Chapter 14
Molecular Oncology
Objectives
 Contrast tissue-specific and tumor-specific
molecular targets.
 List molecular targets that are useful for
diagnosis and the monitoring of solid tumors.
 Explain how microsatellite instability is detected.
 Show how clonality is detected using antibody
and T-cell receptor gene rearrangements.
 Describe translocations associated with
hematological malignancies that can be used
for molecular testing.
Cancer is Caused by Nonlethal Genetic
Mutations Affecting Certain Genes.
 Oncogenes, as proto-oncogenes,
normally promote cell division or cell
survival.
 Oncogene mutations are usually a gain of
function and dominant.
 Tumor suppressors: genes normally arrest
cell division.
 Tumor suppressor gene mutations are
usually a loss of function and recessive
Cancer is Caused by Nonlethal
Genetic Mutations
Molecular Detection of Disease
 Targets:
 Tissue-specific markers (antigens, gene
rearrangements)
 Disease-specific markers (translocations, point
mutations, polymorphisms in tumor suppressor or
oncogenes)
 Viruses (EBV, HCV, HTLV-1)
 Methods:




Hybridization, blotting
Standard PCR, RT-PCR, electrophoresis
PCR with heteroduplex analysis, SSCP
Real-time PCR with gene or patient-specific probes
Gene and Chromosome
Abnormalities Observed in Cancer
 Gene mutations (oncogenes, tumor
suppressor genes)
 Chromosome structural abnormalities
(translocations, deletions, insertions)
 Chromosome number abnormalities
(aneuploidy, polysomy)
Molecular Abnormalities in Solid
Tumors, HER2/neu
 The HER2/neu gene encodes one of a family of
human epidermal growth-factor receptors.
 This gene is frequently amplified in breast
cancer cells, resulting in increased amounts of
HER2 cell surface protein.
 HER2-expressing tumors are sensitive to
herceptin, a monoclonal antibody therapy.
 HER2 protein is detected by
immunohistochemistry (IHC).
 HER2/neu gene amplification is detected by
fluorescence in situ hybridization (FISH).
The EGFR Gene Family
Molecular Abnormalities in Solid
Tumors, EGFR
 The EGFR oncogene encodes another of the same
family of epidermal growth factor receptors.
 This gene is mutated or amplified in several types of
cancer cells.
 Tumors with activating mutations in EGFR are sensitive
to tyrosine kinase inhibitors (TKI).
 EGFR protein is detected by IHC.
 EGFR gene and chromosome abnormalities are
detected by FISH.
 EGFR gene mutations are detected by SSCP, SSPPCR, or direct sequencing.
Molecular Abnormalities in Solid
Tumors, K-ras
 The Kirsten rat sarcoma viral oncogene (K-ras)
encodes a key component of cell signaling.
 Mutations in K-ras are the most common
oncogene mutations in cancer.
 K-ras mutations are associated with tumor
malignancy and may affect response to some
therapies.
 K-ras gene mutations are detected by SSCP or
direct sequencing.
Molecular Abnormalities in Solid
Tumors, TP53
 The 53-kilodalton tumor suppressor gene
(TP53) encodes a transcription factor.
 TP53 is mutated in half of all types of cancer.
 Loss of TP53 function is an indicator of poor
prognosis in colon, lung, breast, and other
cancers.
 Mutant p53 protein is detected by IHC.
 TP53 gene mutations are detected by a variety
of methods, including SSCP and direct
sequencing.
Other Genes Associated with
Solid Tumors
 Ewing sarcoma, EWS
 Synovial sarcoma translocation, chromsome 18;
synovial sarcoma breakpoint 1 and 2, SYT-SSX1, SYTSSX2
 Paired box–Forkhead in rhabdomyosarcoma, PAX3FKHR, PAX7-FKHR
 Ataxia telangiectasia mutated gene, ATM
 Von Hippel-Lindau gene, VHL
 V-myc avian myelocytomatosis viral-related oncogene,
neuroblastoma-derived, MYCN or n-myc
 Rearranged during transfection (RET) protooncogene
Inherited Cancer Gene Mutations
 Inherited tumor suppressor gene mutations are
recessive for the malignant phenotype.
 Tumor suppressor gene mutations are dominant
with respect to increased risk of malignancy.
 Loss of heterozygosity exposes the recessive
mutant allele in a hemizygous state.
 This is explained by the two-hit hypothesis.
Two-Hit Hypothesis
Normal
At risk (inherited mutation)
At risk
Affected
Affected
Loss of
heterozygosity
Loss of Heterozygosity Can Be
Detected by STR Analysis
Loss of a linked heterozygous STR
implicates a concurrent loss of one gene
Loss of the STR allele linked to the
allele.
Normal
fluorescence
normal gene allele is observed by
capillary gel electrophoresis.
Normal
allele
Mutant
allele
Normal
Tumor
fluorescence
Heterozygous STR
Tumor
Inherited Breast Cancer Risk
 BRCA1 and BRCA2 are tumor suppressor genes
encoding proteins that participate in DNA repair.
 Inherited mutations in BRCA1 or BRCA2
significantly increase risk of breast cancer at an
early age.
 Frequently occurring mutations, including 187delAG
and 5382insC in BRCA1 and 6174delT in BRCA2,
are detected by SSP-PCR and other methods.
 Most mutations are detected by direct sequencing
of both genes.
Detection of BRCA1 185delAG by
SSP-PCR
X
180 bp MW + m m + B
Mutation-specific
primer
MW = MW standard
+ = normal
m = mutant
B = reagent blank
The 180 bp
product indicates
the presence of
Mutation.
230 bp
180 bp
120 bp
Agarose gel
Hereditary Nonpolyposis
Colorectal Carcinoma
 Hereditary nonpolyposis colorectal
carcinoma (HNPCC) accounts for about
5% of colon cancer.
 HNPCC is the most common form of
hereditary colon cancer.
 HNPCC is associated with mutations in
genes encoding components of the MMR
system, most frequently MLH1 and
MSH2.
Replication Error (RER)
 Microsatellites (short tandem repeats) are
sensitive to errors during DNA replication.
 These errors are normally corrected by
the mismatch repair system (MMR).
 Components of the MMR system are
encoded by MLH1, MSH2, and several
other genes.
Microsatellite Instability (MSI)
Microsatellite instability is the production of new
alleles from unrepaired replication errors.
T7
TT TT TTT
AAAAAAA
Mismatch normally
recognized
and repaired by
the MMR system.
T6
T7
T TTT TT
AAAAAA
New (T6) allele generated on the
next round of replication.
T7
T7
TTT TTT T
AAAAAAA
Normal (T7) allele
HNPCC and MSI
 85–90% HNPCC tumors have MSI.
 Mutations in genes of the MMR system
(loss of function) are inferred by testing for
MSI.
 MSI analysis determines gene function.
Direct sequencing is used to detect the
actual gene mutation.
HNPCC and MSI
MSI is analyzed by assessing stability of at least
five microsatellite loci as recommended by the
National Cancer Institute.
Marker
BAT25
BAT26
D5S346
D2S123
D17S250
Repeating unit
Mononucleotide
Mononucleotide
Dinucleotide
Dinucleotide
Dinucleotide
HNPCC and MSI
MSI is detected by comparing PCR amplicons of the
microsatellite loci. Unstable loci appear as extra
products in tumor tissue compared to normal tissue.
N = Normal
T = Tumor
Unstable locus
Unstable locus
Stable locus
(Capillary gel electrophoresis)
Molecular Detection of Leukemia
and Lymphoma
 Targets:
 Antibodies, gene rearrangements, translocations,
point mutations, polymorphisms, viruses
 Methods:




Hybridization, blotting
Standard PCR, RT-PCR, electrophoresis
PCR with heteroduplex analysis, SSCP
Real-time PCR with gene or patient-specific probes
Gene Rearrangements (GR)
 Gene rearrangements are normal events
that occur in lymphocytes.
 Antibody genes [immunoglobulin heavy
chain genes, immunoglobulin light chain
genes (k, l)] and T-cell receptor genes (a,
b, g, d) rearrange.
 Rearrangement occurs independently in
each cell.
Immunoglobulin and T Cell
Receptor Gene Rearrangements
Early B cell precursor
Pre-B
B cell
Mature PC
Lymphoid
stem cell
IgH GR
TCR d and g GR
IgH GR + IgL GR
IgH + IgL GR
TCR b and a GR
Cytotoxic T
Early thymocytes
Common
thymocytes
Helper T
Immunoglobulin Heavy Chain
(IgH) Gene Rearrangement
L
VH1
L
VHN
DH
JH
One of each
(germline)
gene segment
is selected and
joined; the
intervening
DNA is looped
out.
L V
DJ C
C
This intron
is removed
by splicing.
Immunoglobulin light chain genes and T-cell
receptor genes rearrange in a similar manner.
Gene Rearrangements
GR may be used to detect leukemias and
lymphomas arising from cells that have
rearranged their immunoglobulin (Ig) or T
cell receptor (TCR) genes.
Clonality
 Normal lymphocyte populations are polyclonal with
respect to Ig and TCR genes.
 A leukemia or lymphoma is monoclonal with regard
to Ig or TCR rearranged genes.
Polyclonal
oligoclonal
Monoclonal
Detection of Monoclonal Lymphocyte
Populations by Southern Blot
Monoclonal populations are detected by
rearranged bands unique to the tumor cell
population.
HIndIII
HInd III
11 kb
BamH1
L
VH1
L
VHN
DH
18 kb
JH
BamH1
EcoR1 BamH1 HindIII
MW G R
G R G R
C
18 kb
EcoR1
EcoR1
Autoradiogram
labeled probe
G = germline (negative)
R = rearranged (positive)
Detection of Monoclonal
Lymphocyte Populations by PCR
Monoclonal populations are detected by sharp bands
unique to the tumor cell population.
JHPCR
Monoclonal
populations will yield
a single PCR
product.
Normal
(polyclonal)
populations will
yield a
polyclonal PCR
product.
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors

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PreB ALL
B-cell leukemia
Acute TCLL
AML/MDS
AML (M2)
APL (M3)
AMML (M4)
AMoL (M5)
t(1;19)
t(2;8), t(8;14), t(8;22), t(11;14)
t(11;14)
t(11q23)
t(8;21), t(6;9)
t(15;17)
t(11;21)
t(9;11)
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors
 CML
 ALL






Burkitt
DLBCL
TCL
Follicular
MCL
MM
t(9;22), t(11;22)
t(9;22), t(12;21), t(8;14), t(2;8),
t(8;22), t(11q)
t(8;14), t(2;8), t(8;22)
t(3q27), t(14;18); t(8;14)
t(8;14)
t(14;18), t(8;14)
t(11;14)
t(14q32)
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors
Translocations and other abnormalities in
chromosome structure and number are
detected by FISH.
14
8
t(8;14) translocation
Translocation detection using FISH breakaway probe.
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors
 Translocations are detected with higher
sensitivity using PCR.
 qPCR may be used to quantify tumor load
during patient monitoring.
 FISH is recommended for initial diagnosis.
PCR is better for monitoring.
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors: t(14; 18)
 t(14;18) is a reciprocal translocation between
the long arms of chromosomes 14; 18 is found
in 90% of follicular lymphoma cases and 20–
30% of large cell lymphomas.
 With translocation, the B-cell leukemia and
lymphoma (BCL2) gene is moved from
chromosome 18 to chromosome 14.
 BCL2 is dysregulated and overexpressed when
moved to chromosome 14.
PCR Detection of t(14;18)
The forward primer hybridizes to chromosome 18 while
the reverse primer hybridizes to chromosome 14.
IgH
Bcl2 gene
JH primers
Any of these primers may
be used.
MBR = major breakpoint
region
MCR = minor cluster
region
M = molecular weight
marker
+ = positive for
translocation
- = negative
MBR primers
MCR primers
M++- -- - +PSN
The band size is
determined by the
chromosomal
breakpoints.
Agarose gel
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors: t(9; 22)
 t(9;22) is a reciprocal translocation between the
long arms of chromosomes 9; 22 is found in
chronic myelogenous leukemia and acute
lymphoblastic leukemia.
 This translocation forms a chimeric gene
between the breakpoint cluster region (BCR)
gene on chromosome 22 and the Abelson
leukemia virus (ABL) gene on chromosome 9.
 The translocated chromosome is the
Philadelphia chromosome.
Translocations Used in Diagnosis and
Monitoring of Hematological Tumors: t(9; 22)
The chimeric gene, BCRABL, produces an
abnormal protein that drives the tumor cell
phenotype.
e1
Fusion
mRNA
(8.5 kb)
Fusion
protein
b1 2 3
a2 3
b3a2
AAAAA
p210 BCRABL
4....
Detection of t(9; 22) by RT-PCR
BCR
Philadelphia chromosome
ABL
Splicing
Reverse transcription
cDNA
BCRABL
cDNA made from patient
mRNA is amplified if the
translocation is present.
Detection of t(9; 22) by RT-PCR
1 = molecular weight
standard
2-5 = positive for
translocation
6 = negative
7-11 = amplification
controls
12 = blank
1 2 3 4 5 6 7 8 9 10 11 12
Translocation
products
(ABL)
Translocation
products
(BCRABL)
The band size is
determined by
different
chromosome 22
breakpoints.
Agarose gel
Quantification by qPCR (TaqMan)
1 2 3 4 5
 For qPCR, use a standard
curve of tumor cells diluted
6 7 8into
9 10 11
12
normal
cells.
 For RT-qPCR, use a
standard curve of
transcripts of known copy
numbers diluted into normal
RNA.
Summary
 Molecular testing analyzes tissue-specific and tumorspecific (mutation) targets.
 Genome, chromosome, and gene mutations are useful
targets for diagnosis and detection of solid tumors.
 Microsatellite instability is a test for function of the
DNA mismatch repair system, which may be mutated
in hereditary colon cancer.
 Ig and TCR gene rearrangements are tissue-specific
markers for certain lymphomas and leukemias.
 Translocations are tumor-specific markers for some
hematological disorders.