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