1. Tumor Markers

2. 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

3. Cancer A simple definition of cancer is: Cancer cells :
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
proto-oncogenes & counterbalanced by tumor-suppressor genes.

4. Development of new blood vessels to supply oxygen & nutrients to cells
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

5. Proto-oncogenes In cancer cells, oncogenes may be:
Proto-oncogenes are genes present in normal human cells & their products (proteins) may play important roles in normal cellular processes (as normal promotion of growth)
These proto-oncogenes may be activated to oncogenes
Oncogenes can 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.

6. Tumour Suppressor Genes
Tumour suppressor genes are genes that encode for proteins (product)
that are involved in protecting cells from unregulated growth
Mutation in tumor suppressor genes may lead to cancer

7. 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:
- Lack of symptoms at early stages.
- Diagnostic procedures (X ray, CT, MRI..) are not suitable for early diagnosis.
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 & follow up of malignant tumors & their secondaries.

8. Tumor Markers Tumor Markers
Substances present in or produced by a tumor itself
OR Produced by host in response to a tumor
Can be used to
Determine the presence of a tumor
OR Differentiate a tumor from normal tissue
Based on
Measurements in blood or secretions
A molecule, a process or a substance that is altered quantitatively or qualitatively in precancerous or cancerous conditions, the alteration being detectable by an assay.

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

10. Characteristics of an Ideal Tumor Marker
Should be highly specific (negative in all negative cases)…. Diagnosis & Screening
Should be highly sensitive (positive in all positive cases)….Diagnosis & Screening
Should provide a lead-time over clinical diagnosis ….Screening
The levels of the marker should correlate with the tumor burden .. Prognosis
It is accurately reflecting any tumor progression or regression….Monitoring
Procedure of estimation of the marker should be reliable
The test used for detection should be relatively cheap

11. Applications of Tumor Markers
1- Diagnosis (D): to help to establish the diagnosis
2- Screening (S): to identify patients with early cancer (before
appearance of clinical manifestations)
3- Prognosis (P): to assess the aggressiveness
4- Monitoring (M): follow up of regression or progression
Evaluate the Response to Treatment (RT)
Detection of Recurrence (R)
5- Determination of Risk: i.e. in genetically predisposed individ.

13. 1- Screening
Screening asymptomatic individuals (in very early cases of cancer)
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 groups with risk factors as:
- Family history
- Environmental exposure
- Geographic prevalence
- Clinical profile

14. 1- Screening cont.
High risk groups: are individuals at increased risks for malignancy
Tumor markers used for screening of high risk individuals:
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

15. 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%

16. To increase sensitivity & specificity of a tumor marker
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

17. 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 (surgery, chemotherapy, radiotherapy, etc)
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.

18. 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)

19. 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

20. 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
Disturbances of renal function: affects levels of some tumor markers
Consequences 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…..).

21. AFP: alpha fetoprotein
B-hCG: beta human chorionc gonadotrpin
CEA: carcinembrionic antigen
CA 125: carbohydrate antigen 125

22. Factors that affect serum concentrations of tumor markers
False negative results occur with:
Insufficient expression of the marker
or production of the marker in some of the tumor cells only
Insufficient blood circulation in the tumor
Production of autoantibodies against the marker
Rapid degradation & clearance of the marker

23. Frequency of Ordering of Markers
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.
Checking the effectiveness (success ) of treatment:
Confirming of the success of surgical removal of a tumor is checked by measuring the marker concentrations ideally after a period not less than 5-6 half-lives (to allow tumor marker levels to fall to normal).
In case of treatment with chemotherapy or radiotherapy the period should be longer

24. 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

25. 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

26. 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)

27. Suggested Recommended Markers for diagnosis/prognosis
Tumor
Tumor markers
Cancer head of the pancreas
CA 19-9
CEA
Colorectal carcinoma
Pheochromocytoma
Vanillylmandelic
Acid (VMA) in urine
Nonseminomatous testicular cancer
AFP
-hCG
Vesicular mole & Choriocarcinoma
Prostate cancer
PSA

28. Oncofetal Proteins

29. 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

30. 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 & declines at term.
The maternal AFP level increase from 12 weeks to peak during the third trimester
It is a marker of hepatocellular carcinoma & nonseminomatous carcinoma of testis
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

31. Alfa-fetoprotein (AFP) cont.
Clinical Application of AFP:
Screening, 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 ~ 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 -hCG)

32. 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
Post-surgery surveillance
Monitor response to chemotherapy

33. Hormones as Tumor marker
The production of hormone in cancer involves 2 separate routes:
The endocrine tissue that normally produces the hormone can produce excessive amounts (e.g. Vinylmandilic acid, VMA is a tumor marer for pheochrmocytoma, tumor of the adrenal medulla)
A hormone can be produced by nonendocrine tissue that normally does not
produce the hormone (e.g. ectopic production of ACTH & PTH)

34. 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 nonseminomatous tumors of the testis (with AFP).

35. Enzymes as Tumor Markers
Generally, an increase in enzymes is not specific or sensitive
to identify type of cancer.
PSA is an exception

36. 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.

37. 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. 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)

38. Carbohydrate Markers Carbohydrate relate tumor markers: are either:
Antigens on tumor cells
Or: Secreted by the tumor cells
( CA = carbohydrate antigens)

39. 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.

40. Receptors as Tumor Markers

41. Estrogen/Progesterone Receptors (ER/PR)
These receptors can play a role in breast carcinogenesis assessment of ER/PR status is crucial for optimal treatment planning (similar to HER-2)
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 (ER+/PR+ ) to poorest (ER−/PR–.)

42. Two classes are involved in the development of cancer
Genetic Markers
Two classes are involved in the development of cancer
Oncogenes: as HER-2/neu
Suppressor genes: as p53, BRCA1 , BRCA2…

43. Human Epidermal Growth Factor Receptor 2 (Her-2/neu)
HER-2 gene product is a transmembrane protein normally involved in
cell growth & differentiation through its interaction with circulating growth
factors
It is identified as an proto-oncogene as its amplification results in protein
over expression that supports rapid cellular proliferation (i.e. becomes oncogene).
Her-2/neu is a marker for breast & ovarian cancers
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
It is a proto-oncogene that upon:
Mutation (especially point mutation)
Or Over expression
will encode an Epidermal Factor Receptor (EGF-R) that mediate tumorigenesis (i.e. It is an activation mutation

44. Tumor suppressor genes
Tumour suppressor genes are genes that encode for proteins that are:
Involved in normal protection of cells from unregulated
growth
If mutated may lead to cancer
Examples of tumor suppressor genes:
P53
BRCA1 & BRCA2

45. p53 Carcinogens causing mutations in p53:
p53 (an example of tumor suppressor genes)
P53 gene is located on ch.17 (together with genes of BRCA1 & Her-2/neu)
Gene product (protein) normally result in cell cycle arrest & induces apoptosis
Upon mutation: loss of function mutation  cancer:
Cancer colon, Brain cancer, Cancer lung & Cancer liver
Carcinogens causing mutations in p53:
Aflatoxin B
Potent hepatocarcinogen (common in China & Africa)
Causes mutations of p53 gene leading to cancer liver
Smoking:
Causes mutations in p53 gene leading to lung cancer

46. BRCA1 & BRCA2
They are genes that are inherited as autosomal dominant trait.
Two genetic loci : BRCA1 on chromosome 17 & BRAC2 on
chromosome 13
If these genes are mutated, cancer occur (mutation is inherited to offspring)
Carriers of BRCA1 gene mutation:
have an 85% risk of developing breast cancer
& 45% risk of developing ovarian cancer