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

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    • 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

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    • 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

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