TUMOR MARKERS

&
MEDICAL GENETICS
•One minimally invasive method for early detection and
therapeutic monitoring of cancer involves the
measurement of tumor markers.

•The pathogenesis of the disease has been associated

historically with (1) chemical carcinogens (diet, fumes,
skin contacts), (2) radiation, (3) viruses, and (4) heredity.

* Mechanistically, all of these act by causing DNA

mutations, culminating in the loss of growth control with
increased cellular proliferation and decreased cellular
differentiation.
•Since neoplastic transformation is associated with
altered gene expression, which in turn affects the
production of enzymes, hormones, receptors, proteins
and metabolites that are released into the circulatory
system, (1)measurement of these substances aids in the
diagnosis and characterization of the disease.

* Similarly, (2) the DNA content of a tumor cell can be

examined for mutations, the absence of suppressor
genes that would prevent tumor formation, and the
presence of oncogenes that turn on tumor growth and
ploidy, which is an indicator of growth rate and
aggressiveness.
•This group of substances has come to be known as
tumor markers.

> Characteristics of an Ideal Tumor Marker:

•A tumor marker is a substance synthesized by the host in
response to a tumor that can be used to detect the
presence of the tumor.

•An ideal tumor marker has the following characteristics:

Specificity for cancer. The substance should be
produced only by the tumor
Sensitivity for cancer. A very small tumor growth or
metastasis produces measurable amounts of marker.
The amount of marker produced correlates well with
the tumor load.

There should be a consistent concentration of the

tumor marker in patients with stable disease.

The half-life of the marker needs to be short enough

so that when production drops, the level of marker
falls off rapidly, producing low or undetectable
concentrations in patients who are in remission.

Levels of the marker (initial and serial) should have

prognostic value.
•The assay for the marker should be analytically sensitive,
specific, accurate, precise, easy to perform, inexpensive,
and rapid.
•Unfortunately, there are no ideal tumor markers.
> Uses of Tumor Markers;

1) Screening for disease in asymptomatic population

2) Diagnosis of disease in symptomatic patients
3) Aid in clinical staging
4) Measurement of tumor burden
5) Therapeutic monitoring and selection
6) Detection of recurrence of disease (relapse).
7) Prognostic indicator
•Tumor markers have limited use in diagnosis because of
the overlap between values obtained in benign and
malignant disease.
•The primary use of tumor markers is to monitor therapy
in patients who have been diagnosed as having a
malignant tumor.

> Biological Sources:

•In general, these are found and measured in body fluids
and tissues.
. Benign tumors are generally well differentiated.

*The cells in a benign tumor are similar to the cells of the

corresponding normal tissue, and therefore the tumor
markers produced are typically the products found in the
normal tissue.

•They may be found in increased amounts in the

circulation, depending on the size of the tumor.

•Malignant tumors may produce substances associated

with a normal cell or they may be different.
•When a normal cell is transformed into a tumor cell,
gene expression changes.

•The affected cell may lose its ability to synthesize

some specific cell product, or it may manufacture
greatly increased amounts.

*The cell may be less specialized than the tissue it

evolved from and assume the characteristics of the
less well-differentiated cells of the embryo,
synthesizing proteins (on-cofetal) found in the
embryo but not in a normal adult.

* Tumors are assumed to be unicellular in origin.

•After the cell is transformed, it loses growth control
and begins to divide rapidly.

•The cells lose contact inhibition and invade the primary

site.

•They then invade the adjacent organs and blood and

lymph systems, which may carry the cells to distant
organs.

•As this invasion process takes place, new proteins can

also be used as markers.
•Patterns of commonly measured chemical analytes may
serve as possible markers of neoplastic disease and often
serve as a clue to the origin of the disease:

1) Receptors (estrogen, progesterone, androgen, and

corticosteroid)
2) Proteins (immunoglobulins, glycoproteins,
carcinoembryonic proteins, or oncofetal antigens)
3) Genetic markers (oncogenes and suppressor genes)
4) Other markers (sialic acid conjugates, polyamines, and
amino acids
5) Enzymes
ENZYMES

•Enzymes may have been the first markers used in

screening for cancer.

•An elevated level of any one of them leads to a more

extensive search for disease, although certainly this does
not always lead to the discovery of a malignant disease.

•Other enzymes are more specific for cancer.

 Acid Phosphatase (ACP)

•ACP is not a reliable screening or early disease diagnostic

tool.
•Elevated levels of the prostatic isoenzyme (PAP) of acid
phosphatase have been used to diagnose and monitor
prostate cancer but may not necessarily be found when
malignancy is present, especially in the early stages of
disease.
•It is sometimes used in conjunction with PSA to
discriminate between patients who have extracapsular
disease and those whose disease is encapsulated’
•Alkaline Phosphatase

*Quantitation of total phosphatase (ALP) is useful in the

diagnosis of primary bone and hepatic malignancies and
metastases to these organs.
* Organ-specific ALP isoenzymes have been identified
(bone, kidney, liver, small intestine, and placenta [Regan
isoenzyme]), and often it is necessary to identify which
isoenzymes are present before ALP can be used as a tumor
marker.
* Frequently, one needs to differentiate the bone from the
liver fraction.
•The isoenzymes may then be quantitated.

•Elevations of ALP correlate well with osteoblastic

activity and be used to monitor therapy and look for
recurrence in patients with osteosarcoma or metastatic
prostate cancer.
•Creatine Kinase

* The BB isoenzyme of creatine kinase (CK-BB, CK-1),

commonly known as the brain fraction, is normally not
observed in serum due to the blood-brain barrier.
•CK-BB is the fetal form of the enzyme and as such is
associated with many malignant diseases, especially
those of epithelial cell origin.
•CK-BB measured in cerebrospinal fluid (CSF) may be
used in estimating lesion size in brain tumors.
•CK-BB isoenzymes are identified and quantitated using
electrophoresis.
•Lactate Dehydrogenase

•Lactate dehydrogenase (LD) is a nonspecific marker.

•Elevated serum LD-1 has been described in germ cell

tumors.

•Similarly, increased serum concentrations of LD-3 are

observed in patients with leukemias and lymphomas,
whereas the LD-5 isoenzyme is frequently elevated in
patients with colon, breast, lung, liver, stomach cancers,
and a variety of adenocarcinomas such as that of
prostate.
•Neuron-Specific Enolase
•Neuron-specific enolase (NSE) is a specific marker for
tumors associated with the neuroendocrine system.

•Serum and/or CSF elevations of this marker are

observed.
•NSE may be quantitated by RIA and the levels correlate
well with the tumor burden
•5’Nucleotidase and Gamma Glutamyltransferase
•5’Nucleotidase (5’NT) and gamma glutamyltransferase
(GGT) are both more sensitive markers for liver cancer
than ALP.
•However, neither of them is specific for cancer.
•They may both be elevated in cases of cirrhosis and GGT
is elevated in a large percentage of pancreatic cancers.

•Amylase, Lipase, Trypsin, and Ribonuclease

Amylase (AMS), lipase (LPS), trypsin (TPS), and
ribonuclease (RNase) are enzymes associated with the
pancreas.
•Terminal Deoxynucleotidyl Transferase
•It is quantitated using an immunoassay (RIA or EIA) and
is used to predict prognosis and responsiveness to drugs
as well as to help classify the leukemias.

•Collagenase and Cathepsin D

•Collagenase and cathepsin D are part of a larger class of
compounds known as proteases.
•Synthesized and secreted by malignant cells, these
compounds are responsible for degrading the extracellular
matrix and thus permitting their invasion and metastasis.
•Both can be quantitated in extracts from surgically
removed solid tumors.
•The results correlate well with the 5-year survival time
for patients and hence are used in developing a
prognosis.
•Collagenase is measured in bone malignancies and
carhepsin D is often quantitated in breast cancer
patients.

•Histaminases
•Histaminases or diamine oxidase (DAO) has been used
as a tumor marker in cases of medullary thyroid cancer
to confirm a high level of calcitonin, which is normally
found in this disease.
•Muramidase
•Muramidase or lysozyme is sometimes used in the
monitoring of monocytic and myelomonocytic leukemias.
HORMONES
* Hormones may be secreted in abnormally increased
concentrations by tumors of the endocrine glands
normally responsible for their production (eutopic
production) or by tumors of other organs that normally do
not produce the hormone (ectopic production).

•The synthesis and secretion of these hormones can

cause severe symptoms and even life-threatening effects.
•

•The paraneoplastic endocrine syndromes, which result

from the excessive hormonal production, are often the
first indication of malignancy and the quantitation of the
hormone(s) in question can support or establish a
diagnosis.
 •Hormones are regularly employed to monitor a patient
following surgery and other therapeutic modalities but
they have not been use to screen an asymptomatic
population.

•It is important to note that benign as well as malignant

tumors secrete excessive amounts of hormone.
•A tumor may secrete multiple hormones, some of which
may be synergistic and some antagonistic to each other.

•A tumor may secrete intact hormones, hormone

precursors, fragments, and subunits of hormones.
RECEPTORS
•Cell receptors are protein structures located on external
cell membranes and within the cell.
•The function of the receptor is to recognize and bind a
specific ligand such as a hormone or neurotransmitter.

•The ligand-receptor complex then initiates a biologic

response.
•The production of receptors is regulated in part by
ligand concentration.
•Because malignancy causes changes in receptor function
and quantity, receptors can be used as tumor markers.
•Estrogen and Progesterone Receptors
•Estrogen (ER) and progesterone (PgR) receptors are
assayed clinically to determine which breast cancer patients
may respond to endocrine therapy.
•If tumor growth is stimulated by estrogen, an antiestrogen
such as tamoxifen may be used to limit the growth of the
tumor.
•Estrogen and progesterone receptors have also been used
to predict the response of patients with endometrial cancer
to hormonal therapy.
•Cell receptor studies are done on a tissue sample
obtained from tumor biopsy or excision.

•OTHER RECEPTORS

•Androgen (AR) and progesterone (PgR) receptors have

been measured to predict the outcome of antiandrogen
therapy in prostate cancer patients.

•Similarly, glucocorticoid receptors as a prognostic

indicator are being evaluated in cases of lymphoblastic
leukemia.
•Epidermal growth factor receptor (EGFR) is a
glycoprotein known to bind epidermal growth factor
(EGF) and transforming growth factor alpha (TGF-α).
•The absence of this receptor correlates well with a good
response to tamoxifen.
• High levels of the receptor seem to indicate a poor
prognosis in terms of relapse and patient’s survival.

•Laminin receptor is another potential marker for breast

cancer.
• Laminin is a major constituent of basement membrane.
•Laminin receptors allow malignant cells to attach to
basement membranes, which are then dissolved, and the
cells destroyed as cancer invades other tissue.
•Malignant cells have more unoccupied laminin
receptors than do normal cells.
PROTEINS
•Many glycoproteins can be classified as oncofetal
antigens or as carbohydrate markers.
•Oncofetal proteins (oncofetal antigens) are proteins
that are normally expressed by the fetus but not by the
healthy adult.
•Since these proteins are synthesized in high
concentration by tumor cells in cancer patients, they
were named oncofetal antigens or proteins.
•Historically, alpha-fetoprotein and carcinoembryonic
antigen were the first of these proteins discovered.
•Alpha-Fetoprotein

•Alpha-fetoprotein (AFP), so named because it migrates

electrophoretically between alpha1 and albumin, is the
most abundant protein present during fetal
development.

•The level gradually drops until at the age of about 1

year, AFP is at normal adult levels (0 to 15 ng/mL).

•AFP is associated with various germ cell tumors and

hepatoma,

•It is useful in characterization and staging of disease

and in monitoring therapy.
 * In adults, high blood levels (over 500
nanograms/milliliter) of AFP are seen in only three
situations:
· HCC (Hepatocellular CA)
· Germ cell tumors (cancer of the testes and ovaries)
· Metastatic cancer in the liver (originating in other
organs)

•AFP is a good marker for primary hepatoma because it is

elevated in approximately 60-80% of the cases.

•It shows potential as a screening tool for Down’s

syndrome.
•Carcinoembryonic Antigen

•Carcinoembryonic antigen (CEA) is one of the older

oncofetal proteins still in use.
* It is not specific for the colon but is found in a variety of
malignant and nonmalignant conditions such as breast, GI,
lung, ovary, pancreas, and prostate cancers.

•Additionally, it is elevated in alcoholism, inflammation of

the bowel, cystic fibrosis, and in heavy cigarette smokers.
•Despite the nonspecificity of CEA, it is useful in establishing
prognosis and in monitoring therapy.
• CEA measured in body fluids other than serum (ascetic
fluid, fluid from cyst, urine, or lavage from any cavity) may
also aid in diagnosis.
 CARBOHYDRATES
•The carbohydrate tumor markers are glycoproteins
defined by their antigenic property.

•They are cell surface antigens or secreted antigens that

are expressed by tumor cells, and against which a
monoclonal antibody or antibodies has been produced.

•These cancer antigens or epitopes are generally more

specific than natural markers, and are defined by the
monoclonal antibody system used to quantitate them.

•The carbohydrate tumor markers can be subdivided

into mucins and blood group antigens.
•Examples of mucin markers are CA15-3 and CA125,
whereas CA19-9 and CA72-4 and examples of blood
group antigen markers.
•It should be noted, however, that patients who
genetically do not express a particular blood group
antigen will test negative for certain tumor antigens even
though they have the tumor.

•Additionally, a given cancer epitope may be present in

more than one type of cancer and two or more epitopes
may be expressed by a single tumor.

* The use of mouse monoclonal antibodies in cancer

therapy has led to the development of a subset of
patients who have human antimouse antibodies (HAMA),
which interfere with mouse-derived monoclonal
antibody immunoassays.
•Prostate-Specific Antigen
•The function of PSA is to cause liquefaction of seminal
coagulum.
•Because of its tissue specificity, it became the first tumor
marker recommended for screening for prostate cancer in
older men.
•The serum level is roughly proportional to tumor volume
and should return to normal within a month after radical
prostatectomy. Serum levels are sensitive to recurrence of
disease.
•Increased PSA density (>0.15) and increased PSA velocity
(>0.75 ng/mL/y) correlate better with prostatic cancer
than with BPH.
•CA15-3
•CA15-3 is a glycoprotein mucin present on mammary
epithelium.

•Assays for CA15-3 are not specific for breast cancer

because they can be elevated in a variety of carcinomas.

• However, they are sensitive for breast cancer and are

currently used to monitor patients following surgery.
•CA549, CA27.29, breast cancer mucin (BCM), and
mucin-like carcinoma-associated antigen (MCA) are
newer breast cancer markers.
•CA19-9
•CA19-9 is a mucin-like oligosaccharide characterized
by antibody first developed from mice immunized to a
human colon cancer cell line (SW-1116).

•It has a high specificity for pancreatic cancer but also

recognizes other gastrointestinal cancers.

•CA19-9 is used clinically to monitor therapy and to

predict disease recurrence.

•Because CA19-9 is related to the Lewis substance,

Lewis-negative patients will not produce CA19-9.
•CA125
•CA125 is a glycoprotein defined by the monoclonal
antibody OC125. It is a good marker for ovarian
carcinomas.

•It has limited use in diagnosis but has been used in

conjunction with CEA to characterize ovarian tumors.

•The highest levels are associated with ovarian cancer.

•Ovarian cancer antigen (OCA) is thought to be similarly

useful.
•CA72-4
•Immunometric assays for CA72-4 are marketed for use
in the detection of all forms of GI cancer, but especially
gastric cancer, which does not react especially well with
other markers.

•Squamous Cell Carcinoma Antigen

•Squamous cell carcinoma antigen (SCC) is one of 14
subfractions of tumor-associated antigen (TA4).

•Fifty-eight percent (58%) of squamous cell carcinomas

(head, neck, ling, esophagus) show elevated levels of
SCC.
•CYFRA 21-1
•Cyfra 21-1 is a marker for non-small cell lung cancer

•P-Glycoprotein

•P-glycoprotein is found in cell membranes of drug-

resistant cells. It is theorized that P-glycoprotein is active in
transporting the drugs out of the cells.
•It is normally found in kidney, liver, adrenal, and GI tract
cells.
•P-glycoprotein can be measured using a monoclonal
antibody, C219.
•Ferritin
Ferritin is the major iron storage protein and is found in
most cells.
Only a small percentage of total body ferritin is found in
plasma where it binds and transports iron (the major iron
transport protein is transferrin).
It is frequently assayed as an indicator of iron status
since the plasma or serum ferritin level is directly
proportional to the body iron stores.
Ferritin is elevated in any disease that causes a profound
disturbance in iron metabolism and erythropoiesis.
•It may be elevated in hepatitis and aplastric anemia, but
it is also elevated in leukemias, lymphomas, myeloma,
neuroblastoma, gastric, colon, pancreatic, lung and breast
cancers, and in melanoma.

•Beta2-Microglobulin
*Beta2-microglobulin (B2M) is an antigen found on the
surface of all nucleated cells.
•It is a subunit of human leukocyte antigen (HLA) and is
elevated in all diseases associated with rapid cell turnover.
•It is used as a marker for leukemias, lymphoma, and
multiple myeloma and it correlates well with B
lymphocyte activity.
•It may also be elevated in patients suffering from
human immunodeficiency virus (HIV) infection.

•C-Peptide
•C-peptide is the connecting peptide between the A and
B chain of proinsulin.
•It is dissociated from these chains to produce active
insulin.
•C-peptide is elevated in patients with increased
endogenous insulin production, for example, insulinoma.
•C-peptide is not elevated in patients receiving
exogenous insulin.

•Some patients with beta cell pancreatic tumors produce

fluctuating levels of insulin but their C-peptide levels of
insulin are uniformly high.
•This permits early diagnosis of recurrent disease.
•Immunoglobulins
•The immunoglobulins have been used as markers in
multiple myeloma and in Waldenstrom’s
macroglobulinemia for many years.

•They are quantitated and characterized using

electrophoresis and immunoelectrophoresis (monoclonal
bands) and by using turbidimetric methods with specific
antisera.

•Bence Jones protein (urinary free immunoglobulin light

chains) are also present in patients with these cancers.
•Thyroglobulin
•Thyroglobulin (Tg) is a large glycoprotein located in the
thyroid where it serves as a precursor to thyroxine (T4)
and triiodothyronine (T3).
•Thyroglobulin is elevated in patients with follicular and
papillary thyroid carcinoma and thyroid adenoma as well
as in a variety of other thyroid conditions.
•Since thyroglobulin is not elevated in patients with
medullary thyroid carcinoma, it serves to help
discriminate between this and other hyperthyroid
diseases.

•Tissue Polypeptide antigen

•Tissue polypeptide antigen (TPA) is an oncofetal protein
related to the cytokeratins.
*It is synthesized during mitosis and therefore is a useful
marker of cellular proliferation.
Quantitation of this marker has proved useful in
monitoring and staging because it correlates well with the
tumor burden.
 MEDICAL GENETICS &
Genetic DISORDERs

> Clinical Laboratory Medicine - Focused on 2

Subspecialties of Genetics:
a) human genetics – the study of heredity in man
b) medical genetics – the study of human genetic
variation of medical significance.
1959 = cytogenetic studies were first utilized in clinical
laboratory studies, making a major advancement in
clinical diagnosis:
a) the ability to detect changes in chromosome
structure,
b) direct correlation of this changes to disease &
phenotypic anomalies in individuals.

> Many of tests have become the “gold standard” for

diagnosis.
There is a growing number of disease genes
being cloned… there is increasing emphasis on
developing molecular direct mutation
analyses.

These analytical studies work well when a

diagnosis is known or suspected…

Clinical applications for cytogenetic analysis =

range from prenatal diagnosis to cancer
evaluation.
Scientists called cytogeneticists can recognize
and identify many of these gross chromosomal
abnormalities by examining chromosomes
through a microscope.

Cytogeneticists use three things to tell

chromosomes apart:
· chromosome size
· the position of the centromere
· characteristic banding patterns of alternating
light and dark bands (caused by staining the
chromosomes with dyes)
The parts of a chromosome:
>Telomere – The ends of the chromosome
> Centromere – The primary constriction of the
chromosome; It also divides the chromosome
into a short arm (p) and a long arm (q)
> Chromatid – A single molecule of DNA
CHROMOSOME BANDS LABELING:
The long arm and short arm are labeled q (for
queue) and p (for petit), respectively.

At the lowest resolution, only a few major bands

can be distinguished, which are labeled q1, q2, q3;
p1, p2, p3, etc., counting from the centromere.

Higher resolution reveals sub-bands, labeled q11,

q12, q13, etc, numbered from the centromere out
toward the telomere.

Sub-sub-bands identified by even higher

resolution are labeled q11.1, q11.2, q11.3, etc.
KARYOTYPE
= the
representation
of entire
metaphase
chromosomes
in a cell,
arranged in
order of size.
Autosomes are
further divided
into seven
groups: A to G
For example, the cytogenetic map location of a
gene termed CFTR (cystic fibrosis transmembrane
conductance regulator) is 7q31.2, which indicates
it is on chromosome 7, q arm, band 3, sub-band 1,
and sub-sub-band 2.

> The ends of the chromosomes are labeled ptel

and qtel. For example, the notation 7qtel refers to
the telomere (the end) of the long arm of
chromosome 7.
Normal male karyotype
Normal female karyotype
MEASURES OF VARIATION
"Genetic variation among individual humans
occurs on many different scales, ranging from
gross alterations in the human karyotype to single
nucleotide changes.
1) SINGLE NUCLEOTIDE POLYMORPHISMS
> Nucleotide diversity
is based on single
mutations called
single nucleotide
polymorphisms
(SNPs).

> The nucleotide

diversity between
humans is about 0.1%,
which is 1 difference
per 1,000 base pairs.
A difference of 1 in 1,000 nucleotides between
two humans chosen at random, amounts to
approximately 3 million nucleotide differences
since the human genome has about 3 billion
nucleotides.

Most of these SNPs are neutral but some are

functional and influence phenotypic differences
between humans through alleles.

It is estimated that a total of 10 million SNPs exist

in the human population of which at least 1% are
functional (see International HapMap Project).
2) COPY NUMBER VARIATION
In full sequences of an individual's genome, the
analysis of diploid sequences, has shown that non-
SNP variation accounts for much more human genetic
variation than single nucleotide diversity.
This non-SNP variation includes copy number
variation and results from 1)deletions, 2)inversions,
3)insertions and 4)duplications.
When copy number variation is included, human to
human genetic variation is estimated to be at least
0.5% (99.5% similarity).
Copy number variations are inherited but can also
arise during development.
1.a) AUTOSOMAL DOMINANT INHERITANCE

Autosomal Dominant Traits are those in which

a single copy of an allele is enough for the trait to
be expressed or shown in the phenotype of the
animal.

Dominant conditions are those that are

expressed in heterozygotes.
1.b) AUTOSOMAL RECESSIVE INHERITANCE

Autosomal recessive traits require that

the Individual have 2 copies of the trait to
express the phenotype.

The genes for autosomal-recessive traits

are also located on the autosomes, but the
mutant, disease-causing alleles are recessive to
the normal alleles; thus, if one normal allele is
present, it is usually sufficient to prevent any
expression of the disease.
3) MITOCHONDRIAL INHERITANCE

- refers to the additional genes in cell’s

mitochondria.

- Because mitochondria are almost

exclusively passed from parent to child in the egg
and not in the sperm, a hallmark of
mitochondrial inheritance is transmission from
an affected woman to all of her children.
> X-linked dominant inheritance works differently
depending upon whether the mother (left image)
or father (right image) is the carrier of a gene that
causes a disease or disorder.

All daughters of an affected father will also be

affected but none of his sons will be affected
(unless the mother is also affected).

In addition, the mother of an affected son is also

affected (but not necessarily the other way
round).
 X--linked recessive inheritance expressed in

(1)males (who are necessarily

hemizygous for the gene mutation because
they have only one X chromosome); and

(2) females who are homozygous for the gene

mutation

> Carrier females do not usually express

the phenotype.
Normal placenta
-fetal side

Normal placenta
-maternal side

a 3N complement may be
derived from dispermy, the
fertilization of a haploid egg by
two sperm, and this generally
results in a partial hydatidiform
mole.
Hydatidiform mole
A ring chromosome is a chromosome whose arms
have fused together to form a ring.

A ring chromosome is denoted by the symbol r.

 Ring chromosomes may form in cells following

genetic damage by mutagens like radiation, they may
also arise spontaneously during development.

> Symptoms seen in patients carrying ring chromosomes

are more likely to be caused by the deletion of genes in
the telomeric regions of affected chromosomes, rather
than by the formation of a ring structure itself.
> Complex rearrangements including segmental
microdeletions and microduplications have been seen in
numerous ring chromosomes providing important clues
regarding the mechanisms of their formation.
 Disorders arising from the formation of a ring
chromosome include ring chromosome 20 syndrome
where a ring formed by one copy of chromosome 20 is
associated with epilepsy; ring chromosome 14 and ring
chromosome 13 syndrome are associated with mental
retardation and dysmorphic facial features; ring
chromosome 15 is associated with mental retardation,
dwarfism and microcephaly.

 Ring formation of an X-chromosome causes Turner

syndrome.
 microcephaly

dwarfism
Translocations are rearrangements involving two or
more nonhomologous chromosomes.

Each chromosome breaks off and the pieces exchange

places, establishing two (or more derivative
chromosomes.

As with inversions, the majority of translocations are

balanced, and only when the important structural gene is
disrupted does the translocation have clinical
ramifications.

Again, the primary danger of a balanced translocation

is that carriers are at increased risk for chromosomally
abnormal liveborn children.
In the first meiotic division, translocated chromosomes
assume a cross-shaped structure in order for the alleles
to pair properly.

At anaphase, the altered change in chromosome

complement can be noted directly.

Monosomy X becomes 45, X and gain of a sex

chromosome would be 47, XXY, 47XXX, or 47,XYY.

On the other hand , if the sex chromosome change is

acquired as in some cancer cell lines , a +or – indicating
this would be required (i.e. 45, X-Y for a male whose Y
chromosome has been lost as a result of his disease.
Examples :

Deletion: 46,XX,del(4)(p15)
= Terminal deletion of the short arm of 4 at band 15

Duplication: 46,XX, dup(11)(q23)

= Terminal duplication of the long arm of 11 at band 23

Translocation: 46,XY,t(4;9)(q21.2;p22)
= Translocation between 4 and 9

Inversion: 46,XY,inv(9)(p11q21.1)
= Pericentric inversion in 9 between bands p11 & q21.1
•Cytogenetic abnormalities may be found in apparently
“normal” individuals as well as in patients with phenotypic
anomalies or with a diagnosed genetic disorder.
•Diagnosis may occur at any stage of life. When the same
set of features is seen in several unrelated individuals, it
may be possible to establish a syndrome.
• The characteristics associated with a syndrome are
assumed to have a common basis, which often can be
shown to be a specific chromosome abnormality.

•However, although a syndrome is defined by a certain set

of characters, there is variability in affected individuals and
not all will show an identical phenotype.
PRENATAL CYTOGENETICS
•Studies have shown that 1 to 13 conceptuses has a
chromosomal abnormality, but of these, only 6 per 1000 are
liveborn, indicating most errors are recognized and
eliminated.
•For example, of all 45,X conceptions, 95% will
spontaneously terminate.
•The most common chromosome abnormalities detected in
prenatal testing are the liveborn trisomies and the sex
chromosome aneuploidies.
•Because these are usually the result of a meiotic
nondisjuction error, there is a very low risk of recurrence.
POSTNATAL
Approximately 0.6% of newborns have a chromosome
abnormality.
If the child dies shortly after birth, cytogenetic analysis
may provide information critical in understanding the
demise.
CHILDHOOD AND ADULT
•A common misconception about genetic disorders is that
because they are inherited, the diagnosis will be obvious at
birth.
•In fact, the full clinical presentation of many disorders
takes time to develop and may not be fully expressed until
later in life.

•In addition to considering the full range of cytogenetic

possibilities, molecular and biochemical options must also
be taken into account.
CANCER GENETICS

Another area of medicine in which cytogenetics is

becoming increasingly important is oncology.

>Excellent clinical data exist for leukemias and lymphomas,

including specific chromosome rearrangements that are
directly associated with tumorigenesis.

> If treatment is successful, most chromosome

abnormalities are no longer evident in the bone marrow
and as long as the karyotype appears to be “normal,” the
patient would be said to be in remission.
•However, if the treatment does not eliminate the aberrant
cell line completely, remission may be merely an interlude in
which the disease-causing cells are suppressed to such low
levels that they are not detectable by routine karyotype
analysis.
•Then at relapse, the same chromosome anomalies will
reappear and may be accompanied by additional
abnormalities and/or more complex cell lines, finding
consistent with disease progression.
•An increase in complexity over time is known as karyotype
evolution and, in general, poor prognosis and severity of
disease is directly correlated to the number and type of
chromosome abnormalities seen.
 CHROMOSOMAL ANEUPLOIDY SYNDROMES

Autosomal Aneuploidies
> The most common cause of mental retardation is trisomy
21 or Down syndrome.
This disorder has a birth incidence of 1 in 700 and is
characterized by hypotonia, flat facies, slanted palpebral
fissures, small ears, protruding tongue, transverse palmar
crease, heart defects, and hypogonadism.
> Approximately 92.5% of all Down syndrome individuals
have 47 chromosomes including three copies of
chromosome 21 resulting from a nondisjuction error in
meiosis.
However, just under 3% of patients tend to express a
milder phenotype and have been shown to be mosaics with
two cell lines that may include a 46, XX or 46, XY line.
In addition, about 5% of Down syndrome patients have
only 46 chromosomes, since the extra 21 is part of a
Robertsonian or other translocation.
> A child with a translocation is often indicative of the
presence of a translocation carrier parent, so, in these cases,
karyotype analysis of both parents is generally
recommended to determine if the couple is at increase risk
of having additional Down syndrome children in future
pregnancies.
•The other two liveborn trisomies are trisomy 13 and
trisomy 18. Trisomy 13, Patau syndrome, has a birth
incidence of 1 in 4000 to 1 in 10,000.

•Approximately 1 in 8000 newborns are diagnosed with

trisomy 18 or Edwards syndrome.
 Patau syndrome

Down syndrome

Edwards syndrome
Sex Chromosome Aneuploidies

•The aberrant karyotypes of individuals with three X

chromosomes [47,XXX females] or with one X and two Y
chromosomes [47 XYY males] often go undetected
throughout life.

•Some individuals have generalized learning difficulties.

•Klinefelter syndrome (47,XXY) males tend to be tall and

thin with relatively long legs.
•The most common reason for referral is postpubertal
hypogonadism; female-like breast development; and
infertility due to small testicles, hyalinized testicular tubules,
and azoospermia.

•The most commonly recognized sex chromosome

aneuploidy presenting with a female phenotype is Turner
syndrome: 45,X (Fig. 62-23B)

•The single X chromosome is usually maternal in origin,

indicating that a paternal meiotic nondisjunction is the most
common source of error.
•The phenotype for Turner syndrome is highly variable.

•Affected individuals are typically short (,4’11”) with

gonadal dysgenesis and learning difficulties.
•In general, however, most liveborn Turner syndrome
patients can live productive lives, and mildly affected
individuals may not know that they are affected with the
disorder until puberty.
Klinefelter syndrome

Turner syndrome
STRUCTURAL CHROMOSOMAL ANOMALIES

•Wolf-Hirschhorn syndrome also known as 4p-syndrome,

is due to an apparently terminal deletion of the short arm
of chromosome 4 [del(4)(p16)] (Fig 62-14).
•The characteristic facial appearance has been likened to
‘Greek warrior’s helmet’ due to the arched eyebrows and
long nose.
•Cri du chat or 5p- syndrome [del(5)(p15)] is characterized
by a distinctive high-pitched, cat-like cry in infancy.
 Wolf-Hirschhorn syndrome
Cri du chat
MICRODELETION SYNDROMES AND CONTIGOUS GENE
SYNDROMES
It is a very small deletion, usually only a fraction of a
single chromosome band.

Microdeletions can be confused with molecular

deletions, so it is important to recognize that although
microdeletions are small, they are significantly larger (>500
kb) than the typical molecular deletion (1 base pair [bp] to
several hundred bp, which can only be resolved by
molecular technology.
> Certain syndromes are actually due to deletions that
encompass portions of several adjacent, unrelated genes.
The size of the deletion and number of genes affected
may vary such that the phenotype expressed may differ
significantly between individuals.
>These syndromes are collectively called contiguous gene
syndromes and represent a subset of the larger category of
microdeletion syndromes
Miller- Dieker syndrome clearly demonstrates the
overlap between microdeletion and contiguous gene
syndromes.
> The disorder has been associated with a microdeletion of
the distal short arm of chromosome 17 (17p13.3).
> Cardinal clinical features are lissencephaly (smooth
brain) and craniofacial anomalies.
The best known of the microdeletion syndromes are
Prader-Willi syndrome (PWS) and Angelman syndromes
(AS).
They both share the same interstitial deletion of the
proximal long arm of chromosome 15 [del(15)(q11.2q11.2)],
but the presentation is significantly different.
Prader-Willi patients are small and hypotonic at birth, but
change within the first year of the life & begin to gain weight
rapidly.
If not placed on a controlled diet, they can become quite
obese due to overeating.
AS patient, are severely mentally retarded.
Miller- Dieker syndrome

Prader-Willi syndrome

Angelman syndrome
> Williams syndrome has been associated with a deletion of
the elastin gene (ELN) on the proximal long arm of
chromosome 7 (7q11.23).

The disorder is characterized by cardiac abnormalities,

hypertension, hoarse voice, premature aging of the skin,
and behavioral anomalies.

> Velocardiofacial syndrome is possibly the most common

microdeletion syndrome in humans, but is often not
recognized due to a broad spectrum of clinical features
and a presentation that can be quite mild.
 OTHER CYTOGENETIC PHENOMENAL FRAGILE X
SYNDROME
Fragile X syndrome
= the second leading cause of mental retardation and is
the primary cause of inherited mental retardation.
 The mutation was identified as an amplification of a
trinucleotide repeat sequence.

Breakage Syndromes
Chromosomal breakage syndromes (table 62-7) are a
set of autosomal recessive disorders that were originally
grouped together due to the common finding of
chromosome instability or fragility. Inability to repair the
DNA can lead to:
 (1) breakage or increased recombination that can be
characterized by chromosome instability
(2)additional mutations and defects in the DNA
sequence that can lead to cancer.
Velocardiofacial syndrome
Williams syndrome

Fragile X syndrome

Breakage Syndromes
(Ambras Syndrome) = gene MAP2K6
 •GENETIC MARKERS
•Direct analysis of nuclear DNA is useful in determining
diagnosis (benign versus malignant), determining
prognosis (survival time), monitoring patients for increase
aneuplasia, and predicting or evaluating therapeutic
response
•Normal nonproliferating cells (in G1 or G0 cell cycle
phase) are diploid, whereas DNA doubles during the
synthesis phase (S phase) of proliferation and
remains doubled (cell cycle G2) until mitosis (M
phase).
•Cancer cells may have an abnormally increased
amount of DNA (aneuploidy) owing to increased
number of chromosomes, increase DNA per
chromosome, and minutes (small pieces of DNA
from viruses).
•Additionally, cancer cells may have an abnormally
high percentage of their cells that are in S phase.
•Both the ploidy (DNA) could be determined by flow cell
cytometry, giving a measure of tumor aggressiveness.
•There are two types of genetic tumor markers: (1)
oncogenes, and (2) anti-oncogenes (suppressor genes).
• The oncogenes are derived from normal cellular genes
(proto-oncogenes).

* The oncogenes are activated by mutational events that

cause increased transcription of growth and proliferation,
promoting protein products or products which
antagonize (suppress) normal cellular apoptosis.
•Mutations include: insertions, deletions, translocations,
inversions, and point mutations.
•Many oncogenes are of viral origin and many human
oncogenes are associated with hematologic malignancies
but a few are associated with solid tumors.
* The suppressor genes code for normal protein products
that down regulate (control) cellular proliferation.
* The loss of these genes, and consequently their protein
products, allows continuous transcription of growth,
proliferation, and metastasis, promoting protein
products.
ONCOGENES
•Located on Chromosome 1, N-ras (“neuroblastoma
RAS viral (v-ras) oncogene homolog”).

•The c-erbB-2 gene (HER-2/neu) codes for a

transmembrane receptor with protein kinase activity.
•The protein product functions as a growth factor
receptor and the over expression of the gene is strongly
associated with breast and ovarian cancer.
•Assays exist for both the gene and the protein product,
and results correlate well with prognosis.
* The c-myc gene codes for a protein product (p62) that
binds to DNA and regulates transcription.
•Overexpression of the gene is associated with small cell
carcinoma of the lung, breast carcinoma, gastric and
other GI cancers, and promyelocytic leukemia.

•Translocation of the gene is seen in Burkitt’s lymphoma

and a variety of others B and T cell lymphomas.

* The Philadelphia chromosome is the result of a

translocation of the abl gene from the distal portion of
chromosome 9 to the bcr region of chromosome 22.
•The result of this abnormal gene splicing is to produce
an abl-like tyrosine kinase protein product that reacts
faster and with a wider group of substrates.

•The c-abl/bcr gene is involved in signal transduction and

associated with chronic myelogenous leukemia.
•The N-myc oncogene was first observed in brain cancer.

•It is the result of abnormal DNA amplification and its

protein product functions as a transcription regulator.

•It is associated with neuroendocrine tumors.

•SUPPRESSOR GENES
•The DCC gene is located on chromosome 18 and
encodes a protein that is similar to cell adhesion
proteins.
•It is responsible for down regulating proliferation in
gastrointestinal cells.
•The loss of this gene causes late stage colon cancer.
•The gene for p53, located on chromosome 17, codes
for a protein that regulates transition into S phase of
the cell cycle.
•Mutation or loss of this gene leads to continuous
cellular proliferation.
•Abnormal or absent p53 gene and its product are
associated with several cancers.
•The NFI gene is located on chromosome 17 and acts as a
ras GTpase activator.

•The loss of this gene causes neurofibromas, sarcoma,

and glioma.

•The RB gene was first described in patients with

inherited retinoblastoma.

•The gene is located on chromosome 13 and the protein

product (pRB) is an important transcription inhibitor and
regulates transition from G1 to S phase.
•The majority of familial (inherited) breast cancer
patients have a mutation or loss of one of these genes.

•OTHER MARKERS
•Lipid-associated Sialic acid
* Sialic acid is a family of acylated derivatives of
neuraminic acid usually found on the terminal end of the
carbohydrate portion of glycoprotein or glycolipid in cell
membranes.
•The carbohydrate portion may influence cell-to-cell
interaction, affecting cohesion, adherency, and
antigenicity.
•These characteristics change following malignant
transformation of a cell.
•It is used for monitoring patient response to therapy and
recurrence of disease.

•Hydroxyproline
•Hydroxyproline (Hyp) is an amino acid that is elevated in
patient’s with bone metastases.
•It is a marker of invasion and metastasis and is measured
in urine using HPLC.
•Polyamines and acetylated polyamines
•Polyamines (putrescine, spermidine and spermine) are
stabilizing agents that associate with cell membranes
and nucleic acids.
• The polyamines are metabolic products produced by
all proliferation cells and acetylated in the liver.
•Since their concentration in urine appears to parallel
the rate of proliferation, they have been used to
monitor therapy and recurrence of disease.
•Blood Cell Surface Antigens
•Antibodies to lymphoid and myeloid cell surface
antigens together with flow cell cytometry can be used
to confirm the diagnosis of leukemias and lymphomas
and to discriminate between different subtypes.
•This is an aid in diagnosis and gives reliable prognostic
information.
•This is because one of the two RB gene alleles is
absent in the germ line, and therefore it only takes the
loss of one (the other) allele to produce a tumor.

•The WT1 gene is located on chromosome 11 and codes

for a nuclear protein that acts as a transcription factor.
• Loss of this gene causes Wilm’s tumor
(nephroblastoma), a childhood kidney cancer.
The BRCA1 and BRCA2 suppressor genes are currently
under investigation as markers of genetic susceptibility
for breast cancer.
“For whoever wants to save their life will lose
it, but whoever loses their life for me will find
it, says the Lord .” Matthew 16:25 NIV