in the energy-gener- ating process of the cell. Ontogenetically the NSE isoen- zyme appears in the final stages of neuronal differentiation and is hence a good nerve cell maturation marker. Reference Interval Abnormal levels are usually higher than 9ng/mL. Clinical Applications NSE is found elevated primarily in small-cell lung cancer (SCLC) and neuroblastomas. Other neuroendocrine tumors with elevated levels of NSE include insulinomas, medullary thyroid carcinomas, pheochromocytoma, and gut carcinoids. The main clinical application of NSE is in FIGURE 5 Action of neuron-specific enolase.
  • 17. 849CHAPTER 9.13 Cancer Markers the monitoring of these tumors for response to chemo- therapy or to detect early relapse. SCLC is the most aggressive of the lung cancers, and most of the patients have already progressed to metastasis by the time of diagnosis. However, SCLC responds par- ticularly well to chemotherapy compared to other lung cancers. NSE levels can help to classify the type of lung cancer when used in conjunction with histology, enabling the appropriate course of therapy to be initiated. Monitor- ing NSE levels can also assist in determining the effective- ness of chemotherapy and to predict relapse of the disease. Neuroblastoma is a common childhood cancer, which is often malignant. In addition to monitoring, NSE levels help to differentiate between neuroblastoma and Wilm’s tumor, which originates in the kidney. Both these condi- tions may present as a palpable abdominal mass and ele- vated levels of NSE are suggestive of neuroblastoma. Chromogranin A appears to be a better marker for car- cinoid tumors. Limitations Careful specimen handling is essential for this immunoas- say because of the presence of NSE in erythrocytes and other blood cells. Avoid samples that are hemolyzed and exhibiting an absorbance of at least 0.3 at 500nm. Assay Technology Usually tested using a polyclonal–monoclonal immuno- metric assay. Type of Sample Nonhemolyzed serum samples are required and repeated freezing and thawing is to be avoided. Frequency of Use Uncommon. SQUAMOUS CELL CARCINOMA ANTIGEN Squamous cell carcinoma (SCC) antigen is a 48kDa glyco- protein, originally isolated from a squamous cancer of the uterine cervix. Immunohistochemically, SCC was found to be a cytoplasmic protein of normal and cancerous squa- mous cells. More than 90% of cancers of head and neck cancer and 80% of cervical cancers are SCCs. Studies have indicated that SCC antigen is a good marker for monitoring the effectiveness of treatment of SCCs. Although not approved by the FDA for routine clinical use, the development of a tumor marker assay for squamous cancers of the head, neck, lung, and cervix heralds an important step in the management of these cancers. Function Not known. Reference Interval A study conducted by ARUP Laboratories established an upper 97th percentile of 2.2ng/mL (males and females). Clinical Applications SCC antigen is the first commercially available tumor marker for squamous cancers. The serum levels of SCC antigen are elevated in a significant percentage of patients with squamous cancers of the cervix, head, neck, and lung, and the level of the tumor marker increases with the stage of the disease. The specificity appears to be good for squa- mous cancers, and adenocarcinomas do not give rise to abnormal concentrations of this marker. Some benign gynecological and pulmonary diseases are responsible for higher SCC values than the normal reference interval. SCC antigen levels tend to be normal in early-stage squa- mous cancers. About 40% of stage III and 60% of stage IV head and neck cancers exhibit SCC antigen levels above the reference interval for normals. Cervical squamous car- cinomas in similar stages have shown a higher proportion (80%) of patients with elevated antigen levels. The degree of differentiation of the tumor does not appear to be related to the level of SCC antigen. Monitoring patients with squamous cancers has demonstrated that the assay can detect recurrence and provide a prognosis. Limitations Several nonmalignant benign diseases of the skin (e.g., eczema) and lungs (e.g., tuberculosis), sarcoidosis, and other conditions can result in elevated SCC antigen levels. Assay Technology Currently, the assay methodology employs radiolabeled SCC antigen and polyclonal antibody in a competitive assay format. Type of Sample Serum. Frequency of Use Uncommon. CYFRA 21-1 Tissue polypeptide antigen (TPA) is a pan-carcinoma marker. This antigen was discovered in 1957 as an insolu- ble residue from human carcinomas. TPA is now known to belong to a class of cytoskeletal proteins called cytokera- tins or intermediate filaments. Cytokeratins 8, 18, and 19 react with anti-TPA antibodies. These cytokeratins are cytoplasmic proteins and are found in all normal epithelial cells and cells lining the ducts and their sacs. Thus, various tumors arising from different organ sites are known to express TPA, which is also released into the serum by cell destruction. TPA assays represent the first-generation cytokeratin tumor marker tests. CYFRA 21-1 is a second- generation monoclonal immunoassay, detecting 21-1 frag- ments of cytokeratin 19. Function The cytoskeleton is responsible for the physical three- dimensional architecture of the cell. During cell
  • 18. 850 The Immunoassay Handbook division, the cytoskeleton assumes a crucial, dynamic, and functional role. The precise function of individual cytokeratins is yet to be fully understood, but as an inter- mediate filament, it has an obvious role in defining the structure of the cytoskeleton and its dynamics during cell division. Reference Interval ARUP Laboratories quote a reference interval of less than 2.3ng/mL for CYFRA 21-1. Other methods are likely to have different reference intervals. Clinical Applications CYFRA 21-1, which measures cytokeratin 19, is elevated in most lung tumors of the non-small cell category, with the highest sensitivity in lung squamous cell cancers. Limitations G Cytokeratin markers are not suitable for diagnosis of carcinoma but are used to monitor patients, often along with other organ-specific tumor markers. G Elevations in TPA are seen in the last trimester of preg- nancy and in various benign diseases of the lung, liver, stomach, and pancreas. G Monitoring of patients with cytokeratin markers during therapy is more complex than using other markers. Further work is needed to resolve the nature of these soluble serum fragments of cytokeratin parent mole- cules, which are more insoluble in nature. Assay Technology An enzyme immunoassay is available from Fujirebio Diag- nostics. The test is also available from Abbott Diagnostics for the Architect® analyzers. Type of Sample Serum. Frequency of Use Uncommon. HUMAN CHORIONIC GONADOTROPIN See PREGNANCY: HUMAN CHORIONIC GONADOTROPIN for further information on this marker. Reference Interval The reference value for pregnancy is typically >25mIU/ mL. However, for the use of beta-hCG for oncology appli- cations, a cutoff of 5mIU/mL is typical for females and 3mIU/mL for males. This may vary between methods. Clinical Applications hCG is a major analyte in the diagnosis of pregnancy, and this aspect is covered in a separate chapter. Choriocarcino- mas and male germ-cell tumors are characterized by ele- vated levels of hCG and its subunits. Increases in hCG levels have also been found in cancers of the breast, lung and small intestine, and in some prostate cancers. The combined measurement of hCG and AFP levels has been shown to be superior in confirming diagnosis and manag- ing non-seminomatous germ-cell tumors. Monitoring of germ-cell tumors is effective with these markers, which mirror the clinical progression or regression of the disease. Cancers secreting hCG often produce abnormal forms of the molecule. These include altered glycosylation of the peptide and secretion of α chains. Some scientists have attempted to develop an hCG assay that is specific for can- cerous conditions by exploiting these anomalous features. This type of assay has not yet become commercially available. Limitations See PREGNANCY. Assay Technology See PREGNANCY. Types of Sample Serum or plasma. Frequency of Use Not very common as a tumor marker. HER-2/NEU (HER-2, C-ERB B-2) ONCOPROTEIN The human epidermal growth factor receptor (HER-2) oncogene encodes a transmembrane tyrosine kinase recep- tor that is a key indicator marker for invasive breast cancer and target for therapy. This protein, with other members of the HER family, acts as a switch that causes cancerous growth through cell proliferation, motility, resistance to apoptosis, invasiveness, and angiogenesis. It communicates molecular signals from outside the cell to the inside, turn- ing genes on and off. It is elevated in approximately 20% of breast cancers and, if positive, indicates a poorer prog- nosis, and that treatment with anti-HER-2 therapy may be effective. Several therapies, some still in clinical trials, are aimed at suppressing the activity of the HER family of proteins, including Herceptin® (trastuzumab), which is a humanized mAB therapeutic approved for the treatment of metastatic breast cancer patients with overexpression of the HER-2/neu growth factor antigen. Function The HER-2/c-erb B-2 protein has a critical role in convey- ing messages into the cell as a member of the growth factor family, via tyrosine kinase-dependent cascade events. When an activating ligand binds to this protein, it dimer- izes by associating with another member of the HER family or with another HER-2 protein molecule. The specific tyrosine residues on the intracellular portion of HER-2 that are phosphorylated, determined by the ligand and dimerization partner, define the signaling pathway that is activated. The wide variety of ligands and crosstalk between cellular pathways provide a diverse signaling capability.
  • 19. 851CHAPTER 9.13 Cancer Markers Reference Interval See published guidelines from the American Society of Clinical Oncology—College of American Pathologists. Clinical Applications If the HER-2 protein is being overexpressed by the tumor, the prognosis is poorer, and treatment applied to suppress the HER-2 receptor will be most effective. The measure- ment of the levels of the HER-2 protein expressed by the gene is an important marker in the prognosis of breast can- cer patients. Some stomach tumors are responsive to HER-2 receptor treatments, and this test may be carried out on advanced stomach cancers. As with many other prognostic tumor markers, increased levels indicate poor prognosis, early relapse, and shorter duration of survival. See Ross et al., 2009. Assay Technology Tissue biopsies are examined using slide-based methods such as immunohistochemistry, fluorescence in situ hybrid- ization (DNA hybridization using fluorescent-labeled probes) and chromogenic in situ hybridization. Immuno- histochemistry is the most commonly used method. For example, Ventana Medical Systems (Tucson, Arizona) manufactures the Ventana Pathway™ immunohistochem- istry assay, and Dako (Glostrup, Denmark) manufactures the Dako HercepTest™. There are also blood tests using ELISA (immunometric/sandwich) methodology. Frequency of Use This marker is one of the new tumor markers and its clini- cal use is likely to increase in the future. BLADDER TUMOR ANTIGEN Bladder cancer is the sixth most frequent cancer in women and the fourth most common in men. Men are three times more likely to get bladder carcinoma than women. Smok- ing and exposure to chemicals appear to be risk factors. Most bladder cancers (90%) are transitional cell carcino- mas of epithelial origin. The rest are squamous cell can- cers, adenocarcinomas, or undifferentiated carcinomas. Traditional detection methods are cytoscopy and urinary cytology, analogous to the PAP smear tests for cervical cancer. As early as 1945, Papanicolaou and Marshall described the detection of cancer cells of the urinary tract by urinary cytospin sediments. Several bladder tumor marker tests have been developed including bladder tumor antigen (BTA), urinary cytokeratin, NMP22, and fibrin/ fibrinogen degradation products (FDP). The BTA antigen appears to be the complement factor H or a closely-related protein. The apparent molecular weight of the BTA antigen is predominantly 150kDa, although some degraded fragments have also been identi- fied. Several different cancer cell lines have been shown to secrete BTA into the medium. Function The BTA antigen has a complement factor C3b binding site, and it degrades C3b in the presence of complement factor I. The structural relationship to serum complement factor H (hCFH) was deduced by partial sequence analysis. It is speculated that the secretion of complement factor- like activities may confer a selective in situ growth advan- tage to cancer cells by blocking the complement-mediated lytic activity. The function of hCFH is to interact with complement factor C3b and inhibit the formation of mem- brane attack complex, thus preventing cell lysis. The hCFH appears to have a role in the regulation of the alter- nate complement pathway. Reference Interval A cutoff value of 14U/mL has been suggested, based on the mean plus 3 S.D., derived from urine of healthy indi- viduals. Some tests are qualitative only. Clinical Applications The BTA antigen is useful in monitoring transitional cell carcinomas, which represent the bulk of bladder cancers. The sensitivity is excellent for noninvasive high-grade tumors and relatively high for low-grade and in situ tumors. NMP22 does not appear to offer any advantage over BTA antigen. Limitations G Urine samples (fresh, refrigerated, or frozen) are used in this assay, and care should be taken in serial mea- surements due to variable amounts of voided urine. G It is not as good as cystoscopy for finding bladder can- cer. Many clinicians prefer to use cystoscopy to follow up bladder cancer treatment. G BTA antigen is not diagnostic. Renal stones, nephritis, renal cancer, urinary tract infections, cystitis, or recent trauma to the bladder or urinary tract can cause false positives. Assay Technology The BTA TRAK enzyme immunoassay is a dual monoclo- nal sandwich assay. The same pair of monoclonals are used in the new qualitative BTA Stat test, which was approved by the FDA in 2000 as the first home-use device for cancer marker recurrence. This qualitative test is similar to the lateral flow dipstick pregnancy test based on the principle of immunochromatography. This test is an adjunct to cytoscopy, and based on the results of the BTA Stat test, the urologist has the choice to use either a rigid or flexible cytoscope. If the results are positive, the rigid cytoscope could be used under general anesthesia to remove the recurrent tumor during examination. With a negative BTA Stat result, the flexible exploratory cytoscope could be used with only a local anesthetic. Frequency of Use The BTA TRACK™ EIA and BTA Stat tests (B.D.S. Inc., Redmond, WA, USA) are relatively new assays, and hence, their use is currently limited. The regulatory endorsement of the qualitative test may increase its home use in the future.
  • 20. 852 The Immunoassay Handbook IMMUNOCHROMATOGRAPHY ASSAYS FOR TUMOR MARKERS In recent years, there has been a growing number of qualita- tive or semiquantitative cancer marker assays developed on the user-friendly lateral flow, immunochromatography for- mat. This format can accommodate immunometric or com- petitive immunoassays. In the immunometric format, one of the antibodies is labeled with either colloidal gold, colored latex or colloidal carbon to generate the pink, blue, or black lines, respectively. In the competitive assay format, it is the analyte that is labeled. The sample moves along the device by capillary flow, solubilizing the label, and carrying it across a band of immobilized antibody, on a color-con- trasted white membrane. A detailed description of several of these types of assays (for other analyte applications) can be found in the PRODUCT TECHNOLOGY chapters (Part 7). Also, see LATERAL FLOW IMMUNOASSAY SYSTEMS. While most of these types of tests provide qualitative information on the presence or absence of a given analyte, some degree of quantitative information is now possible with the recent development of several instruments developed to measure the intensity of the end point band. In the future, it is likely that such tests would be performed in primary health care centers, such as the physician’s office, to initiate appropriate therapeutic courses of action or referral to secondary and tertiary health care centers for subsequent follow-up. Currently a few tumor marker assays are available in the immunochromatography format. They are: G Ideal Rapid UBC™ test (IDL Biotech, Sweden)—A one- step self-testing assay for urinary bladder cancer (UBC) detecting cytokeratin fragments. A quantitative ELISA assay format is also available from the same source. G BTA Stat™ test (B.D.S. Inc., Redmond, Washington, USA)—An FDA-approved home test for monitoring recurrence of urinary bladder cancer. This assay mea- sures the presence of urinary complement factor H and related proteins. G One-Step FOB™ test (TECO Diagnostics, California, USA)—Fecal occult blood test detects the presence of human hemoglobin in feces. This test utilizes mono- clonal and polyclonal antibodies specific to human hemoglobin and, hence, has less interference from dietary source hemoglobin, vitamin C, or iron. G AFP Card™ test (TECO Diagnostics, California, USA)—This is a one step assay detecting AFP and could have applications in hepatomas. G PSA test (PSA-CHECK-1®, PROSTA-CHECK®, Veda Lab, France)—The 1992 recommendation of the American Cancer Society to use an annual PSA test in conjunction with digital rectal examination for every male above the age of 50 years has likely resulted in the development of several rapid PSA immunochromatog- raphy tests. The test from Veda Lab claims to rapidly discriminate PSA values above or below 4ng/mL, the accepted cutoff for apparently healthy males. FREE LIGHT CHAIN ASSAYS The first biochemical cancer marker to be discovered was the Bence Jones proteins in urine. These are the mostly monoclonal homogeneous kappa or lambda light chains of immunoglobulins derived from the malignant B cells. Until now, the traditional method of detecting free light chains (FLCs) has been either by electrophoresis of pro- teins or by immunofixation electrophoresis of urine. The urinary tests have several limitations such as low sensitiv- ity, need for stringent urine collection over 24h, subse- quent concentration prior to assay, and the metabolism of the light chains by the proximal tubules, thus potentially masking the malignant condition to some extent. More recently, serum FLC assays have been developed that are more sensitive and can be automated (Bradwell, 2003). FDA-approved immunoassay tests are now available as an adjunct for the diagnosis and monitoring of multiple myeloma. The kits are also available for use on two auto- mated systems (Beckman Coulter IMMAGE® and Sie- mens Dade-Behring BNII). Function The light chain is part of the two-chain immunoglobulin molecule and is found in all types of immunoglobulin with some rare exceptions such as camel immunoglobulins, which have only one chain. The light chain has a constant region and three variable regions that have unique amino acid sequences in different antibodies. Together with the three variable regions of the immunoglobulin heavy chain, the six unique domains of every immunoglobulin combine to form the specific antigen-combining site, called the paratope. The paratope is the complementary face that binds to the specific epitope (antigenic determinant) face on the antigen. Clinical Applications Serum FLC levels are elevated in 85% of patients with nonsecretory multiple myelomas, 95% of intact immuno- globulin multiple myeloma, and 100% of light-chain mul- tiple myeloma. The most important application of the FLC assays is in the detection of nonsecretory multiple myelomas when serum or urine electrophoretic tests for monoclonal proteins are in the normal range. The half-life of FLC in serum is ~2–4h compared to intact IgG. Hence, serial measurements of the FLC can be a quicker indicator of the outcomes of a therapeutic regimen. The serum lev- els of FLC are also seen in amyloid disease, wherein the light chains can form polymeric deposits. Decreasing lev- els of serum FLC as a result of chemotherapy is a very good indicator of long-term survival. Assay Technology The immunoassays are based on the development of anti- bodies specific to masked unique light chain epitopes. The developed kits using these antibodies are based on latex- enhanced nephelometric and turbidimetric methods. Unlike the classical electrophoretic tests, concentration of sample is not necessary. Limitations The FLCs are not specific to cancer, and only 20% of B-cell chronic lymphocytic leukemias have abnormal levels. Most monoclonal gammopathies are of undeter- mined significance and 60% have elevated FLC.
  • 21. 853CHAPTER 9.13 Cancer Markers Frequency of Use This is a relatively new test and in view of its advantages of sensitivity and in measuring clinically useful levels in serum, it is likely to replace the urine-based methods in the future. Novel Experimental and Other Minor Markers This section includes a brief overview of recent immunoas- says for tumor markers that have not yet achieved wide- spread acceptance. Many of the antigens have been identified by the reactivity of monoclonal antibodies developed by various investigators. It appears that a number of these immunoassays determine the levels of novel mucinous anti- gens. Much remains to be learned about the biochemistry of these complex molecules, and their utility in a clinical set- ting is yet to be established. Nevertheless, some of the early results show promise in the monitoring of cancer patients. An entirely new class of tumor marker gene-based assays are emerging, which is beyond the scope of this chapter and book. Several minor tumor markers, e.g., des-gamma- prothrombin, calcitonin, ACTH, TA-4, creatine kinase ββ, inhibin, LDH, TSH, and catecholamines, are used in spe- cific clinical niches and are summarized in Table 2. CANCER ANTIGEN 72.4 Centocor developed this novel panadenocarcinoma muci- nous marker, utilizing a pair of monoclonal antibodies CC-49 and B72.3. MAb B72.3 reacts with sialyl 2–6Gal- NAc-O-Serine/threonine (Sialyl Tn), which is considered another oncofetal antigen (Fig. 4). This assay appears to be useful in gastric carcinoma. S-100 ANTIGEN The S-100 antigen of neuroendocrine origin is an acidic protein and is a homo- or heterodimer of A and B sub- units. The S-100 B serum assay (Sangtec IRMA, Diasorin AB, Sweden) is a melanoma marker, useful in monitoring of patients and as a prognostic marker. Elevation of this antigen in surgically treated and disease-free melanoma patients suggests early recurrence. BONE ALP Metra Biosystems (Mountain View, California, USA) introduced an assay to measure bone ALP. This assay may be more specific in identifying bone metastasis, instead of measuring total ALP, which is also elevated in liver metas- tasis, hepatomas, and prostate cancer. NMP-22 The NMP network is the residual framework seen in the nucleus after exhaustive extraction to remove membrane, chromatin, and cytoskeletal proteins. The NMP-22 assay (Matritech, Massachusetts, USA) for bladder cancer iden- tifies the nuclear mitotic apparatus protein, associated with the mitotic spindle during mitotis. CHROMOGRANIN A Chromogranin A (CgA) is a secretory, acidic protein of the neuroendocrine granules, of 45kDa. It is elevated in carci- noid tumors, neuroblastoma, and SCLC. It is abnormal in one out of three people with these conditions and in two of three if metastasis has occurred. It can be elevated in some advanced cases of prostate cancer with neuroendocrine features. The cutoff is typically 50ng/mL, but it varies with the method used. See GASTROINTESTINAL TRACT chap- ter for more detail on this analyte. TELOMERASE Telomeres are the specialized nucleoprotein ends of all eukaryotic chromosomes, composed of tandem repeats of the nucleotide sequence TTAGGG. Progressive cell divi- sions shorten the telomere length, which is thought to be the biological clock that triggers senescence. However, in stem cells and cancer cells, this is regenerated by the enzyme telomerase. Cancer cells appear to escape this TABLE 2 Minor Tumor Markers Useful in Niche Clinical Applications Tumor Markers Clinical Application 1. Des-gamma- carboxyprothrombin Hepatoma vs cirrhosis 2. Calcitonin Bone metastasis, meduallary carcinoma of thyroid 3. Adrenocorticotropic hormone (ACTH) Neuroendocrine tumors 4. Creatine kinase BB Neuroendocrine tumors 5. TA-4 Squamous cell carcinoma 6. Cancer-associated serum antigen (CASA) Mucinous ovarian cancer 7. Inhibin Granulosas and mucinous cystadeno-carcinoma 8. Lactate dehydrogenase (LDH) Germ cell tumors 9. Thyroid stimulating hormone and thyroglobulin Thyroid cancer 10. Catecholamines Neuroblastomas, pheochromo- cytoma, carcinoid tumors 11. Tumor-associated trypsin inhibitor (TATI) Renal and gastric carcinomas 12. Neopterin Prognostic in myeloma and hematological malignancies 13. Epidermal growth factor receptor (EGFR) SCC and prognostic in breast cancer 14. Ferritin Advanced adenocarcinomas 15. 5-hydroxyindoleacetic acid (5-HIAA) Carcinoid tumors 16. Lipid-associated sialic acid Nonspecific marker in several cancers 17. Parathyroid hormone- related peptide (PTH-RP) Tumors with hypercalcemia 18. Terminal deoxynucleotidyl transferase Classification of leukemias 19. Urinary gonadotropin peptide (UGP) Ovarian cancer
  • 22. 854 The Immunoassay Handbook replicative senescence by upregulating telomerase activity. In approximately 85% of cancer cells, telomerase activity is increased, whereas its production is repressed in somatic cells, except for proliferating progenitor cells and activated lymphocytes, and hence this is emerging as an exciting new marker. A PCR-ELISA has been developed by Boeh- ringer Mannheim, Germany, to measure telomerase activ- ity. A biotin-labeled specific primer is elongated by telomerase and subsequently amplified by PCR, hybrid- ized to the telomerase repeat-specific detection probe. A streptavidin-coated microtiter plate is used in the final ELISA detection step. UBC ANTIGEN The quantitative UBC antigen is a new test that is useful in the management of UBC. Unlike most other tumor mark- ers whose levels in body fluids reflect the tumor burden, the UBC antigen levels are indicative of the course of the disease measured in terms of tumor cell activity. Hence, this test is indicated for monitoring tumor recurrences, and its levels are correlated with the stage and grade of cancer. Both IRMA and ELISA versions are available for diagnosis and follow-up, supplemented by a point-of-care urine dipstick test (UBC® Rapid, IDL Biotech, Sweden). NOR-/METANEPHRINE RIA Normetanephrine and metanephrine are O-methylated metabolites of the catecholamines, noradrenaline and adrenaline, respectively. These metabolites are elevated in pheochromocytoma, ganglioneuroma, and other neuro- genic tumors. An enzyme immunoassay for the detection of these two metabolites in heparin plasma has been devel- oped (DLD Diagnostika GmbH, Germany). PML PROTEIN A new cancer marker for acute promyelocytic leukemia (APL) has been identified along with the development of a monoclonal probe (Dako Inc., Denmark) for identification of the promyelocytic leukemia protein (PML). In APL cells, the nuclei exhibit a microgranular pattern with numerous small dots, while the normal hematopoietic cells exhibit the speckled pattern with 5–10 nuclear dots. The architecture of the PML nuclear granules is distorted in APL cells, bearing the characteristic reciprocal 15:17 chro- mosomal translocation. It is important to diagnose APL from the other types since this leukemia subtype is success- fully treated often with chemotherapy and all-trans-retinoic acid. TUMOR M2 PYRUVATE KINASE Tumor M2 pyruvate kinase was defined by Eigenbrodt around 1985. In cancers, the active tetrameric form of the M2 isoenzyme of pyruvate kinase is converted to an inactive dimeric form by direct interaction with oncoproteins to channel glucose carbons into DNA synthesis. Circulating tumor M2 pyruvate kinase is more commonly elevated in esophageal, gastric, and colorectal cancer patients than con- ventional tumor markers. Fecal M2 pyruvate kinase is a sensitive marker of colorectal cancer. As a fecal marker for colorectal cancers, fecal tumor M2 pyruvate kinase has a sen- sitivity of 73–92% at a cutoff value of 4U/mL as against 50% sensitivity for the guaiac fecal test. The marker can also be measured in plasma. Limited information exists as yet on the utility of tumor M2 PK as a prognostic marker, as a marker of malignant transformation, or in assessing tumor recur- rence or response to treatment. Large multicenter trials are, therefore, needed to define its clinical role (Kumar, 2007). ADAM8 Genes involved in pulmonary carcinogenesis have been identified by examining gene expression profiles of non- SCLCs to identify molecules that might serve as diagnos- tic markers or targets for the development of new molecular therapies. A gene encoding ADAM8, a disintegrin, and metalloproteinase domain-8 was one candidate for the identification of such molecules investigated by Ishikawa and group. Tumor tissue microarray was applied to exam- ine the expression of ADAM8 protein in archival lung can- cer samples from patients. Serum ADAM8 levels of 105 lung cancer patients and 72 controls were also measured by ELISA. A possible role of ADAM8 in cellular motility was examined by Matrigel™ assays. ADAM8 is abundantly expressed in the great majority of lung cancers examined. A high level of ADAM8 expression is significantly more common in advanced stage IIIB/IV adenocarcinomas than in adenocarcinomas at stages I–IIIA. Serum levels of ADAM8 were significantly higher in lung cancer patients. Hence, ADAM8 could be useful as a diagnostic marker and also a therapeutic target. HE4 HE4 is part of a family of protease inhibitors that func- tions in protective immunity, which is overexpressed in ovarian cancers, especially in serous and endometrioid his- totype. It is secreted by the cell and then detectable in the sera of patients with ovarian carcinoma by an enzyme immunoassay. Preliminary studies of HE4 suggest a higher specificity than CA 125 in different benign and malignant conditions, excluding renal failure. Patients with renal fail- ure have very high HE4 serum levels, undistinguishable from ovarian cancer. For this reason, patients with this pathology were excluded in our study. Excluding this dis- ease, slightly elevated HE4 serum levels were found in only one third of patients with effusions or in 5% of patients with chronic liver diseases. PSME3 Initial validation studies by Roessler et al. confirmed the relevance of PSME3 as a tumor-associated protein. Poly- clonal antibody to recombinantly expressed PSME3 was generated, and upregulation of the protein in colorectal cancer tissue was confirmed by western blot analysis and immunohistochemistry. Importantly, the marker could also be measured in serum using a highly sensitive immu- noassay and was significantly elevated in serum of colorec- tal cancer patients compared with healthy individuals and patients with benign bowel disease.
  • 23. 855CHAPTER 9.13 Cancer Markers ARF The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which par- ticipate in the two main cell cycle control pathways, p16INK4a-Rb and p14ARF-p53111–115. p14ARF (p19Arf in mice) is a 14kDa (19kDa) protein predominantly localized in the nucleolus. It blocks the cell cycle in both G1 and G2 phases and inhibits the growth of incipient cancer cells by indirectly activating p53. It also inhibits ribosomal RNA processing and interacts with topoisomer- ase I. Arf triggers sumoylation of many cellular proteins, including Mdm2 and nucleophosmin (NPM/B23), with which p19Arf physically interacts in vivo. This occurs equally well in cells expressing or lacking functional p53. Thus, Arf’s p53-independent effects on gene expression and tumor suppression might depend on Arf-induced sumoylation. KI67 Proliferative markers have been broadly evaluated as prog- nostic factors for early-stage breast cancer patients. Ki67, a nuclear nonhistone protein, was identified after immuni- zation of mice with the Hodgkin’s lymphoma; 10–13 Ki67 is expressed only in cells in the proliferative phases of the cell cycle (G1, S, G2, and M phases). Ki67 is vital for cell proliferation, since downregulation of Ki67 using anti- sense nucleotides prevents cell proliferation. Ki67 is tightly controlled and regulated, implying a fundamental role in cell proliferation. However, it has been very difficult to determine its function because of its lack of obvious homology with known proteins. Another suggested role of Ki67 is organizing DNA, based on its localization to extra- nucleolar sites during early G1; these sites contain centro- meric and satellite DNA. Ki67 is also known to bind to DNA. MacCallum and Hall suggested a structural role for Ki67 within the nucleolus, based on its ability to interact with other proteins and bind with RNA and DNA. They also suggested that Ki67 is an essential factor in the syn- thesis of ribosomes during cell division. Further studies should be conducted to elucidate the roles of Ki67 in cell proliferation and tumorigenesis. VASCULAR ENDOTHELIAL GROWTH FACTOR VEGF is composed of a family of five isoforms (VEGFA, VEGFB, VEGFC, VEGFD, and PLGF) that act as ligands for tyrosine kinase receptors (VEGF-Rs). Upon binding of VEGF to its receptors (primarily VEGFR2), intracellular signaling pathways, including MEK-ERK and PI3K-Akt, are activated that mediate angiogenic switches. This acti- vation of angiogenesis in both normal and cancerous tissue is dependent on increased endothelial cell proliferation and invasion, increased vessel permeability, and recruit- ment of other support cells that make up the vessel archi- tecture, such as pericytes. VEGF has been implicated as a key mediator of angiogenesis in breast cancer. The most important factor that determines survival of breast cancer patients is dissemination of cancer cells from the primary site into distant organs and establishment of metastatic colonies. Comparison of gene signatures from primary tumor, regional, and distant metastasis indicates that VEGF is only overexpressed in distant metastasis and is associated with poor survival. CYCLIN E Cyclin E is the limiting factor for G1 phase progression and S phase entry. The cyclin E gene is a target of E2Fs, and the protein associates with Cdk2 and activates its kinase activity shortly before entry of cells into the S phase. While there is evidence of the importance of cyclin D1 in mammary tumorigenesis, the role of cyclin E in this respect has only recently been established. Cyclin E is expressed in supra-physiological levels in many human cancers and its genomic locus is frequently amplified. High levels of cyclin E and low levels of the G1-specific cell cycle inhibitor p27KIP1 exhibit a good correlation. Another clue for the importance of cyclin E in breast car- cinoma is the finding of centrosome amplifications in these tumors, which could pave the way for genomic instability. TBX2/3 T-box proteins contain a T-domain that affects dimeriza- tion and DNA binding. TBX2 belongs to the Tbx subfam- ily of T-box transcription factors. TBX2 and TBX3 are closely related T-box proteins that have been implicated in tissue development in different sites, including the mam- mary gland. TBX3 is required for normal mammary development in mouse models and in patients with ulnar– mammary syndrome (UMS). It has been reported that TBX2 is amplified in 8.6–21.6% of sporadic human breast carcinomas, where the protein is overexpressed. Ectopic expression of TBX2 results in DNA polyploidy and cispla- tin resistance. Hence, overexpression of Tbx2 contributes to breast carcinogenesis by accelerating cell proliferation, changing DNA ploidy, and making cells resistant to che- motherapy. More studies are needed to elucidate the mech- anism of TBX2/3 overexpression in breast cancer. TA-90 TA-90 is a protein found on the outer surface of mela- noma cells. Like S-100, TA-90 can be used to look for the spread of melanoma. Its value in following melanoma is still being studied, and it is not widely used at this time. It is also being studied for use in other cancers such as colon and breast cancer. KRAS Cetuximab (Erbitux®) and panitumumab (Vectibix®) are drugs targeting the EGFR protein that can be useful in the treatment of advanced colorectal cancer. These drugs do not work in colorectal cancers that have mutations (defects) in the K-ras gene. Doctors now commonly test the tumor for this gene change and only use these drugs in people whose cancers do not have the mutation. K-ras mutations can also help guide treatment for some types of lung cancer. Tumors with the mutations do not respond to treatment with erlotinib (Tarceva®) or gefitinib (Iressa®).
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Ellis, W.J., Vessella, R.L., Noteboom, J.L., Lange, P.H., Wolfert, R.L. and Rittenhouse, H.G. Early detection of recurrent prostate cancer with an ultra- sensitive chemiluminescent prostate-specific antigen assay. Urology 50, 573– 579 (1997). Gold, P. and Freedman, S.O. Demonstration of tumor specific antigen in human colonic carcinoma by immunological tolerance and absorption techniques. J. Exp. Med. 121, 439–461 (1965). Gold, D.V., Modrak, D.E., Ying, Z., Cardillo, T.M., Sharkey, R.M., Goldenberg, D.M. New MUC1 serum immunoassay differentiates pancreatic cancer from pancreatitis. J. Clin. Oncol. 24, 252–258 (2006). Gong, J.-L., Liang, Y., Huang, Y., Chen, J.-W., Jiang, J.-H., Shen, G.-L. and Ru-Qin Yu, R.-Q. Ag/SiO2 core-shell nanoparticle-based surface-enhanced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools. Biosens. Bioelectron. 22, 1501–1507 (2007). Hammarstrom, S., Shively, J.E., Paxton, R.J., et al. 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