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Chapter 14

Chapter 14. Molecular Oncology. 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.

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Chapter 14

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  1. Chapter 14 Molecular Oncology

  2. 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.

  3. 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

  4. Cancer is Caused by Nonlethal Genetic Mutations

  5. 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

  6. Gene and Chromosome Abnormalities Observed in Cancer • Gene mutations (oncogenes, tumor suppressor genes) • Chromosome structural abnormalities (translocations, deletions, insertions) • Chromosome number abnormalities (aneuploidy, polysomy)

  7. 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).

  8. The EGFR Gene Family

  9. 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.

  10. 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.

  11. 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.

  12. 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, MYCNorn-myc • Rearranged during transfection (RET) protooncogene

  13. 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.

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

  15. Normal Mutant allele allele Normal fluorescence Normal Heterozygous STR fluorescence Tumor Tumor 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.

  16. Inherited Breast Cancer Risk • BRCA1 and BRCA2are 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.

  17. X 180 bp Detection of BRCA1 185delAG by SSP-PCR 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

  18. 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.

  19. 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.

  20. T7 T T T T T T T A A A A A A A T T T T T T T T T T T T T A A A A A A A A A A A A A Mismatch normally recognized and repaired by the MMR system. T6 T7 T7 T7 New (T6) allele generated on the next round of replication. Normal (T7) allele Microsatellite Instability (MSI) Microsatellite instability is the production of new alleles from unrepaired replication errors.

  21. 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.

  22. HNPCC and MSI MSI is analyzed by assessing stability of at least five microsatellite loci as recommended by the National Cancer Institute. MarkerRepeating unit BAT25 Mononucleotide BAT26 Mononucleotide D5S346 Dinucleotide D2S123 Dinucleotide D17S250 Dinucleotide

  23. 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 Unstable locus Unstable locus Stable locus (Capillary gel electrophoresis)

  24. 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

  25. 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.

  26. Immunoglobulin and T Cell Receptor Gene Rearrangements

  27. 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.

  28. 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.

  29. Polyclonal Monoclonal oligoclonal 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.

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

  31. 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. JHPCR Monoclonal populations will yield a single PCR product.

  32. 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)

  33. 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)

  34. 14 8 t(8;14) translocation Translocations Used in Diagnosis and Monitoring of Hematological Tumors Translocations and other abnormalities in chromosome structure and number are detected by FISH. Translocation detection using FISH breakaway probe.

  35. 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.

  36. 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.

  37. IgH Bcl2 gene JH primers MBR primers MCR primers Any of these primers may be used. MBR = major breakpoint region MCR = minor cluster region M = molecular weight marker + = positive for translocation - = negative M + + - - - - + P S N The band size is determined by the chromosomal breakpoints. Agarose gel PCR Detection of t(14;18) The forward primer hybridizes to chromosome 18 while the reverse primer hybridizes to chromosome 14.

  38. 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.

  39. e1 b1 2 3 a2 3 4.... Fusion b3a2 mRNA (8.5 kb) AAAAA Fusion p210 BCRABL protein 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.

  40. 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

  41. Detection of t(9; 22) by RT-PCR 1 = molecular weight standard 2-5 = positive for translocation 6 = negative 7-11 = amplification controls 12 = blank 1 2 3 4 5 6 7 8 9 10 11 12 Translocation products (ABL) Translocation products (BCRABL) The band size is determined by different chromosome 22 breakpoints. Agarose gel

  42. 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. 1 2 3 4 5 6 7 8 9 10 11 12

  43. 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.

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