Tumour Marker

PS Ogunro

Tumour Marker

• Defination:-A tumour marker is a biomarker found in the blood, urine, or body tissues that can be elevated in cancer, among other tissue types. There are many different tumor markers, each indicative of a particular disease process, and they are used in oncology to help detect the presence of cancer. An elevated level of a tumor marker can indicate cancer; however, there can also be other causes of the elevation.

• Tumor markers can be produced directly by the tumor or by non-tumor cells as a response to the presence of a tumor. Most tumor markers are tumor antigens, but not all tumor antigens can be used as tumor markers.

Definition of Ideal Tumor Marker

1. Be easy and inexpensive to measure in readilyavailable body fluids.

2. Be specific to the tumor studied and commonly associated with it.

3. Have a stoichiometric relationship between plasma level of the marker and tumor mass.

4. Have an abnormal plasma level, urine level, or both,in the presence of micrometastases, that is, at astage at which no clinical or presently availablediagnostic methods reveal their presence.

5. Have plasma levels, urine levels, or both, that arestable and not subject to wild fluctuations.

6. If present in the plasma of healthy individuals, existat a much lower concentration than that found inassociation with all states of cancer.

Obviously much additional research must be donebefore such ideal tumor markers will be found.However, it is important to recognize that theevaluation of an ideal tumor marker shouldrelate to the clinical setting. To do so, it has beensuggested that all tumor markers should alsocomply with the following major criteria.

1. They should prognosticate a higher or lower riskfor eventual development of recurrence.

2. They should change as the current status of thetumor changes over time.

3. They should precede and predict recurrences before they are clinical detectable.

In addition, if a tumor marker is to be used to detect very early stages of cancer, a treatment for that cancer must be available. It might be unethical to detect cancers for which no effective treatment is available.

There are no tumour markers that meet all of these criteria. In addition, if a tumour marker is used to detect early stages of cancer, a treatment must exist.

An ideal tumour marker would be useful for screening, diagnosis, prognosis and staging, monitoring response to therapy, monitoring for recurrence or remission and tumour localisation.

Evaluating the clinical utility of a tumour marker

1. The decision level (cut-off that seperates positive and negative results) for a specific marker / analyte must be established.

2. This is accomplished using the predictive value model (sensitivity, specificity and predictive value) of the test for a positive diagnosis.

3. The ROC curve (see diagram) provides visual display of performance for the entire range of decision levels.

4. The most optimal decision level (i.e. with most optimal sensitivity and specificity) can be pinpointed.

5. Useful when comparing different markers for the same cancer.

Figure: Receiver Operating Characteristic (ROC) Curve: This curve plots the sensitivity vs (1 – specificity) over a series of decision levels. The closer the curve gets to the top left corner, the better is the test at distinguishing diseased from non-diseased

Decision

Classification of tumour markers:

1. Proteins:

a. Enzymes

b. Hormones

c. Other proteins

i. Monoclonal Immunoglobulins

ii. β2-microglobulin

iii. C-peptide

iv. Hormone receptors (estrogen and progesterone receptors)

2. Oncofoetal antigens

3. Carbohydrate markers

4. Genetic markers

a. Oncogenes

b. Tumour suppressor genes

5. Newer “markers”

a. Microarray

b. Proteomics

c. Cell free RNA

PROTEINS

A. ENZYMES

• Plasma enzyme/iso-enzymes activities often increased due to secondary effects of tumour.

• Levels correlate well with tumour burden. Usually non-specific (excl. PSA) and thus not useful in diagnosis of tumour type or organ involved. Benefit in monitoring.

• Iso-enzyme measurement can improve specificity. Foetal form (iso-enzymes) or ectopic production in certain malignancies.

Examples of useful enzymes in malignancy:

i. Alkaline phosphatase

• Useful in assessment of bone or liver metastases. Bone ALP elevated in osteoblastic lesions (e.g. prostate carcinoma), not in osteolytic lesions (e.g. myeloma).Correlates better with extent of liver metastases than other liver enzymes. Elevated GGT and isoenzyme determination (heat stability) confirms liver origin. Regan iso-enzyme (placental ALP) elevated in malignancies such as ovarian, lung, trophoblastic and GIT cancers.

ii. Lactate Dehydrogenase

• Glycolytic enzyme released following cell damage.

• Usually tumours that primarily utilise glycolysisrather than aerobic respiration for their energy requirements. Elevated with many large tumours, including breast, colon and stomach tumours; also in lymphoma, leukaemia and neuroblastoma.

• Levels correlate well with tumour burden.

iii. Creatine kinase

• Occasionally elevated in tumours such as prostate, prostate and small cell carcinoma of the lung. Usually BB iso-enzyme. May cause false elevation in CK-MB level in certain CK-MB assays.

iv. Neuron specific enolase

• Glycolytic enzyme found in neuronal tissue and neuroendocrine system. Elevated in small cell carcinoma of the lung, neuroblastoma, phaechromocytoma, carcinoid, medullarycarcinoma of the thyroid and pancreatic endocrine tumours. Associated with poor prognosis

v. Urokinase-plasminogen activator (uPA) system

• Includes uPA, uPA receptor and uPA inhibitors (PAI-1 & -2). uPA activates plasmin, leading to eventual degradation of extracellular matrix and activation of certain growth factors. Negative prognosticator in breast, colorectal, gastric and oesophageal cancers. PAI-1 regulates uPA, also inhibits apoptosis and promotes angiogenesis. High PAI-1 levels associated with aggressive disease. Analytical methodology: ELISA in serum and tissue extracts; Immunohistochemistry (semi-quantitative, but easier).

vi. Telomerase

• Repairs telomeres (hexanucleotide repeats at the end of a chromosome; shortens with each replication; sets finite life span to normal somatic cells). Telomerase suppressed in most somatic cells at birth. Germline cells and other immortal cells have continued activity, preventing telomere shortening. Found in many tumours incl. that of lung, breast, bladder and cervix. Correlates with tumour progression and prognosis. Analytical methodology: Telomeric Repeat Amplification Protocol (PCR, Elisa, RT-PCR)

MARKERS OF PROSTATE CANCER

vii. Prostatic acid phosphatase

• Tartrate sensitive acid phosphatase produced by epithelial cells of prostate gland. No longer in routine use, replaced by PSA. Clinical use restricted to confirmation of metastatic prostate cancer and prostate cancer staging. Less likely to elevated in BPH than is PSA

viii. PROSTATE SPECIFIC ANTIGEN (PSA)

• Sensitive and stable

• Expressed by normal, benign, hyperplastic and malignant prostatic tissue.

• Thus specific for prostate, but not for malignancy. Single chain glycoprotein. Mw 28.4kDa. Serine protease (similar action to trypsin and chymotrypsin), produced by epithelial cells of aciniand ducts of prostate gland. Secreted into seminal fluid. Function: cleaves seminogelin (after sexual intercourse), liquefying seminal coagulum to release entrapped spermatozoa. Basement membrane prevents PSA escape into circulation before inactivation in ducts

• . Disruption of this barrier (as in cancer) causes PSA overspill directly into circulation before inhibition.

• Two major forms of PSA exist i.e.

a. free = nicked inactive form (this is good PSA)

b. complexed = intact enzymes that form complexes with α1-antichymotrypsin and α2-macroglobulin (this is bad PSA)

Clinical application:

• Total PSA < 4 ug/L is considered normal.

• 4 – 10 ug/L “grey zone”

• > 10 ug/L prostate cancer

• PSA useful in detection, staging and monitoring of prostate cancer. Not specific for prostate cancer. Also elevated in benign conditions such as:

• BPH - overlaps with early organ-confined prostate carcinoma (4 – 10ug/L)

• Prostatitis – returns to normal within 6 weeks (levels usually 4 – 10ug/L)

• Also urethral catheterisation, acute urinary retention, prostate biopsy, prostatic massage, digital rectal examination (2-fold increase), sexual intercourse, even cycling.

Approaches to improve specificity for early prostate CA:a. age-adjusted reference intervals

: 40-49yrs 0-2.5ug/L : 50-59yrs 0-3.5ug/L : 60-69yrs 0-4.5ug/L : 70-79yrs 0-6.5ug/L

b. PSA density-[PSA]/prostate volume by transrectalultrasonography

c. PSA velocity – rate of PSA increase over time. Rate > 0.75ug/L/yr suggestive of prostate cancer

d. Percentage fPSA- especially useful when tPSA falls in the grey zone. fPSA < 15% highly suggestive of prostate CA.

e. Complexed PSA – elevated cPSA associated with prostate carcinoma

• PSA levels correlate with stages of tumourextension and metastases, advanced stages associated with higher PSA levels.

• Organ confined usually < 50ug/L

• Bone metastases seldom < 20ug/L

• PSA in monitoring of treatment:

• Almost exclusively produced by prostatic tissue. Levels should drop below detection limit after prostatectomy – within 2 – 3 weeks (t ½ = 2-3 days).

• Levels increase with cancer progression, decrease in remission and remain unchanged in stable disease.

Analytical methodology:

Immunometric assay. Different assays yield different results due to variations in antibody epitopes, assay calibration, assay reaction time, reagent matrices, assay sensitivity and imprecision. Equimolar assays bind fPSA and cPSA equally (best). Non-equimolarassays favour one over the other (unreliable). Ultrasensitive assays (detection limit 0.01 –0.001ug/L) – facilitates detection of residual prostate cancer

B. HORMONES

Can be elevated either due to:

• Overproduction by endocrine tissue

• Ectopic production (non-endocrine tissue)

Thus elevation of a specific hormone is not specific for a particular tumour.

• Hormones used as tumour markers include:

i. ACTH

Produced by corticotrophic cells of anterior pituitary. Can be pituitary or ectopic production. Level >200ug/L suggestive of ectopic production. Usually small cell carcinoma of lung (also pancreatic, beast, stomach and colon cancer).

ii. Calcitonin

• Produced by C-cells of thyroid. Marker for medullary carcinoma of thyroid. . Valuable in screening, diagnosis, prognosticating and monitoring treatment in familial MEN II. Correlates with tumour volume, invasion and metastases. Pentagastrin / calcium stimulation improves sensitivity.

iii. Human chorionic gonadotropin (HCG)

• Glycoprotein, consisting of 2 subunits, encoded by separate genes.

• Cancer associated with differential production of subunits. Most cancer patients produce both free and intact molecules. HCG markedly elevated in trophoblastic tumours (> 1million IU/L); also in 70% of patients with non-seminomatous testicular tumours (along with AFP). Lower levels of elevations in melanoma, breast, GIT, lung and ovarian cancer; also in benign conditions such as cirrhosis, duodenal ulcer and inflammatory bowel disease.

• Levels correlate with tumour burden and prognosis. Useful in monitoring of treatment and progression of trophoblastic disease. Decline expected after removal of tumour (t ½ = 12-20hrs). Slow decline or persistent elevation implies residual disease. Reference limit in men and non-pregnant women is < 5IU/L

Analytical methodology: sandwich immunometricmethod.

• Intact HCG: measures only intact HCG

• Total β HCG: measures both free and intact β subunits – preferred as tumour marker (tumours produce large amounts of free β subunits)

iv. Other hormones used as tumour markers include:

• ADH: small cell carcinoma lung; adrenal cortex, pancreatic and duodenal cancer

• Gastrin: gastrinoma

• Growth Hormone: pituitary adenoma; renal, lung cancer

• Human placental: trophoblastic, gonads, lung, breast cancer

• Lactogen

• PTHrP: breast, lung cancer

v. Catecholamines and serotonin:

C. OTHER PROTEINS

i. Paraproteins –

ii. β2 microglobulin –

iii. C-peptide – useful in insulinomas

iv. Hormone Receptors – Estrogen (ER) and Progesterone (PR) receptors

Nuclear receptors.Sensitive indicator of potential responsiveness to hormonal therapy in breast cancer. 80% of patients who demonstrate both, will respond to hormone therapy. (60% with only ER)

ONCOFOETAL ANTIGENS

Produced normally in foetal life. High concentrations in foetal serum, decreasing to low levels or disappearing after birth.

Some neoplasms revert to embryonic phenotype, secreting foetal products.

i. α –Foetoprotein (AFP) : - glycoprotein, mw 70kDa t ½ = 5 days, structurally and genetically related to albumin Elevated in neural tube defects (maternal serum and amniotic fluid). Protein crosses from foetal plasma into amniotic fluid via defect. See Pregnancy notes. Mild elevations (<200ug/L) in benign liver conditions such as hepatitis (recovery) and cirrhosis. Very high levels in primary hepatoma (derepression of AFP gene) and less often in other malignant conditions (e.g. testicular tumours). >1000ug/L indicative of cancer

ii. Carcinoembryonic antigen (CEA)

• Part of large family of relate glycoproteins, mw 150 – 300kDa. Elevated in various cancers incl. 70% of colorectal cancers, stomach (50%), pancreas (50%) cancers, occasionally other tumours. May be elevated in benign conditions such as cirrhosis, pulmonary emphysema, rectal polyps, benign breast disease and ulcerative colitis. Not sensitive screening test, useful in clinical staging,

prognosticating and monitoring therapeutic response.

CARBOHYDRATE ANTIGEN (CA) MARKERS

• High molecular weight mucins / blood group antigens found on tumour cell surface or secreted by tumour cells. Useful as tumour markers, more specific than naturally secreted markers such as enzymes and hormones.

i. CA 15-3: Marker for breast carcinoma; also elevated in other malignancies such as pancreatic, lung, ovarian, colorectal and liver cancer. Not suitable for screening; although useful for monitoring therapy and disease progression in metastatic breast cancer.

ii. CA 125: Glycoprotein, mw >200kDa. Marker for ovarian cancer. Useful in screening family members where ovarian Ca is strongly familial. Also elevated in endometriosis and other malignancies.

iii. CA 19-9: Glycolipid, mw 200 – 1000kDa. Normally synthesised by human pancreatic and biliary duct cells, as well as gastric, colon, endometrial and salivary epithelia. Marker for adenocarcinoma of pancreas (↑ in 80% of cases), but rise is too late to be useful in early detection

GENETIC MARKERS

• Malignancy is thought to be the outcome of multiple genetic changes. Two classes of genes are implicated i.e. oncogenes and suppressor genes.

A. Oncogenes

Derived from proto-oncogenes activated by gain-of-function mutations (point mutations, insertions, deletions, translocations or inversions); most encode proteins that activate cell proliferation. Activation leads to cell division and thus malignancy. Oncogenes mostly associated with haematological malignancies (e.g. leukaemia). Presence detectable by demonstration of mutation or the protein product. Useful for predicting prognosis and treatment response. Also in prediction of cancer risk (e.g. RET oncogene mutations in MEN syndromes).

Example of Oncogenes

i. Ras genes (K-ras; H-ras) – encodes

P21 - signal transduction for mitosis.

N-ras mutations in neuroblastomas, acute myeloid leukaemia, pancreas, colon, lung, endometrial and bladder cancers.

ii. C-myc – encodes

p62 – essential for DNA replication; enhances RNA transcription. B & T cell lymphoma, sarcoma and endotheliomas.

iii. HER-2/Neu (c-erb) – encodes

p105 – Epithelial growth factor receptor family. Normally expressed on epithelia. Increased expression in breast, ovarian, GIT tumours. Useful prognosticator in breast cancer. Assoc. with neural tumours.

iv. Bcl-2 – protein product inhibits apoptosis. Normally expressed on long lifespan cells (e.g. neurons) and proliferative rapidly renewing cell lines (e.g. basal epithelial cells). Haematological malignancies (lymphoma, myeloma, chronic leukaemias). Over-expression implies chemotherapy resistance.

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v. BCR-ABL – Philadelphia chromosome fusion gene–balanced translocation (9:22); inhibition of apoptosis; 90% chronic myeloid leukaemia. Useful in diagnosis, treatment and monitoring of CML. In AML, associated with higher risk of relapse.

vi. RET – tyrosine kinase receptor- unregulated activation occurs in papillary thyroid cancer, familial medullary thyroid carcinoma & MEN2.

B. Tumour suppressor genes

• Normally encode for a product that suppresses the expression of malignancy i.e. suppresses cell division or promotes apoptosis. Loss of normal chromosomes or genes may lead to reversion to malignancy. Often associated with solid tumours. Detection of mutations clinically useful in diagnosis and prognosis of cancer; also in prediction of cancer susceptibility (e.g. RB and retinoblastoma).

Examples of Tumour suppressor genes

i. RB – retinoblastoma gene – p105RB-Hypophosphorylated form complexes with transcription factors (e.g. E2F), blocks transcription of genes in S-phase cells. Loss of function leads to increased DNA synthesis and cellular proliferation. Screening for RB mutations determines RB susceptibility in familial form.

ii. P53 – arrests cell-cycle (inhibits G1) via p21, in response to DNA damage. 80% colon cancer patients have deletion of one gene and point mutation in the other allele.

iii. BRCA 1&2

Mutations of these genes associated with breast and ovarian cancer.

BRCA1mutation carriers have 85% risk for breast and 45% for ovarian cancer by age 85yrs.

iv. APC – adenomatous polyposis coli -mutations in 70% colorectal cancer

v. NF1 – neurofibromatosis 1 –neurofibromin (similar to GTPase activating protein); loss of function leads to continuous cell activation. Von Reckinghausen disease, colorectal cancer, melanoma and neuroblastoma

vi. WT1- Wilms tumour suppressor gene

vii. DCC – Deleted in Colorectal Carcinoma – deletion associated with advanced stage and poor prognosis in colon cancer; although better prognosis and tumour differentiation in gastric cancer.

viii. PTEN –mutations cause apoptosis inhibition; Associated with advanced tumour stage and poor prognosis in breast, liver and cervical cancer.

• Others

A. CATECHOLAMINES :- Phaeochromocytomas

B. SEROTONIN:- carcinoid syndrome (rare; usually bronchial tumours or liver metastases) with GIT (borborygmi, diarrhoea, colicky pain, nausea, vomiting); cardiovascular (flushing, pulmonary stenosis and right heart failure); respiratory (bronchospasm) and other features.

NEWER “TUMOUR MARKERS”a. Microarrays (DNA “chip”): In cancer tissue, this is useful

in characterising tumours (e.g. breast tumours) into clinically relevant subgroups, which in turn can act as prognostic markers and therapeutic guides.

b. Proteomics (protein “chip”):These can be used as markers for cancer detection and diagnosis, and as treatment guides. (Makes use of similar microarray technology developed for DNA analysis.)

c. Cell-free nucleic acids:(Specific mutations, e.g. N-rasmutation in acute myeloid leukaemia, have been demonstrated in plasma.) Levels correlate with tumourburden and stage of disease.

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