1 / 39

Molecular Oncology

Molecular Oncology. Chapter 14. Many Mutations Lead to Cancer. Normal Cell. First Mutation. Second Mutation. Third Mutation. Malignant Cells. Fourth or Later Mutation. Cancer is Caused by Nonlethal Genetic Mutations Affecting Certain Genes.

tait
Download Presentation

Molecular Oncology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Molecular Diagnostics Molecular Oncology Chapter 14

  2. Molecular Diagnostics Many Mutations Lead to Cancer Normal Cell First Mutation Second Mutation Third Mutation Malignant Cells Fourth or Later Mutation

  3. 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 recessive

  4. Molecular Diagnostics 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

  5. Molecular 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

  6. Molecular Diagnostics 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)

  7. Molecular Diagnostics The EGFR Gene Family EGF: epidermal growth factor TGF-a: transforming growth factor alpha HER: Heregulins NRG: neuregulins

  8. Molecular Diagnostics 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.

  9. 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. Molecular Diagnostics Molecular Abnormalities in Solid Tumors: K-ras

  10. Molecular 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.

  11. Molecular 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.

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

  13. Molecular Diagnostics 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.

  14. Molecular Diagnostics 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.

  15. Molecular Diagnostics 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

  16. Molecular 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.

  17. 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.

  18. Molecular Diagnostics 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. Mismatch normally recognized and repaired by the MMR system. 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.

  19. Molecular Diagnostics 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. MarkerRepeating unit BAT25 Mononucleotide BAT26 Mononucleotide D5S346 Dinucleotide D2S123 Dinucleotide D17S250 Dinucleotide

  20. Molecular Diagnostics 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. Unstable locus Unstable locus Stable locus (Capillary gel electrophoresis)

  21. Molecular 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

  22. Molecular Diagnostics 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.

  23. Molecular Diagnostics Immunoglobulin and T Cell Receptor Gene Rearrangements TCR=T-cell receptor GR=Gene rearrangements IgH and IgL=immunoglobulin heavy and light chains

  24. Molecular Diagnostics 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. V=variable D=diversity J=joining L=leader C=constant

  25. Molecular Diagnostics 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.

  26. Molecular Diagnostics 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.

  27. Molecular Diagnostics 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.

  28. Molecular Diagnostics Detection of Monoclonal Lymphocyte Populations by PCR Monoclonal populations are detected by sharp bands unique to the tumor cell population. JHPCR Monoclonal populations will yield a single PCR product. Normal (polyclonal) populations will yield a polyclonal PCR product.

  29. Molecular 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)

  30. Molecular 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)

  31. Molecular Diagnostics 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.

  32. Molecular 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.

  33. Molecular 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.

  34. Molecular Diagnostics PCR Detection of t(14;18) The forward primer hybridizes to chromosome 18 while the reverse primer hybridizes to chromosome 14. The band size is determined by the chromosomal breakpoints. Any of these primers may be used. MBR = major breakpoint region MCR = minor cluster region M = molecular weight marker + = positive for translocation - = negative

  35. 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.

  36. Molecular 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.

  37. Molecular Diagnostics 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

  38. Molecular Diagnostics 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 Translocation products (ABL) Translocation products (BCRABL) The band size is determined by different chromosome 22 breakpoints.

  39. 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

More Related