 Cancer  For breast and ovarian cancers: done by identifying germline

- refers to the uncontrolled growth of cells that can develop into a BRCA1 and BRCA2 mutations
solid mass or tumor and spread to other areas of the body  For familial colon cancers: can be identified by the presence
- the second leading cause of death in North America of the adenomatous polyposis coli gene (APC)
- it is estimated that 45% of males and 38% of females will develop
invasive cancer in their lifetime  Prognosis
- Tumorigenesis (Formation): activation of growth factors (eg, - Tumor marker concentration generally increases with tumor
epidermal growth factor [EGF]) and oncogenes (eg, K-ras), in progression, reaching their highest levels when tumors metastasize
combination with inhibition of apoptosis, tumor suppressor, and - Serum tumor marker levels at diagnosis can reflect the
cell cycle regulation genes (eg, BRCA1, p53, cyclins) aggressiveness of a tumor and help predict the outcome for
- Metastasis (Spreading): additional genetic changes are required patients
such as loss of cell adhesion proteins (eg, β-catenin and E-cadherin)  High serum tumor markers might indicate the presence of
and activation of angiogenesis genes (eg, VEGF) malignancy and possible metastasis associated with a poorer
- can be detected and monitored using biologic tumor markers prognosis

 Tumor markers  Laboratory Methods

- may be used for screening, diagnosis, prognosis, therapy
monitoring, and detecting recurrence Immunoassays
- are produced either directly by the tumor or as an effect of the - the most commonly used method to measure tumor markers
tumor on healthy tissue (host) (quantitative)
- encompass an array of diverse molecules such as serum proteins, - can be affected by interference resulting from icterus, lipemia,
oncofetal antigens, hormones, metabolites, receptors, and hemolysis, and antibody cross-reactivity
enzymes - falsely elevated levels in patients if adequate washing steps are not
- elevated enzymes are largely a result of the high metabolic demand included between patient samples
of these proliferative cells (enzyme levels tend to correlate with
tumor burden, making them clinically useful for monitoring the 1. Linearity
success of therapy) - Linear range: is the range of analyte concentrations in which a linear
relationship exists between the analyte and signal
Serum proteins - Linearity: is determined by analyzing (in replicates) specimens spanning
- are also used to monitor cancer therapy: the reportable range
a) β2-Microglobulin - Guidelines for this determination are outlined in the Clinical Laboratory
 is found on the surface of all nucleated cells Improvement Amendments (CLIA)
 used as a nonspecific marker of the high cell turnover that is - Samples exceeding the linear range need to be systemically diluted to
often observed in tumors determine values within the reportable linear range
b) Immunoglobulins
 provide a more specific measure of plasma cell production of 2. Hook Effect
monoclonal proteins observed in hematologic malignancies - caused by antigen excess: excessively high tumor marker
such as multiple myeloma concentrations that result in falsely low measurements
- caused by analyte concentrations excess: result in a lack of
Hormones and hormone metabolites “sandwich” formation (decrease signal)
- are widely used as specific markers of secreting tumors
- valuable in diagnosing neuroblastomas, as well as pituitary and 3. Heterophile Antibodies
adrenal adenomas - also known as human antianimal antibodies (HAAAs) or HAMAs
- interference can be seen in immunoassays if an individual has
Oncofetal antigens circulating antibodies against animal immunoglobulins
- one of the first tumor markers discovered - HAMAs: are most commonly encountered in patients who have
- Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP): are been given mouse monoclonal antibodies for therapeutic reasons
expressed transiently during normal development and are then or have been exposed to mice5 but they may be idiopathic
turned on again in the formation tumors - Nonimmune animal serum is often added to immunoassays to
minimize the effects of heterophilic antibodies
Monoclonal defined antigens
- are directly identified from human tumor extracts or cell lines High-Performance Liquid Chromatography
- are directed toward specific carbohydrate or cancer antigens - the most widely used methodology to detect catecholamines and their
- are best used for monitoring treatment of tumor that secrete these metabolites in plasma and urine
epitopes - Extraction process: analytes are separated from either plasma or urine

Receptors Neuroblastoma
- are used to classify tumors for therapy - the most common malignant tumor in children,
- a “nonserologic” markers - diagnosed by the detection of high levels of plasma catecholamines
- Example: estrogen and progesterone receptors (epinephrine, norepinephrine, and dopamine)
- Treatment: tamoxifen chemotherapy
Pheochromocytoma
- a rare tumor associated with hypertension
 Screening - diagnosed by detecting elevated plasma metanephrines (along with
- no tumor marker identified to date can be used to adequately urine VMA and free catecholamines)
screen asymptomatic populations (exception of PSA)
 most are found in normal cells and benign conditions in Carcinoid tumors
addition to cancer cells - are serotonin- secreting tumors that arise from the small intestine,
 screening would result in detection of false-positives appendix, or rectum
(patients without disease with detectable tumor marker), - Symptoms (carcinoid syndrome): pronounced flushing, bronchial
leading to undue alarm and cost to patients constriction, cardiac valve lesions, and diarrhea
- Susceptibility to cancer: done by molecular diagnostics in patients - involve the detection of 5-hydroxyindoleacetic acid (5-HIAA) [a
with breast, ovarian, or colon cancer serotonin metabolite]
Immunohistochemistry Enzyme Assays
- a few tumor markers are detected directly within solid tissue - cannot be used to identify a specific tumor or site of tumor (exception is
- are present in the cancer cells themselves in a different manner than the PSA)
surrounding tissue  Prostate specific antigen (PSA): a serine protease of the kallikrein
- assays are performed by thinly cutting and placing the tissue in question family that is found exclusively in both diseased and benign
on glass slides prostate glands
 Specific antibodies (and the proper control antibodies) in solution - Enzymes: are found in much higher concentrations intracellularly and
are then incubated with tissue sections to detect the presence (or are release into the system when cells necrosed or underwent changes
absence) of antigens using colorimetric secondary antibodies in permeability
- have the ability to determine whether the antigen in question is in a - Examples of enzymes:
particular cell type (such as a tumor), in the specific subcellular location o Alkaline phosphatase (bone, liver, leukemia, sarcoma),
- Example: Identification of estrogen and progesterone receptors in breast o creatine kinase–BB (prostate, small cell lung, breast, colon,
cancer ovarian)
o Lactate dehydrogenase (liver, lymphomas, leukemia, others)
o PSA (prostate)

 FREQUENTLY ORDERED TUMOR MARKERS

α-Fetoprotein

Introduction and  an abundant serum protein normally synthesized by the AFP increased in :
Description fetal liver that is reexpressed in certain types of tumors o HCC (hepatocellular carcinoma)
 a carcinoembryonic protein o Germ cell tumors
o Benign conditions (pregnancy and liver disease)
o Malignancy (testicular cancer)

Regulation and  a 70-kD glycoprotein related to albumin AFP peaks at: 1/10th concentration of albumin at 30 weeks gestation
Physiology  normally functions as a transport protein Upper limit for serum AFP = 15 ng/mL in healthy adults
 involved in regulating oncotic pressure in the fetus Infants have AFP values that decline to adult levels at 7–10 months

Clinical  is used for the diagnosis, staging, prognosis, and treatment  is used for classification and monitoring therapy for testicular
Application and monitoring of HCC cancer
Interpretation
HCC: a tumor that originates in the liver and is often due to Testicular cancer:
chronic disease such as hepatitis and cirrhosis  Seminomatous tumors: form directly from malignant germ cells
 Nonseminomatous tumors: involve differentiation into embryonal
- Screening high-risk populations: sensitivity: 40%–65%, carcinoma, teratoma, choriocarcinoma, and yolk sac tumors
specificity: 80%–95% (at cutoffs ranging from 20–30 (endodermal sinus tumor)
ng/mL)  AFP is used in combination with β-human chorionic
- Very high levels of AFP (>500 ng/mL) in high-risk individuals gonadotropin (β-hCG) to classify nonseminomatous tumors
are considered diagnostic of HCC
- AFP be used in conjunction with ultrasound imaging every  is used for tumor staging:
6 months in patients at high risk of developing HCC This o Stage I tumors: increased in 10%–20%
includes patients with hepatitis B virus– and/or hepatitis C o Stage II tumors: increase in 50%–80%
virus–induced liver cirrhosis o Stage III: increase in 90%–100%
- High levels of AFP in HCC are associated with poor
prognosis and are exemplified in individuals who do not
respond to therapy or have residual disease following
surgery
- Decrease levels of AFP after treatment is associated with
prolonged survival rates

Methodology Automated immunoassays Sandwich immunoassays

- measure AFP in serum and amniotic fluid - rely on monoclonal or polyclonal antibodies directed toward
different regions of AFP
- Example: Beckman Access, Roche Cobas series, Advia Centaur,
Tosoh AIA series, Clinical Diagnostics Vitros ECi, Diagnostic
Products Corp Immulite Analyzers, and the Abbott ARCHITECT

Application and Primary applications of AFP as a tumor marker: Used as a marker to monitor therapy, detect
Pathophysiology  HCC residual tumor, or detect relapse
 Nonseminomatous testicular cancer
CA 125

Introduction and - was first defined by a murine monoclonal antibody raised - may be useful for detecting ovarian tumors at an early stage and for
Description against a serous ovarian carcinoma cell line monitoring treatments without surgical restaging

Regulation and  is expressed in: - gene encodes a (MW: 200,000–1,000,000 Kd) mucin protein
Physiology 1. Ovary containing a putative transmembrane region and a tyrosine
2. Other tissues of müllerian duct origin phosphorylation site
3. Human ovarian carcinoma cells - not usually found in serum but may be elevated in patients with
endometriosis, during the first trimester of pregnancy, or during
menstruation

Clinical  is the only clinically accepted serologic marker of ovarian - should not be used to screen for ovarian cancer in asymptomatic
Application and cancer individuals
Interpretation - is elevated in a high percentage of ovarian tumors and is
Ovarian cancer recommended as an annual test for women with a family or prior
- 3% of the newly diagnosed malignancies in women history of ovarian cancer
- is among the top five causes of cancer related death - is elevated in:
- includes a broad range of categories:  Stage I disease: 50%
 sex cord tumors  Stage II disease: 90%
 stromal tumors  Stage III or IV disease: > 90% of patients
 germ cell tumors
 epithelial cell tumors (most common)

Methodology Immunoassays Upper limit for serum CA-125 = 35 U/mL

 OC 125 and M11 antibodies:
- these monoclonal antibodies recognize distinct
nonoverlapping regions of the CA-125 epitope

Application and  is predominantly used to monitor therapy and to  is useful both for predicting the success of surgery (debulking
Pathophysiology distinguish benign masses from ovarian cancer procedures) and for determining efficacy of chemotherapy

Example: CA-125 half-life:

 Postmenopausal women with a palpable abdominal Mass: - < 20 days is associated with longer survival
(>95 U/mL) of CA-125 has a 90% positive predictive value - Average half-life: 45 days
for ovarian cancer

CEA (Carcinoembryonic Antigen)

Introduction and - was discovered in the 1960s  can be used to aid in the diagnosis, prognosis, and therapy
Description - is prototypical example of an oncofetal antigen monitoring of colorectal cancer
- it is expressed during development and then reexpressed
in tumors CEA (>10 ng/mL)
- is the most widely used tumor marker for colorectal cancer - are frequently associated with malignancy
- is also frequently elevated in lung, breast, and - not specific for colorectal cancer
gastrointestinal tumors

Regulation and - a large heterogeneous glycoprotein with a MW 200 kD Increased CEA concentrations have been observed in:
Physiology - it is part of the immunoglobulin superfamily and is involved - heavy smokers
in apoptosis, immunity, and cell adhesion (involved in - patients following radiation treatment and chemotherapy
metastasis)
- may be elevated nonspecifically because of impaired - patients with liver damage due to prolonged clearance
clearance or through increased production
Upper normal range for serum CEA = 2.5–5 ng/mL depending
on the assay
Clinical  a tumor marker for colorectal cancer  can be used in combination with histology and the TMN staging
Application and  is used for prognosis of colon cancer in postsurgery system to establish the need for adjuvant therapy
Interpretation surveillance and for monitoring response to chemotherapy
Adjuvant therapy
- is indicated in patients with stage II disease (ie, tumor has
spread beyond immediate colon but not to lymph nodes) who
have high levels of CEA

Methodology - historically used polyclonal antibodies - is available on numerous commercial automated platforms
- currently use monoclonal anti-CEA antibodies  due to high heterogeneity of CEA, it is essential that the same
assay be used for serial monitoring

Application and Before surgical resection: Half-life of CEA: 2–8 days depending on the assay and the individual
Pathophysiology - baseline CEA values are typically obtained to confirm
successful removal of the tumor burden - is not recommended for screening asymptomatic individuals for
colorectal cancer
After surgery and during chemotherapy: - it may be value for detecting recurrence of antigen positive breast
- CEA levels are serially monitored every 2–3 months to and gastrointestinal cancers and to aid in the diagnosis of non–
detect recurrence and determine therapy efficacy small-cell lung cancer

HCG (Human Chorionic Gonadotropin)

Introduction and - a dimeric hormone normally secreted by trophoblasts in the - is elevated in:
Description placenta to maintain the corpus luteum during pregnancy  trophoblastic tumors
 choriocarcinoma
 germ cell tumors of the ovary and testis

Regulation and - a 45-kD glycoprotein consisting of α and β subunits Can be seen in the serum:
Physiology - it is degraded into multiple fragments  Intact molecule (Nicked Hcg) - the free β subunit (β -hCG)
 Hyperglycosylated intact form

Clinical  a prognostic indicator for ovarian cancer GTDs distinct types of tumor:
Application and  a diagnostic marker for classification of testicular cancer 1. Hydatidiform mole
Interpretation  the most useful marker for detection of gestational 2. Persistent/ invasive gestational trophoblastic neoplasia
trophoblastic diseases (GTDs) 3. Choriocarcinoma
 hCG is invariably elevated in women with GTDs17 and 4. Placental site trophoblastic tumors
is often found at higher levels than are observed in
normal pregnancy (ie, > 100,000)  are classified by clinical history, ultrasound, histology, and hCG
 It is particularly helpful marker for monitoring GTD levels
therapy, as levels of hCG correlate with tumor mass
and prognosis

Methodology Automated immunoassays Total β –hCG assays

- use monoclonal capture and tracer antibodies targeted - are the most useful because they detect both intact hormone and
toward free β -hCG
- epitopes in the β subunit and intact hCG

Application and Testicular cancer: Ectopic β –hCG:

Pathophysiology - the free β -hCG subunit is elevated in 60%–70% of patients - is occasionally elevated in ovarian cancer and some lung cancers
with nonseminomas
 hCG can be used in combination with AFP and biopsies to Free β -hCG
diagnose subtypes of testicular cancer - is sensitive and specific for aggressive neoplasms; the free β-hCG is
not detectable in the serum of healthy subjects
PSA (Prostate Specific Antigen)

Introduction and - a 28-kD glycoprotein produced only in the epithelial cells of - a serine protease of the kallikrein gene family and functionally
Description the acini and ducts of the prostatic ducts in the prostate regulates seminal fluid viscosity and instrumental in dissolving the
- cervical mucus cap, allowing sperm to enter

Regulation and - In healthy men, low circulating levels of PSA can be detected - Most of the circulating PSA is complexed to α1-antichymotrypsin or
Physiology in the serum α2-macroglobulin

- 2 major forms of PSA that are found circulating in the blood:  detection of total PSA has been used in screening for and in
(1) free monitoring of prostate cancer
(2) complexed  patients with malignancy have a lower percentage of free PSA

Clinical PSA testing and digital rectal examination (DRE) should be  used to test for prostate malignancy:
Application and performed on men over 50 years of age annually (with a 10-year  Age adjusted cutoff values of PSA
Interpretation life expectancy) and on younger men at high risk, such as those  PSA velocity (rate of rise over time)
with a family history of prostate cancer  Free PSA/total PSA ratios

Standard cutoff values of total PSA = < 4 ng/mL > 4 ng/mL and/or a clinical suspicion of cancer by DRE undergo biopsy to
confirm the presence of prostate cancer and are followed closely
Increased PSA IN: over time
- Prostate infection, irritation, and benign prostatic
hyperplasia
- (enlargement)
- Recent ejaculation or DRE

Methodology Immunoassay Because antibodies recognizing different epitopes may

- detects both free PSA and PSA complexed with α1- recognize the multiple forms of PSA variably, there can
antichymotrypsin but NOT α2-macroglobulin be some discrepant PSA results between manufacturers
- use enzyme, fluorescence, or chemiluminescence on an Known interferences that have been reported for PSA include
automated immunoassay platform both the Hook effect30 and HAMAs31,32

Application and - monitor the progression of prostate cancer after therapy - monitor cancer progression has also been found useful after
Pathophysiology  After radical prostatectomy, serum PSA should become radiation or endocrine therapy
undetectable if the cancer is localized

- Katrina Lotho (PandaMT13)