Molecular Oncology Chapter 14 1 Molecular Diagnostics.

Molecular Oncology

Chapter 14*Molecular Diagnostics

Molecular Diagnostics

Malignant CellsNormalCellFirstMutationSecondMutationThirdMutationFourth orLater MutationMany Mutations Lead to Cancer

*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 recessiveMolecular Diagnostics*


Molecular Detection of DiseaseTargets: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*Molecular Diagnostics


Gene and Chromosome Abnormalities Observed in CancerGene mutations:oncogenes, tumor suppressor genesChromosome structural abnormalitiestranslocations, deletions, insertionsChromosome number abnormalities:aneuploidy, polysomyMolecular Diagnostics*


Molecular Abnormalities in Solid Tumors: HER2/neuThe 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)*Molecular Diagnostics


The EGFR Gene Family*Molecular DiagnosticsEGF: epidermal growth factorTGF-a: transforming growth factor alphaHER: HeregulinsNRG: neuregulins


Molecular Abnormalities in Solid Tumors: EGFRThe 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.*Molecular Diagnostics


Molecular Abnormalities in Solid Tumors: K-rasThe 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: TP53The 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.*Molecular Diagnostics


Inherited Cancer Gene MutationsInherited 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.*Molecular Diagnostics


Normal At risk AffectedAffectedLoss of heterozygosityTwo-Hit HypothesisAt risk (inherited mutation)*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.*Molecular Diagnostics


Inherited Breast Cancer RiskBRCA1 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.*Molecular Diagnostics


Detection of BRCA1 185delAG by SSP-PCR230 bp180 bp120 bpThe 180 bpproduct indicatesthe presence ofMutation.Mutation-specificprimerMW + m m + BAgarose gelMW = MW standard+ = normalm = mutantB = reagent blank*Molecular Diagnostics


Hereditary Nonpolyposis Colorectal CarcinomaHereditary 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 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.



Microsatellite Instability (MSI)Microsatellite instability is the production of new alleles from unrepaired replication errors.Mismatch normally recognizedand repaired by the MMR system.*Molecular DiagnosticsReplication 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 MSI8590% 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.*Molecular DiagnosticsMarkerRepeating unitBAT25MononucleotideBAT26MononucleotideD5S346DinucleotideD2S123DinucleotideD17S250Dinucleotide


HNPCC and MSIMSI 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 locusStable locusUnstable locus*Molecular Diagnostics


Molecular Detection of Leukemia and LymphomaTargets:Antibodies, gene rearrangements, translocations, point mutations, polymorphisms, virusesMethods:Hybridization, blotting Standard PCR, RT-PCR, electrophoresisPCR with heteroduplex analysis, SSCPReal-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.*Molecular Diagnostics


Immunoglobulin and T Cell Receptor Gene Rearrangements*Molecular DiagnosticsGR=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.

One of each gene segment is selected and joined; the intervening DNA is looped out.This intron is removed by splicing.*Molecular DiagnosticsV=variableD=diversityJ=joiningL=leaderC=constant


Gene RearrangementsGR may be used to detect leukemias and lymphomas arising from cells that have rearranged their immunoglobulin (Ig) or T cell receptor (TCR) genes.



ClonalityNormal 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.



Detection of Monoclonal Lymphocyte Populations by Southern Blot Monoclonal populations are detected by rearranged bands unique to the tumor cell population.



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*Molecular Diagnostics


Translocations Used in Diagnosis and Monitoring of Hematological TumorsPreB 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)*Molecular Diagnostics


Translocations Used in Diagnosis and Monitoring of Hematological TumorsCMLt(9;22), t(11;22)ALLt(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)DLBCLt(3q27), t(14;18); t(8;14)TCLt(8;14)Folliculart(14;18), t(8;14)MCLt(11;14)MMt(14q32)*Molecular Diagnostics


Translocation detection using FISH breakaway probe.Translocations Used in Diagnosis and Monitoring of Hematological TumorsTranslocations and other abnormalities in chromosome structure and number are detected by FISH.*Molecular Diagnostics


Translocations Used in Diagnosis and Monitoring of Hematological TumorsTranslocations 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 2030% 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.



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



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.



BCRABLSplicingReverse transcriptioncDNABCRABLPhiladelphia chromosomecDNA made from patient mRNA is amplified if the translocation is present.Detection of t(9; 22) by RT-PCR*Molecular Diagnostics


1 = molecular weight standard2-5 = positive for translocation6 = negative7-11 = amplification controls12 = blankThe band size is determined by different chromosome 22 breakpoints.Translocationproducts(BCRABL)Translocationproducts(ABL)Detection of t(9; 22) by RT-PCR*Molecular 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.


*National Cancer InstituteUnderstanding Cancer and Related Topics Understanding Gene TestingNCI Web site: http://cancer.gov/cancertopics/understandingcancer*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 cells 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. *****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 thedevelopment of various cell types such as gastric mucosa, the heart, and the nervous system.*****************Immunoglobulin heavy chain gene on chromosome14 consists of a series of variable (V), diversity (D),and joining (J) gene segments (germline configuration). The Vsegments are accompanied by a short leader region (L). Oneof each type of segment, V, D, and J, is selected and combinedby an intrachromosomal recombination event, first Dand J, and then V and D. The C (constant) segments arejoined through splicing or a secondary recombination event,class switching.****Immunglobulin heavy chain gene rearrangementby PCR with amplification from the variable region.Forward primers complementary to the variable regionand reverse primers complementary to the joining region areused to amplify the diversity region (top). In a polyclonalspecimen, amplification products in a range of sizes will result.These products produce a dispersed pattern on an ethidiumbromidestained agarose gel (lanes 4, 8, 11). If at least 1% ofthe sample is representative of a monoclonal generearrangement, that product will be amplified preferentiallyand revealed as a sharp band by gel electrophoresis(lanes 3, 6, 9, and 10).***********

Molecular Oncology Chapter 14 1 Molecular Diagnostics.

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Transcript of Molecular Oncology Chapter 14 1 Molecular Diagnostics.