1. Molecular Diagnostics
Molecular Oncology
Chapter 14

2. Many Mutations Lead to Cancer
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Gene Testing
Molecular Diagnostics
Many Mutations Lead to Cancer
Normal
Cell
First
Mutation
Second
Mutation
Third
Mutation
Malignant Cells
Fourth or
Later Mutation
All cancer is genetic, in that it is triggered by altered genes. Genes that control the orderly replication of cells become damaged, allowing the cells to reproduce without restraint.
Cancer usually arises in a single cell. The cell’s progress from normal to malignant to metastatic appears to involve a series of distinct changes in the tumor and its immediate environment, and each is influenced by different sets of genes.
NCI Web site:

3. Molecular Diagnostics
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 and promot apoptosis.
Tumor suppressor gene mutations are usually a loss of function and recessive

4. Molecular Detection of Disease
Molecular Diagnostics
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

5. Gene and Chromosome Abnormalities Observed in Cancer
Molecular Diagnostics
Gene and Chromosome Abnormalities Observed in Cancer
Gene mutations:
oncogenes, tumor suppressor genes
Chromosome structural abnormalities
translocations, deletions, insertions
Chromosome number abnormalities:
aneuploidy, polysomy

6. Molecular Abnormalities in Solid Tumors: HER2/neu
Molecular Diagnostics
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)
fluorescence in situ hybridization (FISH)

7. The EGFR Gene Family EGF: epidermal growth factor
Molecular Diagnostics
The EGFR Gene Family
EGF: epidermal growth factor
TGF-a: transforming growth factor alpha
HER: Heregulins
NRG: neuregulins
The ErbB family of growth factor receptors includes the HER2 receptor and EGFR. The factors that bind to these receptors on the cell surface begin a cascade of events including autophosphorylation and phosphorylation of other proteins by the receptors. EGF, NRG heregulins are small peptides that are active in the
development of various cell types such as gastric mucosa, the heart, and the nervous system.

8. Molecular Abnormalities in Solid Tumors: EGFR
Molecular Diagnostics
Molecular Abnormalities in Solid Tumors: EGFR
The EGFR oncogene encodes another member 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, SSP-PCR, or direct sequencing.

9. Molecular Abnormalities in Solid Tumors: K-ras
Molecular Diagnostics
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.

10. Molecular Abnormalities in Solid Tumors: TP53
Molecular Diagnostics
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.

11. Inherited Cancer Gene Mutations
Molecular Diagnostics
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.

12. At risk (inherited mutation)
Molecular Diagnostics
Two-Hit Hypothesis
Normal At risk Affected
At risk (inherited mutation)
Affected
Loss of heterozygosity

13. Loss of Heterozygosity Can Be Detected by STR Analysis
Molecular Diagnostics
Loss of Heterozygosity Can Be Detected by STR Analysis
Loss of a linked heterozygous STR implicates a concurrent loss of one gene allele.
Loss of the STR allele linked to the normal gene allele is observed by capillary gel electrophoresis.
Heterozygous STR
Normal
allele
Mutant
fluorescence
Tumor

14. Inherited Breast Cancer Risk
Molecular Diagnostics
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.

15. Detection of BRCA1 185delAG by SSP-PCR
Molecular Diagnostics
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

16. Hereditary Nonpolyposis Colorectal Carcinoma
Molecular Diagnostics
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 mismatch repair (MMR) system involved in replication errors repair, most frequently MLH1 and MSH2.

17. Replication Error (RER)
Molecular Diagnostics
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.

18. Microsatellite Instability (MSI)
Molecular Diagnostics
Microsatellite Instability (MSI)
Microsatellite instability is the production of new alleles from unrepaired replication errors.
Mismatch normally recognized
and repaired by the MMR system.
New (T6) allele generated on the next round of replication.
Normal (T7) allele
Replication errors result from slippage during DNA replication.
If the error is not repaired, the next round of replication will create a new allele (top, right) of the original locus.
Additional uncorrected errors will produce more alleles.

19. HNPCC and MSI 85–90% HNPCC tumors have MSI.
Molecular Diagnostics
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.
MSI is analyzed by assessing stability of at least five microsatellite loci as recommended by the National Cancer Institute.
Marker Repeating unit
BAT25 Mononucleotide
BAT26 Mononucleotide
D5S346 Dinucleotide
D2S123 Dinucleotide
D17S250 Dinucleotide

20. (Capillary gel electrophoresis)
Molecular Diagnostics
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.
Unstable locus
Unstable locus
Stable locus
(Capillary gel electrophoresis)

21. Molecular Detection of Leukemia and Lymphoma
Molecular Diagnostics
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

22. Gene Rearrangements (GR)
Molecular Diagnostics
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.

23. Immunoglobulin and T Cell Receptor Gene Rearrangements
Molecular Diagnostics
Immunoglobulin and T Cell Receptor Gene Rearrangements
TCR=T-cell receptor
GR=Gene rearrangements
IgH and IgL=immunoglobulin heavy and light chains

24. Immunoglobulin Heavy Chain (IgH) Gene Rearrangement
Molecular Diagnostics
Immunoglobulin Heavy Chain (IgH) Gene Rearrangement
Immunoglobulin light chain genes and T-cell receptor genes rearrange in a similar manner.
One of each gene segment is selected and joined; the intervening DNA is looped out.
This intron is removed by splicing.
Immunoglobulin heavy chain gene on chromosome
14 consists of a series of variable (V), diversity (D),
and joining (J) gene segments (germline configuration). The V
segments are accompanied by a short leader region (L). One
of each type of segment, V, D, and J, is selected and combined
by an intrachromosomal recombination event, first D
and J, and then V and D. The C (constant) segments are
joined through splicing or a secondary recombination event,
class switching.
V=variable
D=diversity
J=joining
L=leader
C=constant

25. Molecular Diagnostics
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.

26. Molecular Diagnostics
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 Monoclonal
oligoclonal

27. Detection of Monoclonal Lymphocyte Populations by Southern Blot
Molecular Diagnostics
Detection of Monoclonal Lymphocyte Populations by Southern Blot
Monoclonal populations are detected by rearranged bands unique to the tumor cell population.
EcoR1 BamH1 HindIII
MW G R G R G R
Autoradiogram
G = germline (negative)
R = rearranged (positive)

28. Detection of Monoclonal Lymphocyte Populations by PCR
Molecular Diagnostics
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.
Immunglobulin heavy chain gene rearrangement
by PCR with amplification from the variable region.
Forward primers complementary to the variable region
and reverse primers complementary to the joining region are
used to amplify the diversity region (top). In a polyclonal
specimen, amplification products in a range of sizes will result.
These products produce a dispersed pattern on an ethidium
bromide–stained agarose gel (lanes 4, 8, 11). If at least 1% of
the sample is representative of a monoclonal gene
rearrangement, that product will be amplified preferentially
and revealed as a sharp band by gel electrophoresis
(lanes 3, 6, 9, and 10).
Normal (polyclonal) populations will yield a polyclonal PCR product.

29. Molecular Diagnostics
Translocations Used in Diagnosis and Monitoring of Hematological Tumors
PreB ALL t(1;19)
B-cell leukemia t(2;8), t(8;14), t(8;22), t(11;14)
Acute TCLL t(11;14)
AML/MDS t(11q23)
AML (M2) t(8;21), t(6;9)
APL (M3) t(15;17)
AMML (M4) t(11;21)
AMoL (M5) t(9;11)

30. Molecular Diagnostics
Translocations Used in Diagnosis and Monitoring of Hematological Tumors
CML t(9;22), t(11;22)
ALL t(9;22), t(12;21), t(8;14), t(2;8), t(8;22), t(11q)
Burkitt t(8;14), t(2;8), t(8;22)
DLBCL t(3q27), t(14;18); t(8;14)
TCL t(8;14)
Follicular t(14;18), t(8;14)
MCL t(11;14)
MM t(14q32)

31. Translocation detection using FISH breakaway probe.
Molecular Diagnostics
Translocations Used in Diagnosis and Monitoring of Hematological Tumors
Translocations and other abnormalities in chromosome structure and number are detected by FISH.
t(8;14) translocation
Translocation detection using FISH breakaway probe.

32. Molecular Diagnostics
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.

33. Molecular Diagnostics
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.

34. Molecular Diagnostics
PCR Detection of t(14;18)
The forward primer hybridizes to chromosome 18 while the reverse primer hybridizes to chromosome 14.
Any of these primers may be used.
MBR = major breakpoint region
MCR = minor cluster region
M = molecular weight marker
+ = positive for translocation
- = negative
The band size is determined by the chromosomal breakpoints.

35. Molecular Diagnostics
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.

36. Molecular Diagnostics
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.

37. Detection of t(9; 22) by RT-PCR
Molecular Diagnostics
Detection of t(9; 22) by RT-PCR
Philadelphia chromosome
BCR
ABL
Splicing
Reverse transcription
cDNA
cDNA made from patient mRNA is amplified if the translocation is present.
BCRABL

38. Detection of t(9; 22) by RT-PCR
Molecular Diagnostics
Detection of t(9; 22) by RT-PCR
= molecular weight standard
2-5 = positive for translocation
= negative
7-11 = amplification controls
12 = blank
Translocation
products
(ABL)
Translocation
products
(BCRABL)
The band size is determined by different chromosome 22 breakpoints.

39. Quantification by qPCR (TaqMan)
For qPCR, use a standard curve of tumor cells diluted into normal cells.
For RT-qPCR, use a standard curve of transcripts of known copy numbers diluted into normal RNA.
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