Tumor Markers

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Tumor Markers
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Overview

• Cancer remains the second leading cause of death
  in the USA, behind
• heart disease, with an estimated 1,437,180 new
  cases 565,650
• deaths in 2008 alone.
• It is expected that malignancies of prostate
  breast origin will be
• the most common causes of new diagnoses in men
  women,
• respectively, with tumors of the lung bronchus
  the leading cause
• of cancer-related death in both genders

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Cancer

• A simple definition of cancer is
• A relatively autonomous growth of tissue, that
  can develop into a solid
• mass or tumor spread to other areas of the
  body
• Cancer cells
• Are not subject to regulatory system of cell
  growth
• Infiltrate adjacent tissue (in contrast to benign
  tumours)
• Form metastases due to lymphogenic or
  haematogenic spread
• The proliferation of normal cells is thought to
  be regulated by growth promoting
• oncogenes and counterbalanced by
  tumor-suppressor genes.

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Cell cycle Phases of cell activity divided into
G, S M (Growth, DNA synthesis Mitosis
respectively)
Apoptosis
Programmed cell death
Development of new blood vessels to supply oxygen
nutrients to cells
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Proto-oncogenes

• Proto-oncogenes are genes present in normal human
  cells their products (proteins) may play
  important roles in normal cellular processes (as
  growth)
• These proto-oncogenes may be activated to
  oncogenes
• Oncogenes convert normal cells into tumor
  (cancer) cells.
• In cancer cells, oncogenes may be
• Identical to normal genes
• but regulation of their expression in
  cancer cells is abnormal
• Or
  
• Show very small structural differences
  from their counterparts in normal cells.
• These differences will produce agents
  that leads to transformation of normal cells to
  cancer cells.

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Tumour Suppressor Genes

• Tumour suppressor genes are genes that encode for
  proteins (product)
• Involved in protecting cells from unregulated
  growth
• Their mutation may lead to cancer

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How to Diagnose Cancer?

• A golden dream of an oncologist is to diagnose
  cancer at an early stage.
• Diagnosis of carcinoma in the early stages is a
  difficult task due to several reasons including
  lack of symptoms at early stages.
• On the other hand most diagnostic procedures (X
  ray, CT, MRI..etc) are not suitable for early
  diagnosis.
• These methods only detect tumors of at least 1-2
  cm in size (1x109 cells) or more.
• Tumor markers are considered as one of the highly
  valuable tools for detection and follow up of
  malignant tumors their secondaries.

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Tumor Markers

• Tumor Markers
• Are substances present in or produced by a tumor
  itself
• OR
• Are produced by host in response to a tumor
• Can be used
• To determine the presence of a tumor
• OR
• To differentiate a tumor from normal tissue
• Based on
• measurements in blood or secretions

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Tumor Markers

• Tumor markers include a variety of substances
  like
• Cell surface antigens
• Cytoplasmic proteins
• Enzymes
• Hormones
• Oncofetal antigens
• Receptors
• Oncogenes their products (proteins)

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Characteristics of an Ideal Tumor Marker

• Should be highly specific to a given tumor type
• Should provide a lead-time over clinical
  diagnosis
• Should be highly sensitive i.e. non-detectable
  in physiological or benign diseases
• The levels of the marker should correlate
  reliably with the tumor burden
• It is accurately reflecting any tumor
  progression or regression
• with a short half-life
• The test used for detection should be cheap for
  screening at mass level
• It should be of such nature as to be acceptable
  to the target population

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Applications of Tumor Markers

• 1- Diagnosis (D) to help to establish the
  diagnosis
• 2- Screening ( S ) to identify patients with
  early cancer
• 3- Prognosis ( P ) to assess the
  aggressiveness
• 4- Monitoring (M)
• Evaluate the Response to Treatment (RT)
• Detection of Recurrence (R)
• 5- Determination of Risk

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1- Screening

• Screening asymptomatic individuals
• Use of tumor markers, to date, has generally not
  been an effective strategy due to
• Most of the clinically used tumor markers are
  found in normal cells benign conditions in
  addition to cancer cells.
• The relatively low prevalence of individual
  cancer types.
• The screening role is generally very useful if
  restricted to population at high risk because of
• - Family history
• - Environmental exposure
• - Geographic prevalence
• - Clinical profile

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1- Screening cont.

• High risk groups
• Are individuals at increased risks for malignancy
• Alpha fetoprotein (AFP)
• For patients with liver cirrhosis or
  chronic hepatitis who are at risk for
• development of hepatocelluar carcinoma
• Prostate-specific antigen (PSA)
• For men older than 50 years in conjunction
  with a physical examination
• (clinical profile) for early detection of
  cancer prostate
• Calcitonin
• For first degree relatives of a patient
  (family history) with
• medullary carcinoma

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1- Screening cont.

• Sensitivity Specificity of Screening Tumor
  Markers
• Ideal tumor marker for screening asymptomatic
  population should be
• 100 sensitive Always positive in patients with
  the disease
• 100 specific Always negative in individuals
  who do not have the disease
• For examples
• If a test gives positive results in 99 patients
  out of 100 patients
• Its sensitivity is 99
• If a test gives negative results in 90 normal
  individuals out of 100 normal individ.
• Its specificity is 90

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1- Screening cont.

• In reality, an ideal tumor marker which gives
  100 specificity 100
• sensitivity does not exist.
• To increase sensitivity specificity of a tumor
  marker
• Combination of multiple tumor markers
• Combination of tumor markers with other
  procedures
• e.g. combination of Carbohydrate Antigen
  125 (CA 125) with
• ultrasonography for early detection of
  ovarian malignancy

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2- Prognosis

• For cancer patients, determination of prognosis
  is based on determination of aggressiveness of
  tumor, which , in turn determines how a patient
  should be treated.
• As the serum concentrations of tumor markers
  increases with progression and usually reaches
  the highest levels when tumors become
  metastasized, the serum levels at diagnosis are
  likely to reflect the aggressiveness of the tumor
  and predict the outcome.
• High levels of serum tumor marker measured during
  diagnosis would indicate the presence of a
  malignant or metastatic tumor associated with
  poor prognosis.

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4- Monitoring of Disease, Response of Treatment
Detection of Recurrence

• Constitutes the most common clinical use of serum
  tumor markers.
• Markers usually increase with progressive
  disease, decrease with remission do not change
  significantly with stable disease.
• After treatment (surgical resection, radiation or
  chemotherapy), tumor markers are routinely
  followed serially (to monitor the response to
  treatment recurrence).
• It is desirable to monitor the patient using a
  highly sensitive tumor marker test to detect
  recurrence as early as possible
• The appearance of most of the circulating
  tumor markers have a lead time of several month
  (3-6 months) prior to the stage at which many of
  the physical procedures can be used for
  detection of the cancer
• So, rising tumor marker levels may detect
  recurrence of disease before any clinical or
  radiological evidence of disease is apparent
  (biochemical recurrence)

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5- Determination of Risk

• Usually involves genetic probes that evaluate any
  specific genetic abnormality or mutation noted to
  indicate an increased risk of a particular
  malignancy.
• Examples of such abnormalities would include
• - Carriers of Philadelphia chromosome for
  hematological malignancies
• - Carriers of BRCA 1 or 2 genes, which
  confer a higher risk of breast or
• ovarian malignancies

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Samples used for tumor marker estimations

• 1- Tissue (tissue tumor marker)
• Solid tumors
• Lymph nodes
• Bone marrow
• Circulating tumor cells in the blood
• 2- Body fluids (serological tumor markers)
• Ascitic fluid
• Pleural fluid
• Serum
• Urine

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Factors that affect serum concentrations of tumor
markers

• False positive results occur with
• Inflammatory conditions
• Benign conditions
• Presence of liver diseases Causes disturbances
  in metabolism and excretion of some tumor markers
  as AFP, TPA, CEA, CA 19-9, CA 15-3..
• Disturbances of renal function affects levels of
  some tumor markers as beta-2-microglobulin,
  calcitonin, PSA, CEA, CA 19-9, CA 15-3
• As a consequence of diagnostic and therapeutic
  procedures digito-rectal examination,
  mamography, surgery, radio and chemotherapy
• As a consequence of different physiological
  conditions as in pregnancy may affect ßHCG,
  AFP..).

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AFP alpha fetoprotein B-hCG beta human chorionc
gonadotrpin CEA carcinembrionic antigen CA 125
carbohydrate antigen 125
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Factors that affect serum concentrations of tumor
markers

• False negative results occur with
• Insufficient expression of a certain antigenic
  determinant or production in only some of tumor
  cells
• Insufficient blood circulation in the tumor
• Production of immune complexes with
  autoantibodies
• Rapid degradation clearance of antigens

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Frequency of Ordering

• As a general guideline, the time interval between
  serial determinations should be 3 months.
• But in case of an abnormal value, a repeat
  estimate can be ordered within 2 to 4 weeks
  irrespective of the initial reading.
• The success of surgical removal of a tumor as
  determined by tumor marker concentrations is
  ideally ascertained after a period not less than
  5-6 half-lives, to allow tumor marker levels to
  make a plateau or fall to normal.
• This period may be even longer in case of
  treatment with chemotherapy or radiotherapy,
  wherein the therapeutic effects themselves are
  manifested after a lag period.

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GUIDELINES FOR ORDERING/ INTERPRETING TUMOR
MARKER TESTS

• Never rely on the result of a single test
• Order every test from the same laboratory
• Consider half-life of the tumor when interpreting
  the result
• Consider how the Tumor Marker is removed or
  metabolized
• Consider presence of Heterophile
  AntibodiesInterfere with testing due to the
  presence of circulating antibodies against animal
  immunoglobulin (in the test materials)

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METHODS FOR DETECTION

• Immunoassay is the most common measurement method
• Challenges
• Markers often above linearity
• Lipemia, hemolysis and antibody cross reactivity
  cause interferences

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Examples of Frequently Ordered Tumor Markers

• Alpha-fetoprotein
• CA-125
• CEA
• hCG
• PSA
• Her-2/neu
• p53
• BrCa1
• BrAa2
• CA-15.3
• CS-19.9
• Estrogen progesterone receptor
• VMA

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Suggested Recommended Markers for
diagnosis/prognosis
Tumor Tumor markers
Hepatoma (HCC) AFP
Cancer ovary CA-125 Inherited ovarian cancer BrCa1(on chromosome 17, which is the same chromosome having the p53 Her-2/Neu)
Breast Cancer CA15-3 CEA Her-2/neu Estrogen and progesterone receptors If inherited BrCa1, and BrCa2 (on chromosome 13)
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Suggested Recommended Markers for
diagnosis/prognosis
Tumor Tumor markers
Cancer head of the pancreas CA 19-9 CEA
Colorectal carcinoma CA 19-9 CEA
Pheochromocytoma Vanillylmandelic Acid (VMA) in urine
Nonseminomatous testicular cancer AFP ?-hCG CEA
Vesicular mole Choriocarcinoma ?-hCG
Prostate cancer PSA
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Oncofetal Proteins
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Oncofetal Proteins

• These are proteins produced during fetal life.
• They are present in high concentrations in the
  sera of fetuses decrease to low levels or
  disappear after birth.
• They reappear in individuals with cancer.
• This demonstrates that certain genes are
  reactivated as a result of the malignant
  transformation of cells.
• There are several oncofetal antigens as AFP CEA

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Alfa-feto Protein (AFP)

• AFP is a glycoprotein.
• It is one of the major proteins in the fetal
  circulation
• The fetal AFP reaches the peak at 14 weeks of
  gestation and declines at term.
• The maternal AFP level increase from 12 weeks to
  peak during the third trimester
• It is a marker of hepatocellular carcinoma
  germ cell ( non-seminoma ) carcinoma.
• Other causes of increased AFP due to
  non-malignant causes
• - Pregnancy
• - Non- cancerous liver diseases
  hepatitis cirrhosis
• Except in pregnancy , AFP level greater than
  1000ug/L indicates cancer

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Alfa-fetoprotein (AFP) cont.

• Clinical Application of AFP
• Diagnosis, prognosis monitoring of
  hepatocellular carcinoma
• (i.e. hepatoma).
• Used with ultrasound imaging every 6 months in
  patients at high risk of developing HCC (e.g.
  patients with hepatitis B virus hepatitis C
  virus-induced liver cirrhosis).
• AFP is used for early detection (in the lead
  period) which is 3 - 6 months before clinical
  manifestations of the cancer appear.
• AFP is not completely specific for HCC
• AFP may be increased in pregnancy benign liver
  disease.
• AFP is used as a tumor marker for nonseminomatous
  testicular cancer in combination with another
  tumor marker ?-human chorionic gonadotropin
  (?-hCG)

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Carcinoembryonic Antigen (CEA)

• It is the most widely used tumor marker for
  colorectal cancer.
• The main clinical use of CEA is as a tumor marker
  for colorectal cancer for
• Prognosis
• Postsurgery surveillance
• Monitor response to chemotherapy

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Hormones as Tumor marker

• The production of hormone in cancer involves 2
  separate routes
• 1.The endocrine tissue that normally produces the
  hormone can produce excessive amounts e.g.
  pheochromcytoma tumor of adrenal medulla ( TM
  VMA)
• - 2. A hormone can be produced by non endocrine
  tissue that normally does not produce the hormone
  e.g. ectopic production of ACTH

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Human Chorionic Gonadotropin (hCG)

• hCG is a hormone normally secreted by
  trophoblasts in the placenta during pregnancy.
• It is a glycoprotein consisting of ? and ?
  subunits.
• It is the most useful marker for detection of
  gestational trophoblastic diseases (GTDs) that
  include
• Hydatiform mole (vesicular mole)
• Choriocarcinoma
• It is also elevated in nonseminomas tumors of the
  testis.

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Enzymes as Tumor Markers

• Generally, an increase in enzymes is not specific
  or sensitive
• to identify type of cancer.
• PSA is an exception

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Prostate Specific Antigen (PSA)

• PSA is a glycoprotein produced only in the
  epithelial cells of the acini ducts of the
  prostate.
• There are 2 major forms of PSA that are
  found in the blood
• Free Complexed to ?1-antichymotrypsin or
  ?2-macroglobulin.
• Total PSA is used in screening in monitoring of
  prostate cancer
• Free PSA can help to differentiate levels of PSA
  that are in the grey zone i.e. not high enough
  to diagnose cancer prostate, but not low enough
  to rule out the diagnosis of cancer prostate
• Patient with cancer prostate have a lower
  of free PSA.

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Prostate Specific Antigen (PSA)

• Annual PSA testing for screening of prostate
  cancer is indicated for
• In men over 50 years old
• In younger men at high risk e.g. Those with a
  family history of prostate cancer.
• To increase the accuracy of the PSA testing, it
  is essential to use age-adjusted cutoff values of
  PSA
• The best clinical use first clinical
  applications of PSA testing was to monitor for
  the progression of prostate cancer after therapy
  (e.g. radical prostatectomy)
• Causes other than prostate cancer that can ?
  elevated PSA
• Prostate infection
• Prostate irritation
• Benign prostatic hyperplasia (enlargement)

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Carbohydrate Markers

• Carbohydrate relate tumor markers
• are either
• Antigens on tumor cells
• Or Secreted by the tumor cells
• ( CA carbohydrate antigens)

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Carbohydrate Antigen-125 (CA-125)

• CA-125 may be useful for detecting ovarian
  tumors
• Only clinically accepted serologic marker of
  ovarian cancer
• Diagnose ovarian tumors at early stages
• Distinguish benign masses from ovarian cancer
• Monitoring treatments
• CA-125 is not considered specific enough for
  ovarian cancer, as it may be elevated in patients
  with
• Endometriosis
• During the first trimester of pregnancy
• During menstruation.

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Carbohydrate Antigen 15.3 (CA 15-3)

• These are large glycoproteins e.g. mucins that
  are normally found on a variety of epithelial
  cell types, including breast,
• It is most useful in used in disease monitoring
  of metastatic breast cancer

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Receptors as Tumor Markers
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Estrogen/Progesterone Receptors (ER/PR)

• These steroid receptors can play a role in breast
  carcinogenesis and similar to HER-2, assessment
  of ER/PR status is crucial for optimal treatment
  planning
• Decreased recurrence rates disease-related
  mortality have been demonstrated with tamoxifen
  for ER cancers, which are not seen when the
  tumors are ER.
• Receptor status also has significant prognostic
  implications, with best to poorest survival
  ranging from ER/PR to ER-/PR.

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Genetic Markers

• Two classes are involved in the development of
  cancer
• Oncogenes as HER-2/neu
• Suppressor genes as BRCA1 , BRCA2, p53

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Human Epidermal Growth Factor Receptor 2
(Her-2/neu)

• HER-2 gene product is a transmembrane protein
  normally involved in
• cell growth and differentiation through
  its interaction with circulating
• growth factors
• It is identified as an oncogene as its
  amplification results in protein
• over expression that supports rapid
  cellular proliferation (tumorogenesis)
• Her-2/neu is a marker for breast ovarian
  cancers
• It is now routinely measured in breast cancer to
  determine the type of therapy
• Breast cancer positive for Her-2/neu is
  responsive to treatment (Herceptin)
• Breast cancer negative for Her-2/neu is NOT
  responsive to treatment

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Tumor suppressor genes (e.g. p53)

• Tumour suppressor genes
• are genes that encode for proteins that
  are
• Involved in protecting cells from unregulated
  growth
• If mutated may lead to cancer
• An example is p53
• P53 gene is located on chromosome 17 (together
  with the genes of
• BRCA1 Her-2/neu
• Encodes a protein of 53 kDa that normally result
  in cell cycle arrest
• induces apoptosis
• Upon mutation loss of function mutation ? cancer

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p53

• Mutations in p53 gene
• Transition mutations Purine is substituted
  by a purine i.e.
• adenine by guanine or pyrimidine by
  pyrimidine i.e. cytosine by
• thymine.
• Occurs in C - G dinucleotide (hot spot)
• Cancer colon Brain cancer
• Transversion mutations Purine is substituted
  by pyrimidine i.e.
• guanine by thymine or pyrimidine by purine
  i.e. cytosine by adenine.
• Occurs in G to T mutations at codon 249 of
  the gene.
• Cancer lung cancer liver

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p53

• Aflatoxin B
• Potent hepatocarcinogen to which many
  individuals are exposed to it in China Africa.
• It causes transversion mutations of p53 gene
  leading to cancer liver
• Smoking exposure to agent benz (a) pyrene
• cause transversion mutations in p53 gene
  leading to lung cancer
• So, detection of specific type of mutation in
  genes from human tumours may
• reveal the cause of cancer

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BRCA1 BRCA2

• It is inherited as autosomal dominant trait.
• Two genetic loci BRCA1 on chromosome 17 BRAC2
  on
• chromosome 13
• BRCA 1 encodes for a protein that act as a
  trancription factor
• Carriers of BRCA1 gene mutation have an 85
  risk of developing
• breast cancer 45risk of developing
  ovarian cancer