Clinical Chem Proteins & Tumour Markers

Learning outcomes

To understand the importance of plasma proteins in the human body
To know and understand the clinical relevance of plasma protein test for diagnosis of diseases
To understand the uses of tumor marker in cancer diagnosis
To describe patient management and the use of tumor markers in colon and breast cancers

Plasma Proteins

Two major groups: albumin & globulins

Individual plasma protein conc affected by:

nutritional status
physiologic changes
synthesis rate
extracellular distribution
clearance rate

Most synthesized in the liver, except immunoglobulins & protein hormones

Prealbumin (Transthyretin)

^^small transporter protein^^ that migrates before albumin in classic serum protein electrophoresis
^^transports thyroid hormones^^
also binds to retinol-binding protein for the ^^transportation of vitamin A (retinol)^^
synthesised in the liver & choroid plexus of the central nervous system * stimulated by glyuocorticoid hormones, androgens & NSAIDs
[[Laboratory Investigations[[ * immunonephlometry * immunoturbidimetry
Clinical revelance * indicates protein nutrition
higher than normal: severe renal failure, corticosteroid use, oral contraceptive use
lower than normal: malnutrition, liver disease, serious infection, trauma, inflammation, serious or long term illness, hyperthyroidism

Albumin

small protein found in blood, CSF, interstitial fluid, urine & amniotic fluid
synthesised in liver: * stimulated by hormones, e.g. insulin, cortisol & growth hormone * inhibited by proinflammatory substances * regulated by colloidal osmotic pressure and protein intake
catabolised mainly in the muscle, liver & kidneys
Primary function: * maintain the colloidal osmotic pressure in the intravascular & extravascular compartments * serves as a transport protein for fatty acids, phospholipids, cholesterol, amino acids, hormones, bilirubin, drugs, toxins, metallic ions & gas
Laboratory investigations * measured colorimetrically by using bromocresol green dye
Clinical relevance * related to liver or kidney disease, or underlying nutritional deficits * hyperalbuminemia: dehydration, increased insulin level, blood transfusion, exogenous albumin administration, anabolic steroid use, androgen / growth hormone administration * hypoalbuminemia: overhydration, hepatic disease, nephrotic syndrome, protein losing states, inflammation, poor nutrition

α2-Macroglobulin

serine protease inhibitor * inhibits different types of proteinases * inhibits enzymes in the kinin, complement, coagulation & fibrinolytic pathways
carrier protein for cytokines, growth factors & cations
modulates immunologic & inflammatory reactions
very large glycoprotein synthesised mainly in the liver
Laboratory investigations * serum protein electrophoresis * immunologic assay
Clinical Relevance * increased level: nephrotic syndrome, related to estrogen & age * decreased level: acute phase response, pancreatitis, prostate cancer

Immunoglobulins

functions as antibodies
Laboratory investigations * serum protein electrophoresis * immunoelectrophoresis * immunoturbidimetry
Clinical relevance * Immunoglobulin deficiency * most manifest in infancy * those in adults are due to another primary disorder or immunosuppresive therapy * most common types: selective IgG immunodeficiency, selective IgA immunodeficiency, X-linked agammaglobulinemia

\ * Polyclonal hyperimmunoglobulinemia * the blood level of all immunoglobulins is increased * sometimes, a particular immunoglobulin is increased more compared to others * autoimmune diseases → particularly IgG * primary biliary cirrhosis → particularly IgM * acute hepatitis → particularly IgG

\ * Monoclonal immunoglobulinemia * involves only one specific type of immunoglobulin * increased concentration due to increased size of a particular clone of plasma cells (sharp peak on serum protein electrophoresis) * multiple myeloma (plasma cell cancer) * IgG (most common), followed by IgA & IgD; IgM * malignant plasma cells secrete M proteins

Tumour Markers

Production:

directly by the tumours; or
as an effect of the tumours on healthy tissue; or
other cells of the body in response to cancer

Traditional concept on tumour markers:

proteins or other substances that are present at a higher amount in cancers
found in blood, urine, stool, tumours, or other tissues & body fluids of cancer patients

Carcinoembryonic Antigen (CEA)

]]expressed in embryonic tissue of gut, pancreas & liver]]
elevated level in many different types of cancer * 60-90% of colorectal carcinoma * 50-80% of pancreatic carcinoma * 25-50% of gastric and breast carcinoma
also elevated in several benign disorders, i.e., alcoholic cirrhosis, hepatitis & ulcerative colitis
lacking sensitivity & specificity for early detection of cancers
Tumour burden * CEA is measured before and after surgical resection to confirm successful removal of tumour burden
Monitor the efficacy of treatment * CEA should drop within the reference interval in 1-4 months if the treatment is successful
Detect recurrence - serially monitored every 2-3 months

α1-fetoprotein (AFP)

major glycoprotein in fetal plasma
serum level declines rapidly after birth; undetectable level within several months after birth
synthesised by embryonic yolk sac and fetal liver
produced and secreted by certain malignant tumours * utilised as tumour marker
Elevated level in adult males and non-pregnant females * hepatocellular carcinoma, germ cell tumours, ovarian tumours * Diagnosis * clearly, marked elevation of α1- fetoprotein level + presence of liver mass * Prognostication * elevated level indicates aggressive tumour & poor prognosis in hepatocellular carcinoma
Normally detected in maternal serum, and peaks at 12-15 weeks of pregnancy * prenatal surveillance - elevated in neural tube defects * part of triple or quadruple screening test for fetal abnormalities
Non-cancerous conditions, e.g., hepatitis & liver cirrhosis, also reports an elevation in α1- fetoprotein level

Tumour Markers (pt 2)

Important characteristics:

tumour-specific
absent in healthy individuals
readily detectable in body fluids

5 broad uses:

screening
diagnosis
prognosis
treatment outcome prediction/monitoring treatment
relapse

Screening

Current widely-used screening methods

mammography, cytology, fetal occult blood test

Serum tumour markers

lack of sensitivity or specificity * high proportions of false negatives/positives
may be elevated in chronic inflammation or those without cancers
usually elevated when the cancer is already well-establish

Diagnosis

Ideally, the sensitivity & specificity should approach 100%

To date, none of the serum tumour markers reach this level of diagnostic performance

poor diagnostic indicator

Gold standard - histology

Prognosis

Concentration level of tumour markers increases with tumour progression

May reflect the aggressiveness of tumour and predict outcome

May reflect the size of tumour and predict survival

Determination & monitoring of treatment

Presence or absence of tumour marker indicates the responsiveness towards a particular treatment

determine the most suitable treatment approach

Monitor the efficacy of therapy

after surgical resection, radiation, chemotherapy or targeted therapy
direct quantitative relationship between tumour marker concentration and tumour load

Criteria used to assess effectiveness of treatment:

no change: tumour marker >50% of value at t=0
improvement: tumour marker <50% of value at t=0
response: tumour marker <10% of value at t=0
complete response: tumour marker undetectable

Relapse

Monitor relapse in patients whom the initial treatment is effective/tumour is eliminated successfully after treatment

An increased level of tumour marker level/appearance of detectable tumour marker

indicates re-emergence and/or spread of the tumour to new sites

Allow earlier identification and treatment, e.g., prostate specific antigen (PSA) in prostate cancer

Laboratory Measurement

Immunoassays * most commonly used * automated testing & relative ease of use
High-performance liquid chromatography * used to detect small molecules, e.g., catecholamine metabolites * more labour intensive & requires more experience and skill
Immunohistochemistry & immunofluorescence * solid tumour tissue markers from fine-needle aspirate or biopsy samples * determine particular cell type & subcellular location

Two main considerations:

lack of standardisation between different assays * use of same methodology or same kit for monitoring
vary in concentration by orders of magnitude * take note of dilution protocols and risk of antigen excess

DNA Markers

Oncogenes

usually encode for proteins function in cell growth and division
activated by point mutations, insertions, deletions, translocations or inversions, gene amplification
responsible in the transformation of tumour cells and abmormal cell proliferation
i.e., RAS, HER2, Myc, cyclin D

Tumour suppressor genes

inhibit cell proliferation, limit the growth and development of tumours
repair DNA damage and initiate apoptosis f abnormal cells
lost or inactivated by mutations in cancers
examples: TP53, BRCA1, BRCA2

Utility in Cancer Patient Management

Breast Cancer

Serum-based tumour marker: cancer antigen 15-3 (CA15-3)

the product of MUC1 gene
higher level is found in serum of breast cancer patients
used as prognostic indicator, in determining the suitable therapy and treatment monitoring in metastatic breast cancer

Tissue-based tumour markers

biopsy
expression of genes associated with breast cancer, e.g., * estrogen receptor * progesterone receptor * HER2 protein receptor
ER/PR-positive patients have a better prognosis with hormone therapy
HER2-amplified tumours is likely to be successfully treated with trastuzumab

DNA markers

mutations in breast cancer susceptibility genes, e.g., * BRCA1 * BRCA2
related to hereditary breast cancers (>90%)
BRCA1 & BRCA2 augment the risk of breast cancer by 65% & 45%, respectively
BRCA1 tumours show more aggressive clinicopathological features than BRCA2

Colorectal Cancer

Serum-based tumour marker: CEA

neither sensitive nor specific to be used in screening
widely used in prognostication, monitoring patient’s response to treatment and recurrence detection
also elevated in inflammatory bowel diseases, pancreatitis, alcoholic cirrhosis, during pregnancy and in smokers

DNA markers

mutations in KRAS, BRAF and NRAS genes via real-time PCR
microsatellite instability status (MSI) via DNA sequencing
protein expression of DNA mismatch repair genes, i.e., MLH1, MSH2, MSH6, PMS2, via immunohistochemistry