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Molecular Oncology PowerPoint Presentation

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Description : Check out this medical presentation on Pathology, which is titled "Molecular Oncology", to know about tissue-specific and tumor-specific molecular targets.

Tags : Pathology, Molecular Oncology, tissue-specific, tumor-specific, molecular targets

Published on : Aug 06, 2021 | Views : 3350 | Downloads : 2

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Chapter 14 Molecular Oncology
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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.
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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
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Cancer is Caused by Nonlethal Genetic Mutations
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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
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Gene and Chromosome Abnormalities Observed in Cancer Gene mutations (oncogenes, tumor suppressor genes) Chromosome structural abnormalities (translocations, deletions, insertions) Chromosome number abnormalities (aneuploidy, polysomy)
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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).
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The EGFR Gene Family
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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, SSP-PCR, or direct sequencing.
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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.
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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.
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Other Genes Associated with Solid Tumors Ewing sarcoma, EWS Synovial sarcoma translocation, chromsome 18; synovial sarcoma breakpoint 1 and 2, SYT-SSX1, SYT-SSX2 Paired box–Forkhead in rhabdomyosarcoma, PAX3-FKHR, 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
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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.
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Normal At risk Affected Affected Loss of heterozygosity Two-Hit Hypothesis At risk (inherited mutation)
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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.
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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.
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Detection of BRCA1 185delAG by SSP-PCR 230 bp 180 bp 120 bp The 180 bp product indicates the presence of Mutation. Mutation-specific primer MW + m m + B Agarose gel MW = MW standard + = normal m = mutant B = reagent blank
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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.
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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.
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Microsatellite Instability (MSI) Microsatellite instability is the production of new alleles from unrepaired replication errors.
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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.
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HNPCC and MSI 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
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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 (Capillary gel electrophoresis) Unstable locus Stable locus Unstable locus
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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
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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.
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Immunoglobulin and T Cell Receptor Gene Rearrangements
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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.
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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.
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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.
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Detection of Monoclonal Lymphocyte Populations by Southern Blot Monoclonal populations are detected by rearranged bands unique to the tumor cell population.
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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
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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)
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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)
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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.
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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.
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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.
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PCR Detection of t(14;18) The forward primer hybridizes to chromosome 18 while the reverse primer hybridizes to chromosome 14.
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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.
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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.
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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
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1 = molecular weight standard 2-5 = positive for translocation 6 = negative 7-11 = amplification controls 12 = blank The band size is determined by different chromosome 22 breakpoints. Agarose gel Translocation products (BCRABL) 1 2 3 4 5 6 7 8 9 10 11 12 Translocation products (ABL) Detection of t(9; 22) by RT-PCR
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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.
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Summary Molecular testing analyzes tissue-specific and tumor-specific (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.