Oncogenes and their mutations
Tumor suppressors and their mutations
stimulate growth without mitogen
promote cell division or cell survival
-provide a gain of function
- mutations are usually a gain of function and dominant
Tumor suppressors-genes normally arrest cell division
-gene mutations are usually a loss of
function and recessive
-loss of function
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 can be detected by STR analysis and observed by capillary gel electrophoresis. small peak
-if a person is heterozygous they have one regular allele and one that causes cancer. Normally the regular allele can compensate for the cancer one but if its lost then its protectiveness is lost and it the cancer allele will be expressed.
Molecular Detection in Disease
* Targets
- Tissue-specific markers (antigens, gene rearrangements)
- Disease-specific markers (translocations, point mutations, polymorphisms in tumor suppressor or oncogenes)
- Viruses (EBV-nasopharyngeal, HCV liver cancer)
Molecular Detection in Disease
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 - whole genome, polysomy one or more chromosome )
Molecular Abnormalities in Solid Tumors: HER2/neu
-human epidermal growth-factor receptors
-amplified in breast cancer cells expressing increased HER2 cell surface protein
-HER2 tumors are sensitive to Herceptin - monoclonal AB therapy
viewed by FISH or immunohistochemistry
Molecular Abnormalities in Solid Tumors: EGFR - oncogene
-epidermal growth factor receptors
- amplified in several cancer cells
- EGFR tumors with mutations sensitive to tyrosine kinase inhibitors
-EGFR protein detected by IHC but gene and chromosome abnormalities are detected by FISH
-the gene mutations detected by single strand conformation polymorphism (SSCP), SSP-PCR, or direct sequencing - Sanger
SSCP- region of interest is amplified, denatured in heat with Hi-Di formamide, chilled and electrophoresis
Molecular Abnormalities in Solid Tumors: K-ras oncogene
-Kristen rat sarcoma gene encodes part of cell signaling
-mutations in K-ras are most common oncogene mutations and associated with tumor malignancy .
-the mutations are detected by SSCP or direct sequencing
Molecular Abnormalities in Solid Tumors: TP53-53-kilodalton tumor-suppressor gene
-encodes a transcription factor
-TP53 mutated in most types of cancer
-loss of TP53 function indicated poor prognosis in colon, lung, breast cancer. (stops cell cycle, causes oxidative stress, stops DNA repair
-mutant p53 protein detected by IHC
* TP53 gene mutations are detected by a
variety of methods, including SSCP
Inherited Breast Cancer Risk
BRCA1 and BRCA2 are tumor-suppressor genes encoding proteins that participate in DNA repair
-mutations in these genes increase risk of breast cancer detected by SSP=PCR or direct sequencing
-the mutation specific primer is 180 bp because 180 bp indicates presence of mutation
Replication Error
* Microsatellites
- Microsatellites (short tandem repeats) are sensitive to errors during DNA replication when there is a repeat of 1-3 nucleotides
-when DNA poly attaches to the wrong base forming a new allele - micro site instability - These errors are corrected by the mismatch repair system (MMR- ) encoded by MLH1, MSH2 but mutations cause the allele to not be repaired
Microsatellite instability is the production of new alleles from unrepaired replication
errors
Hereditary Nonpolyposis Colorectal Carcinoma (HNPCC)
-most of colon cancer
-HNPCC most common hereditary colon cancer
-associated with mutations in genes MLH1 MSK2
-HNPCC tumors have microsatellite instability MSI
-mutations in the genes for MMR system - loss of function are caught by testing for MSI
* MSI analysis determines gene function. Direct sequencing is used to detect the actual gene mutation. Test 5 micro satellite loci
-extra products will be seen on the gel under the T=Tumor section vs the normal which indicates extra allele because there is instability due to MMR mutation
Molecular Detection of Leukemia - bone & Lymphoma - lymph nodes
Targets
* Methods
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 in lymph
-normal events
* 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
-uses recombinase enzymes
Immunoglobulin Heavy Chain (IgH) Gene Rearrangement
One of each gene segment is selected and joined; the intervening DNA is looped
out
-L is the lead section precedes mark AB for secretion
-C is constant region spliced out of introns
* Immunoglobulin light chain genes and T-cell receptor genes rearrange in a similar manner
-introns are removed by splicing
Clonality
- Normal lymphocyte populations are polyclonal with respect to immunoglobulin or T-cell receptor genes
- A leukemia or lymphoma is monoclonal with regard to immunoglobulin or T-cell receptor rearranged genes
Detection of Monoclonal Lymphocyte Populations
Southern Blot
-detected by rearranged bands unique to the tumor cell population
-18 kb bp difference
-should always have a germline band
PCR
-detected by sharp bands unique to the tumor cell population. monoclonal will have single PCR product and polyclonal will have a few
Translocations (exchange for large amount of DNA) Used in Diagnosis and Monitoring of
Hematological Tumors
: t(14; 18)
- Translocations in chromosome structure and number are detected by FISH but PCR better for monitoring
- qPCR may be used to quantify tumor load during patient monitoring
: t(14; 18)
-reciprocal translocation between the long arms of chromosomes 14;18 found in lymphoma cases
* With translocation, the B-cell leukemia and lymphoma (BCL2) gene is moved
from chromosome 18 to chromosome 14
* BCL2 is dysregulated and overexpressed allowing damaged cells to proliferate
PCR detection
* The forward primer hybridizes to chromosome 18 and reverse primer to chromosome 14- heavy chain
-band size determined by breakpoints
-PCR product only if translocation occurs
Translocations Used in Diagnosis and Monitoring of Hematological
Tumors: t(9;22)
- found in chronic myelogenous leukemia and acute lymphoblastic leukemia
-forms a chimeric gene -BCRABL between the breakpoint cluster region (BCR) gene on chromosome 22 and the Abelson leukemia virus (ABL) gene on chromosome 9 - translocated chromosome is the Philadelphia chromosome
detected by RT-PCR - due to length of introns
-cDNA made from patient and mRNA is amplified only if translocation in present with wells 2-5 positive . the band size is determined by chromosome 22 breakpoints
-RT-qPCR, standard curve of transcripts of known copy numbers diluted into normal RNA for tumor progression
qpcr- uses dilutions to make a curve
