TUMOUR MARKERS

AND FOETAL
TESTING

Prof. S. Mukanganyama-
Metastatic tumor
Department of Biochemistry
HBC 408 L18
 OUTLINE
2
 Basic knowledge of cancer
3

• What is Cancer?
• How does cancer occur?
• How many types of
cancers?
• Current therapeutic
strategies for cancers
 Cancer
4

 Neoplasia (Cancer): a new growth

 an abnormal mass of tissue whose growth
exceeds and is uncoordinated with that of
normal tissues.
 Loss of responsiveness to normal growth
control.
 Cancer = Tumour = Neoplasm - study -
Oncology
 Malignant cells proliferate to produce progeny
that are also malignant.
 Cancer
5

 Malignancy are encoded in the genes of the

cancer cell i.e. is a genetic disease
 Cells derived clonally from a single precursor
cell
 Transformed cells
 Replicate without control
 "Parasites" -compete with normal tissues for
metabolic needs e.g. flourish in patients who
are otherwise wasting
 Partial autonomy
 Proteins within the cell control the cell cycle
 Signals affecting critical checkpoints determine whether
6
the cell will divide (cyclins, kinases)

G1 checkpoint

Control
system

M checkpoint G2 checkpoint
  Cells continue dividing until they touch one
another
7

= density-dependent inhibition

Cells anchor to dish surface and

divide.

When cells have formed a

complete single layer, they stop
dividing (density-dependent
inhibition).

If some cells are scraped away,

the remaining cells divide to fill
the dish with a single layer and
then stop (density-dependent
inhibition).
  Growth factors are proteins secreted by cells
that stimulate other cells to divide
8

After forming a single layer, cells

have stopped dividing.

Providing an additional supply of

growth factors stimulates further
cell division.
  Growth factors bind to specific receptors on the
plasma membrane to trigger cell division
9

Growth factor

Plasma membrane

Relay
Receptor proteins G1 checkpoint
protein
Signal
transduction Cell cycle
pathway control
system
10
 BENIGN TUMOURS
 Localised and cannot spread

 Amenable to surgical removal

 Well differentiated cells, resemble very closely

their normal counterparts

 Develop an enclosing fibrous capsules that

separate them from host tissue
12

Expansile Growth pattern

 Malignant Tumours
13

 True " cancers" –crab-Adhere to any part they

seize upon.
 Invade and destroy structures, spread to distant
sites (metastasise) to cause death.
 Nuclear -cytoplasmic ratio 1:1 instead of the
normal 1:4 or 1:6.
 Giant cells with either one enormous nucleus or
several nuclei.
 Nuclei variable + bizarre in size and shape
 No recognisable patterns of orientation to each
other.
14

Invasive growth pattern

 Malignant tumors can invade other tissues and
may kill the organism
15

Lymph
vessels

Tumor

Glandular
tissue

Metastasis

1 A tumor grows 2 Cancer cells invade 3 Cancer cells spread

from a single neighboring tissue. through lymph and
cancer cell. blood vessels to other
parts of the body.
16

Cancer Cell Do Not Grow Faster

Than
Normal Cells
Rather, Their Growth is Just
Uncontrolled
17

1 fertilized egg
50x1012

1016 cell divisions/lifetime

Proliferation Differentiation Death

 Cellular equilibrium
18

Proliferation Differentiation Death

Transit
Renewing
Proliferating

18
 Cancer: disruption of cellular
19
equilibrium

Proliferation Differentiation Death

 Invasion and Metastasis
20

 Abnormal cells
proliferate and spread
(metastasize) to other
parts of the body

 Invasion - direct
migration and
penetration into
neighboring tissues

 Metastasis - cancer
cells penetrate into
lymphatic system and
blood vessels
 What causes Cancer?
21

 Cancer is caused by
alterations or mutations
in the genetic code
 Can be induced in
somatic cells by:
 Carcinogenic

chemicals
 Radiation

 Some viruses

 Heredity - 5%
 EARLY EVIDENCE THAT
CHEMICALS CAUSE
22
CANCER
• 1775 scrotal cancer &
soot exposure in
English Chimney
Sweepers.
 Catholic nuns - Increased susceptibility to
breast cancer
23
 Targets of carcinogens
24

a) Growth -promoting proto-oncogenes-

Genes involved in normal transmission of
growth-promoting signals from the cell surface to
the nucleus (e.g. ras, src) or are transcription
factors (e.g. myc, fos)

b) Growth-inhibiting cancer suppresser

genes.- “tumour suppresser genes”.
both copies of the gene (e.g. Rb, p53,BRCA1 or
BRCA2) must be either lost, mutated or
inactivated.
 What are the genes responsible for
tumorigenic cell growth?
25

Normal
Proto-oncogenes +
Cell growth
and
Tumor suppressor genes - proliferation

Cancer
Mutated or “activated” ++
oncogenes Malignant
transformation
Loss or mutation of
Tumor suppressor genes

25
Functions of Cellular Proto-Oncogenes

26 1. Secreted Growth Factors

2. Growth Factor Receptors

4. Nuclear
Proteins:
Transcription
3. Cytoplasmic Factors
Signal Transduction
Proteins
5. Cell Growth
Genes
26
27

A generic signalling
pathway

27
 Oncogenes
28

proto-oncogene = ras
Oncogene = mutated ras
Always activated
Always stimulating
proliferation
29
 p53
30

 Phosphyorylated p53 activates transcription of p21 gene

 p21 Cdk inhibitor (binds Cdk-cyclin complex --> inhibits kinase
activity)
 Cell cycle arrested to allow
DNA to be repaired
 If damage cannot be repaired
--> cell death (apoptosis)

 Disruption/deletion of p53 gene

 Inactivation of p53 protein
--> uncorrected DNA damage
--> uncontrolled cell proliferation --> cancer

30
 Activation mechanisms of proto-oncogenes

31

proto-oncogene --> oncogene

31
 TUMOR SUPPRESSOR GENES
32
Disorders in which gene is affected

Gene (locus) Function Familial Sporadic

DCC (18q) cell surface unknown colorectal

interactions cancer

WT1 (11p) transcription Wilm’s tumor lung cancer

Rb1 (13q) transcription retinoblastoma small-cell lung

carcinoma

p53 (17p) transcription Li-Fraumeni breast, colon,

syndrome & lung cancer

BRCA1(17q) transcriptional breast cancer breast/ovarian

tumors
BRCA2 (13q) regulator/DNA repair
32
33
 THE CAUSES OF GENOMIC CHANGES IN
CANCER
34
UV

Carcinogenic Replication Errors

chemicals
Radiation

Normal cell Viruses

Damaged DNA

Rearrangements
Point mutations (translocation, deletions,
amplifications)

Alters DNA of genes controlling cell

Cancer cell proliferation. (Proliferation becomes
abnormal)
 Factors Believed to Contribute
35
to Global Causes of Cancer
36
Fruits and Vegetables Decrease
Cancer Risks
37

 Cancer rates could

decline by up to
20% if everyone
consumed 5 fruits
and vegetables a
day!*
 Cancer fighting
substances:
 Antioxidants
 Dietary fiber
*American Institute for Cancer Research, 1998.
 Carotenoids
 Flavenoids
 Risks For Cancer
38

 Lifetime risk – 25% the probability that an

individual, over the course of a lifetime, will
develop cancer or die from it
 Relative risk – measure of the strength of the
relationship between risk factors and a particular
cancer
 Smoking – 30% of all cancer deaths, 87% of lung
cancer deaths
 Obesity – 50% higher risk for breast cancer in
postmenopausal women, 40% higher risk in
colon cancer for men
 Risk Factors of Cancer
39
 Occupational And Environmental
40
Factors

 Asbestos
 Nickel
 Chromate
 Benzene
 Arsenic
 Radioactive substances
 Cool tars
 Herbicides/pesticides
 Social, Psychological Factors and
41
Chemicals In Foods

 Stress has been implicated in increased

susceptibility to several types of cancers
 Sleep disturbances, diet, or a combination of
factors may weaken the body’s immune system
 Sodium nitrate when ingested forms a potential
carcinogen, nitrosamine
 Sodium nitrate is still used because it is
effective in preventing botulism
 Pesticide and herbicide residues
 Current strategies to combat
cancers
42

• Mechanics -- surgery,1600BC
• Physics -- radiotherapy,1896
• Chemistry -- chemotherapy,1942
• Biology -- immunotherapy,1976
 How Far Have We Come ?
43

Five year survival rate:

 1913 - 10%

 2003 - 66%

 Advances in cancer
research continue
 Evidence for immune reactivity to
tumors
44

 Tumors that have severe lympho -reticular

infiltration have a better prognosis than those
that do not.

 Certain tumors regress spontaneously

 There is an increased incidence of primary and

secondary malignancies (particularly lympho-reticular
tumors) in immunodeficient patients
 Evidence for Anti-tumor immunity
45

 Antibodies and immune T lymphocytes have been

detected in patients with tumors.
 The young and the very old have an increased
occurrence of tumors.
 Finally, animals can be specifically immunized against
various types of tumors
 Tumours that have severe mononuclear cell infiltration
have a better prognosis than those that lack it.
 Certain tumours regress spontaneously (e.g.,
melanomas, neuroblastomas), suggesting an
immunological response
46
 TUMOUR IMMUNITY
47

 If the immune system is to respond to a tumour,

it must recognise tumour antigens that are
viewed as nonself
 Tumour Antigens - intracellular components that
are broken down into peptides 8-11 amino
acids in lengths and presented bound to MHCI
 Larger extracellular components shed from
tumour cells and presented on class II
molecules
 TUMOR ASSOCIATED ANTIGENS
48

 A number of alterations in gene expression

occur in cells during tumorigenesis.
 Tumorigenesis may lead to expression of new
antigens (neoantigens) or alteration in existing
antigens that are found on normal cells.
 Include membrane receptors, regulators of cell
cycle and apoptosis, or molecules involved in
signal transduction pathways.
 Antigenic changes:
49

 Antigenic changes observed in malignant cells

include reappearance of fetal antigens (onco-
fetal antigens)
 Some of these antigens may be secreted while
others are membrane-associated molecules.
 Neo-antigens that contribute toward tumor
rejection are referred to as tumor associated
transplantation antigens (TATA)
 Main types of tumour antigens
50
50

 Tumor-specific transplantation antigens (TSTA)

which are unique to tumor cells and not
expressed on normal cells-Responsible for
rejection of the tumor.

 Tumor associated transplantation antigens

(TATA) -Expressed by tumor cells and normal
cells.
 Tumor-specific transplantation
antigens
51

 Chemical- , UV- or virus-induced tumours express

neo-antigens
 Majority of these tumours often weakly immunogenic
or non-immunogenic.
 TSTAs cannot be identified easily.
 May be secreted while others may be membrane-
associated molecules
 Onco-fetal antigens
52

 Onco-fetal antigens may appear due to de-repression

of genes that were only expressed early in life.
 Two major onco-fetal antigens are
1- alpha-fetoprotein (AFP)
AFP is produced only as a secreted protein
2- Carcino-embryonic antigen (CEA )
found both on cell membranes and in secreted fluids.
 Since secreted antigens contribute little toward
immunity against tumors, the role of these neo-antigens
in immuno-surveillance is questionable
 Tumor-associated developmental
antigens or onco-fetal antigens
53

 Include alpha-fetoprotein (AFP) and carcino-

embryonic antigen (CEA) found secreted in the
serum.
 AFP found in patients with hepatocellular
carcinoma whereas CEA is found in colon
cancer.
 In diagnosis- AFP is produced only as a secreted
protein whereas CEA is found both on cell
membranes and in secreted fluids.
 Alpha-fetoprotein (AFP)
54

 Normal range in humans is 0-20 ng/ml.

 Level rises considerably in patients with
hepatomas and non-seminal testicular
carcinoma.
 A 5-fold or higher rise in this protein is used for
monitoring hepatomas and testicular cancers.
 AFP level may also be raised in some non-
malignant conditions, such as cirrhosis, in
hepatitis and other forms of liver damage.
 Point Mutations in oncogenes or
tumour suppressor genes
55

 Resulting amino acid changes would be novel

compared with normal proteins
 Altered peptides can bind to class I MHC proteins
and recognised by T cells e.g. Point mutations of
p53 gene can lead activation of some T cells
 Adult expression of developmental
genes
56

 Some genes only expressed during the

embryonic development and not in adult
life
 Either the immune system never
encountered peptides from these gene
products or tolerance waned with time
 These genes maybe re-expressed
Changes in carbohydrate structures
57

 Some tumours express altered carbohydrate

structures

 Potentially recognised by some T cells

especially those expressing the gd form of the T
cell receptor
 Carcino embryonic antigens
58

 CEA levels in normal people range up to 2.5

ng/ml,
 They increase significantly in certain
malignancies, particularly colo-rectal cancers.
 They may also rise in some non-malignant
conditions (such as chronic cirrhosis,
pulmonary emphysema and heavy smoking).
 Levels that are 4-5 times normal have been used
to predict recurrence of colo-rectal tumors.
 Prenatal Genetics
59

• Describe the strategies used to screen for

Down syndrome

• Understand the advantages and disadvantages

of screening versus diagnostic genetic testing

• Aneuploidy-the presence of an abnormal

number of chromosomes in a cell, for example
when having 45 or 47 chromosomes when 46
is expected in a human
 Risk Factors for Aneuploidy
60

 Advanced maternal age

 Previous child with DS
 1% recurrence risk until >40 yrs

 Robertsonian translocation carrier-(centric fusion of two

acrocentric chromosomes) occur with a prevalence of ∼1 in 1000 in
the general population
 two different D group chromosomes (chromosomes 13, 14 and
15), two different G group chromosomes (21 and 22)
 At meiosis, these rearrangements form trivalents, segregation of
which may result in gametes nullisomic or disomic for one of the
chromosomes involved in the rearrangement and consequently
to a zygote with trisomy or monosomy for one of the
chromosomes involved
 What is a chromosome
translocation?
61

 Robertsonian translocations occur when one chromosome

becomes attached to another.
 Only five of the 23 pairs of chromosomes take part in
Robertsonian translocations (these are chromosomes 13, 14,
15, 21 and 22).
 Robertsonian translocations happen during the making of
eggs and sperm, or shortly after an egg and sperm come
together to make a baby (at conception).
 A person who carries a balanced Robertsonian translocation
is not usually affected by it, and is often unaware of having it.
 The only time it may cause the person problems, is when he
or she comes to have children.
 The child may inherit an unbalanced form of the translocation.
62
63
 Maternal Serum Screening
General Concepts
64

 Used to adjust risk for Down syndrome based

on maternal age
 Voluntary
 Screening tests are not diagnostic tests and
cannot detect all chromosome abnormalities or
congenital anomalies
 Sensitivity or detection rate <100%
 5% Positive Screen Rate is considered
acceptable
 Maternal Serum Screening
65

 Advantages  Limitations
 Avoids an invasive  Provides a revised risk
test assessment not a
 Avoid potential for diagnosis
fetal loss  Sensitivity <100%
 Identifies a fetus at  Misses other
risk chromosome
 Disadvantages abnormalities
 Anxiety

 False reassurance
66

Anencephalic newborn Cleft lip and palate Microtia

• Congenital abnormalities (birth defects) comprise >

1/5 of all fatalities among newborns/infants.
 Of these, the largest portion consists of cardiac
abnormalities followed by lung abnormalities and
chromosomal aberrations.
67
  Patau's syndrome (47, Y-13-13: 90% lethality in the first year of life).
 Edwards's syndrome (47, Y-18-18: 90% lethality in the first year of life).

 Down’s syndrome (47, Y-21-21)

68
 Second Trimester Maternal Serum
69
Screening for Aneuploidy
 Performed at 15-20 weeks
 Singleton gestation
 Adjusts age risk based on levels of
 AFP
 hCG “Triple”
“Quad”
 Unconjugated estriol (uE3)
 Inhibin-A
 Detection rate in women
 <35: 60-75% for DS
 >35: 75% or more
 >80% for trisomy 18
 Positive screening rate 5%
 Alpha-fetoprotein (AFP)
70

 Glycoprotein of
unknown function
 Used to screen for
trisomy 21
 15-20 weeks gestation
 Detection rate 20-25%

What is alpha-fetoprotein (AFP) blood test?

 alpha-fetoprotein (AFP)
71

 Most widely used biochemical blood test

 a protein normally made by the immature liver
cells in the foetus.
 At birth, infants have relatively high levels of
AFP, which fall to normal adult levels by the first
year of life.
 Also, pregnant women carrying babies with
neural tube defects may have high levels of AFP
 -anabnormal foetal brain or spinal cord that is
caused by folic acid deficiency during pregnancy.
 In which situations are high
72
blood levels of AFP seen?
 In adults, high blood levels (>500 ng/ml) of
AFP in these situations:
 HepatocellularCarcinoma (HCC)
 Germ cell tumors (cancer of the testes and
ovaries)
 Metastatic cancer in the liver (originating in other
organs)
 pregnant women carrying babies with defects
 Maternal Serum Alpha-
73
Fetoprotein Screening (MSAFP)
 Examines the level of alpha-fetoprotein in the
mother’s blood during pregnancy.
 Not a diagnostic test.
 It is often part of the triple screen test that
assesses whether further diagnostic testing
may be needed.
 When is MSAFP performed?
74

 May be performed between the 14th and 22nd weeks of

pregnancy, however most accurate during the 16th to 18th
week.
 Levels of AFP vary during pregnancy so accurate pregnancy
dating is imperative for more reliable screening results.
 All pregnant women should be offered the MSAFP
screening, but it is especially recommended for:
 Women who have a family history of birth defects

 Women who are 35 years or older

 Women who used possible harmful medications or drugs

during pregnancy
 Women who have diabetes
 Human Chorionic Gonadotrophin
75

 Serum levels in DS often >2.5 MoM

 hCG or free b-hCG used
 Elevated levels found in hydatiform molar
pregnancies
 Hydatidiform mole, or molar pregnancy, results
from too much production of the tissue that is
supposed to develop into the placenta
 tissues develop into an abnormal growth, called a
mass
 partial molar pregnancies associated with triploidy
 Molar pregnancy (Mass)
76

 There are 2 types of these masses:

 Partialmolar pregnancy. There is an abnormal
placenta and some fetal development.
 Complete molar pregnancy. There is an abnormal
placenta and no fetus.
 Both forms are due to problems during
fertilization.
 Maternal Serum Screening for
Trisomy 21 and 18
77

Serum marker Trisomy 21 Trisomy 18

AFP

hCG

uE3

Inhibin-A N/A
 Sequential Screening for DS
78

Offer 1st trimester screen

(NT, PAPP-A, hCG)

DS risk >1 in 50 DS risk <1 in 50

2nd trimester screen with

Offer counseling & CVS Integrated Result
(NT, PAPP-A,AFP, hCG, uE3,
Inhibin)
Uses both 1st and 2nd
trimester results to adjust DS risk >1 in 270
maternal age risk for DS
and takes advantage of
higher detection rate Offer counseling & amnio
79