Molecular Oncology

Chapter 14

11

Molecular DiagnosticsMolecular Diagnostics

Malignant Cells

NormalCell

FirstMutation

SecondMutation

ThirdMutation

Fourth orLater

Mutation

Many Mutations Lead to Cancer

22 Molecular DiagnosticsMolecular 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

Molecular DiagnosticsMolecular Diagnostics33

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

44 Molecular DiagnosticsMolecular 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

Molecular DiagnosticsMolecular Diagnostics55

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)

66 Molecular DiagnosticsMolecular Diagnostics

The EGFR Gene Family77 Molecular DiagnosticsMolecular Diagnostics

EGF: epidermal growth factorTGF-: transforming growth

factor alphaHER: HeregulinsNRG: neuregulins

EGF: epidermal growth factorTGF-: transforming growth

factor alphaHER: HeregulinsNRG: neuregulins

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.

88 Molecular DiagnosticsMolecular 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.

99 Molecular DiagnosticsMolecular 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.

1010 Molecular DiagnosticsMolecular 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.

1111 Molecular DiagnosticsMolecular Diagnostics

Normal At risk Affected

Affected

Loss of heterozygosity

Two-Hit Hypothesis

At risk (inherited mutation)

1212 Molecular DiagnosticsMolecular 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

allele

fluor

esce

nce

fluor

esce

nce

Normal

Tumor

Normal

Tumor

1313 Molecular DiagnosticsMolecular 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.

1414 Molecular DiagnosticsMolecular Diagnostics

Detection of BRCA1 185delAG by SSP-PCR

230 bp180 bp120 bp

The 180 bpproduct indicatesthe presence ofMutation.

Mutation-specificprimer

X

180 bp MW + m m + B

Agarose gel

MW = MW standard+ = normalm = mutantB = reagent blank

1515 Molecular DiagnosticsMolecular 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.

Molecular DiagnosticsMolecular Diagnostics1616

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.

1717 Molecular DiagnosticsMolecular Diagnostics

Microsatellite Instability (MSI)

Microsatellite instability is the production of new alleles from unrepaired replication errors.

Mismatch normally recognizedand repaired by the MMR system.

New (T6) allele generated on the next round of replication.

Normal (T7) allele

1818 Molecular DiagnosticsMolecular Diagnostics

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.

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.

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.

1919 Molecular DiagnosticsMolecular Diagnostics

Marker Repeating unitBAT25 MononucleotideBAT26 MononucleotideD5S346 DinucleotideD2S123 DinucleotideD17S250 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.

(Capillary gel electrophoresis)

Unstable locus

Stable locus

Unstable locus

2020 Molecular DiagnosticsMolecular 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

2121 Molecular DiagnosticsMolecular Diagnostics

Gene Rearrangements (GR)

Gene rearrangements are normal events that occur in lymphocytes.

Antibody genes [immunoglobulin heavy chain genes, immunoglobulin light chain genes (,)] and T-cell receptor genes (,,,) rearrange.

Rearrangement occurs independently in each cell.

2222 Molecular DiagnosticsMolecular Diagnostics

Immunoglobulin and T Cell Receptor Gene Rearrangements

2323 Molecular DiagnosticsMolecular Diagnostics

GR=Gene rearrangementsIgH and IgL=immunoglobulin heavy and light chains

TCR=T-cell receptor

Immunoglobulin Heavy Chain (IgH) Gene Rearrangement

Immunoglobulin light chain genes and T-cell receptor genes rearrange in a similar manner.

L VH1 L VHN DH JH C

L V DJ C

(germline)One of each gene segment is selected and joined; the intervening DNA is looped out. This intron

is removed by splicing.

2424 Molecular DiagnosticsMolecular Diagnostics

V=variableD=diversityJ=joiningL=leaderC=constant

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.

2525 Molecular DiagnosticsMolecular 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 Monoclonaloligoclonal

2626 Molecular DiagnosticsMolecular Diagnostics

Detection of Monoclonal Lymphocyte Populations by Southern Blot

Monoclonal populations are detected by rearranged bands unique to the tumor cell population.

L VH1 L VHN DH JH C

EcoR1 EcoR1

18 kb

BamH1 BamH118 kb

HIndIII HInd III11 kb

labeled probe

EcoR1 BamH1 HindIIIMW G R G R G R

Autoradiogram

G = germline (negative)R = rearranged (positive)

2727 Molecular DiagnosticsMolecular Diagnostics

Detection of Monoclonal Lymphocyte Populations by PCR Monoclonal populations are

detected by sharp bands unique to the tumor cell population.

Normal (polyclonal) populations will yield a polyclonal PCR product.

Monoclonal populations will yield a single PCR product.

JHPCR

2828 Molecular DiagnosticsMolecular 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)

2929 Molecular DiagnosticsMolecular 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)

3030 Molecular DiagnosticsMolecular Diagnostics

14 8 t(8;14) translocation

Translocation detection using FISH breakaway probe.

Translocations Used in Diagnosis and Monitoring of Hematological Tumors Translocations and other abnormalities in

chromosome structure and number are detected by FISH.

3131 Molecular DiagnosticsMolecular 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.

3232 Molecular DiagnosticsMolecular 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.

3333 Molecular DiagnosticsMolecular Diagnostics

Any of these primers may be used.MBR = major breakpoint regionMCR = minor cluster regionM = molecular weight marker+ = positive for translocation- = negative

The band size is determined by the chromosomal breakpoints.

PCR Detection of t(14;18)

The forward primer hybridizes to chromosome 18 while the reverse primer hybridizes to chromosome 14.

3434 Molecular DiagnosticsMolecular 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.

3535 Molecular DiagnosticsMolecular 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.

3636 Molecular DiagnosticsMolecular Diagnostics

BCR ABL

Splicing

Reverse transcription

cDNA

BCRABL

Philadelphia chromosome

cDNA made from patient mRNA is amplified if the translocation is present.

Detection of t(9; 22) by RT-PCR

3737 Molecular DiagnosticsMolecular Diagnostics

1 = molecular weight standard2-5 = positive for translocation6 = negative7-11 = amplification controls12 = blank

The band size is determined by different chromosome 22 breakpoints.

Translocationproducts(BCRABL)

Translocationproducts(ABL)

Detection of t(9; 22) by RT-PCR

3838 Molecular DiagnosticsMolecular Diagnostics

1 2 3 4 5 6 7 8 9 10 11 12

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.