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Introduction to Molecular Pathology

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  • ONE STEP: RT + DNA polymerase OR rTth (Thermusthemophilus) that works as an RT and DNA polymerase
  • Hybridization probe: Two separate, single-labeled probes anneal to target bringing donor and reporter into proximityDual-labeled hydrolysis probe: When the reporter and quencher fluorophores are in proximity on the probe – there is no signal. Once the 5’nuclease activity of the DNA polymerase hydrolyses the end nucleotides from the probe where the reporter is. The reporter emits a signal.Minor groove binding probes (MGB): like hydrolysis probes, technique to stabilize shorter probes.Molecular beacon probes: Annealing to the target after denaturation allows the reporter fluorophore to escape the quenching effect, therefore giving a signal
  • The QuantiGenePlex 2.0 System measures mRNA levels directly from a number of sources including, cultured cell lysates, tissue homogenates, dried blood spots, formalin fixed paraffin embedded (FFPE) sections or purified RNA.  It utilizes branched DNA (bDNA) technology, which relies on cooperative hybridization between the target mRNA and a specific probe set.  The probe sets consist of three types of oligonucleotides, Capture Extenders (CEs), Label Extenders (LEs), and Blocking Probes (BLs), whose sequences are selected based on the sequence of the target mRNA.  The Capture Extenders (CEs) are oligonucleotides with roughly half of the sequence being complimentary to sections of the target mRNA and the other half complimentary to the Capture probes (CP) immobilized onto the Capture beads.  The Label Extenders (LEs) are also oligonucleotides with half of the sequence being complimentary to the target mRNA and the other half complimentary to a portion of the Pre-amplifier.  The Blocking Probes (BLs) are complimentary to regions of the target mRNA not recognized by either the CE or the LE oligonucleotides and serve to reduce non-specific hybridization. The CEs provide the assay specificity by binding the target mRNA to the capture bead. Signal amplification is the result of LEs hybridizing with the target mRNA and the Pre-Amplifier sequences. The Pre-Amplifier trunk is then hybridized with multiple Amplifiers that form the branches of bDNA.   The “branches” of each amplifier have multiple biotin moieties, which will in turn bind Streptavidin-conjugated R-phycoerythrin (SAPE), the measureable signal for detection.  Using this technology, the amplification occurs with the signal rather than the target.  The signal is proportional to the target mRNA and can be achieved without purification or amplification (Figure 2).  The assay is designed to be compatible in batch or completely automated modes using BioTek’s ELx405 Magnetic Bead Washer.
  • Transcript

    • 1. Molecular Pathology  Testing of nucleic acids within a clinical context  Helpful  Hereditary disorders  Oncology  Infectious diseases
    • 2. Molecular Pathology  Specific purposes  Diagnosis  Prognosis  Prenatal testing  Pharmacotherapy  Pharmacogenetics  Pharmacogenomics
    • 3. Watson and Crick  The structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases  Doctortvrao’s ‘e’ learning series
    • 4. Watson and Crick discovers DNA / Feb 28th 1953
    • 5. Watson and Crick Builds a Model DNA 7th March 1953
    • 6. First Document on DNA published in Nature 25th April 1953
    • 7. Watson and Crick - Awarded Nobel Prize in 1962
    • 8. DNA  A molecule contains two polynucleotide strands that form an an antiparallel double helix.  Nucleotides:  Nitrogenous base (AT GC,U)  Deoxyribose  Phosphate
    • 9. DNA - Structure  The nucleotide, however, remains as the fundamental unit (monomer) of the nucleic acid polymer.There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine (A), and those with thymine (T).
    • 10. DNA A purine always links with a pyrimidine base to maintain the structure of DNA. Adenine ( A ) binds toThymine (T ), with two hydrogen bonds between them. Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.
    • 11. Chemical structure of DNA
    • 12. DNA is Endless structure  The rungs of the ladder can occur in any order (as long as the base- pair rule is followed)  Those 4 bases have endless combinations just like the letters of the alphabet can combine to make different words.
    • 13. DNA  Example  First strand GGGTTTAAACCC  Second strand CCCAAATTTGGG
    • 14. Central Dogma of Molecular Biology
    • 15. DNA makes a Copy of Self  Replication is the process where DNA makes a copy of itself. Why does DNA need to copy? Simple: Cells divide for an organism to grow or reproduce, every new cell needs a copy of the DNA or instructions to know how to be a cell. DNA replicates right before a cell divides.
    • 16. DNA Replication  DNA replication is semi-conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand.This helps reduce the number of copy errors.  So we remained what we were ?
    • 17. Transcription  RNA polymerase II mediates transcription and generates a precursor ss-mRNA identical to the sense (coding) stand except for U forT.  Precursor ss-mRNA is processed in nucleus by spliceosomes that catalyze intron removal and exon ligation with the regulation by exonic and intronic enhancers and silencers with production of different pre m-RNA that go across nucleus
    • 18. DNA to RNA creates functional translations  DNA remains in the nucleus, but in order for it to get its instructions translated into proteins, it must send its message to the ribosome's, where proteins are made.The chemical used to carry this message is Messenger RNA  Doctortvrao’s ‘e’ learning series
    • 19. DNA – RNA – DNA a never ending cycle  RNA has the job of taking the message from the DNA to the nucleus to the ribosome's.  Transcription - RNA is made from DNA  Translation - Proteins are made from the message on the RNA  Doctortvrao’s ‘e’ learning series
    • 20. Translation  m-RNA directs protein synthesis.  Occurs in ribosomes (rRNA+proteins)  Codons (three bases) are read by transfer (tRNA)  There are 64 possible codons, therefore most of the 21 aminoacids are specified by more than 1 codon
    • 21. RNA = Ribonucleic acid.  RNA is similar to DNA except: It has one strand instead of two strands. Has uracil instead of thymine 3.Has Ribose instead of Deoxyribose
    • 22. Gene Expression  DNA level expression control  Transcriptional  Post-Transcriptional  Epigenetics  DNA methylation  Histone modification
    • 23. Gene Expression  DNA level expression control  Transcriptional  House keeping genes  Always on  Transcription factors  Usually lie upstream in the promoter region  Enhancer and silencer elements
    • 24. Gene Expression  Post transcriptional  Export of mRNA out of nucleus  Alternative splicing  mRNA stabilization  mRNA degradation  RNA interference or silencing  miRNA and siRNA
    • 25. Gene Expression  DNA level expression control  Transcriptional  Post-Transcriptional  Epigenetics  DNA methylation  Histone modification
    • 26. What is Gene  The gene, the basic units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus.
    • 27. Modes of inheritance  Mutations of single genes  Patterns: autosomal dominant, autosomal recessive, X- linked, mitochondrial  Anticipation  Increased severity of a certain diseases in successive familiar generations associated with triple repeats  Mosaicism  At least two cell lines derived from a single zygote
    • 28. Modes of inheritance  Genomic imprinting  Different expression of alleles depending on parent origin  Uniparental Disomy  Both copies inherited from one parent  Environmental influence  Chronic diseases
    • 29. Mutations and Polymorphisms  Mutation: change in DNA sequence  Polymorphism: non disease causing change in DNA or a change found at a frequency of ≥ 1% in population  When evaluating changes in DNA sequence use neutral terms: sequence variant, sequence alteration or allelic variant.There may be:  Missense, nonsense, deletions, insertions, frame shifts, duplications, amplifications, trinucleatide repeats.
    • 30. Single Nucleotide Polymorhisms and Haplotypes  SNPs are single base differences in the DNA of individuals  There are ~10 million SNPs in the human genome  IMPORTANCE: Pharmacogenetics  Ex. CYP (cP450)  Alleles of SNPs that are close together tend to be inherited together.  Haplotype: a set of associated SNPs alleles in a region of a chromosome
    • 31. Overview of Molecular Techniques and Instrumentation  Standard or usual specimen flow  Specimen collection (blood, tissue)  Nucelic acid isolation (DNA or RNA)  Nucleic acid quantification (optional)  Nucleic acid storage  Nucleic acid amplification (or other)  Test interpretation  Quality control
    • 32. Nucleic acid isolation (DNA or RNA)  Manual vs. automated  Cell lysis  Dependent of specimen type, nucleic acid being isolated for, desired purity and application to be used in  FFPE yields ~200 pairs  Purification  Organic: phenol-chloroform  Non organic: silica, anion exchange chromatography and magnetic particles  DNA or RNA Isolation  RNA rapidly degrades…
    • 33. Methods  DNA sequencing  Southern Blot  PCR  RT-PCR  RealTime PCR  Methylation-Specific PCR  In-situ PCR  ProteinTruncationTest  Transcription-Mediated Amplification  Strand Displacement Amplification  Nucleic Acid Sequence- Based Amplification  Signal amplification  Branching DNA  Hybrid Capture  Invader  FISH  DNA arrays and chips
    • 34. Gene sequencing  Determining the exact sequence of the four bases in a given DNA template  Two methods  Maxam-Gilbert  Chemical degradation  Sanger  Chain termination  Radiolabeled, Dye-prime or Dye-terminator (cycle sequencing)  Pyrosequencing  Sequnces a short length of DNA (~30-60 bases)
    • 35. Applications of Direct DNA sequences Clinical condition Gene HIV drug resistance HIV-protease, RT Cystic fibrosis CFTR gene Beta thalassemia Beta globin Cancer predisposition • breast BRCA1 •Hereditary non polyposis colon cancer TP53 •MEN PTEN Ret proto-oncogene Congenital hearing loss Connexin 26 HCV genotyping 5’UTR
    • 36. Array-based Comparative Genomic Hybridization  Comparative Genomic Hybridization is done in metaphases in classical cytogenetics (M-CGH)  Resolution 5 Mb  Bacterial Artificial Chromosome (BAC) maps the human genome therefore an Array based-CGH can be created (A- CGH). Different resolutions up to 32,000 (45 kb)  cDNA-CGH  Oligonucleotide-CGH  Can detect Single Nucleotide Pleomorphisms (SNPs) [Gene Chip]
    • 37. Methods  DNA sequencing  Southern Blot  PCR  RT-PCR  RealTime PCR  Methylation-Specific PCR  In-situ PCR  ProteinTruncationTest  Transcription-Mediated Amplification  Strand Displacement Amplification  Nucleic Acid Sequence- Based Amplification  Signal amplification  Branching DNA  Hybrid Capture  Invader  FISH  DNA arrays and chips
    • 38. Southern Blot  Edwin M Southern, 1974  DNA extracted  DNA cut into pieces (Restriction Endonucleases)  Electrophoresis and size separated  Blot (transferred) to a membrane  Anealed with labeled (radioactive, fluorescence, chemiluminescent) probe
    • 39. Southern Blot working protocol
    • 40. Uses of Southern Blotting  Southern blots are used in gene discovery and mapping, evolution and development studies, diagnostics and forensics. In regards to genetically modified organisms, Southern blotting is used as a definitive test to ensure that a particular section of DNA of known genetic sequence has been successfully incorporated into the genome of the host organism.  Used in prognosis of cancer and in prenatal diagnosis of genetic diseases
    • 41. Methods  DNA sequencing  Southern Blot  PCR  RT-PCR  RealTime PCR  Methylation-Specific PCR  In-situ PCR  ProteinTruncationTest  Transcription-Mediated Amplification  Strand Displacement Amplification  Nucleic Acid Sequence- Based Amplification  Signal amplification  Branching DNA  Hybrid Capture  Invader  FISH  DNA arrays and chips
    • 42. PCR  Kary B. Mullis 1983  Target amplification  Single oligonucletide  Multiplexed  Mimics the natural process of DNA replication, therefore, requires:  DNA template, DNA polymerase, dNTPs, buffer, Mg++, two primers to flag the target sequence  Thermal cycler  Denaturation ~95°C  Annealing ~45-60°C  Extension ~72°C
    • 43. PCR  Denaturation  Breaks the hydrogen bonds between the ds-DNA  Anealing  Binding to oligonucleotide sequence (probe)  Extension  DNA polymerase (heat stable,Taq [Thermophilus aquaticus]) replicates the selected DNA sequence  Xn = X0 × (1 + E)n E= 0 - 1
    • 44. RT-PCR  To detect or quantify RNA transcripts or viral RNA  RNA is converted to DNA  Reverse transcriptase (Avian MyeloblastosisVirus and Moloney Murine Leukemia virus)  Isothermal reaction with primers: oligo dT, random hexamer primers, or target specific primers  One step vs. two steps
    • 45. PCR or RT-PCR  Product analysis / detection  RealTime  Hybridization  Membrane bound  Reverse line blots  Liquid Bead Array with Flow Cytometry  Electrophoresis  Agarose  Capillary  Cycle sequencer
    • 46. Multiplexed – PCR and ELISA Protein Expression Profiling Cancer Markers Cardiac Markers Cellular Signaling Cytokines, Chemokines, and Growth Factors Endocrine Isotyping Matrix Metalloproteinases Metabolic Markers Neurobiology Transcription Factors/Nuclear Receptors Genomic Research FlexmiR® v2 Custom microRNA Assay FlexmiR microRNA Panels Gene Expression Profiling Genotyping Genetic Disease Cystic Fibrosis Cytochrome p450 Immunodiagnostics AllergyTesting Autoimmune Disease HLATesting Infectious Disease Vaccine Testing Newborn Screening Biodefense/Environmental
    • 47. Luminex
    • 48. RealTime - PCR  Amplifies and detects PCR product fluorescently in each well of PCR plate  Don’t have to run gel afterwards  Use for endpoint detection  Examples  Fast PCR screening without gels  Locate clone or mutant of interest  Genotyping SNPs  Genotype individuals using allele specific primers
    • 49. RealTime - PCR  The crossing threshold or cycle threshold (Ct) is the amplification cycle number at which fluorescence is obtained  Ct is proportional to the amount of staring template (interrogated sequence) in the sample  Excellent for Q-PCR
    • 50. PCR Advantages  Sensitivity  Specificity  Speed  Versatility  Automated  No need for intact DNA/RNA Disadvantages  Target sequence needs to be known  Target needs to be conserved among individuals (polymorphisms)  Oligonucleotide length  Can fail in the detection of chromosomal abnormalities like translocations, inversions, large addition or deletions  Contamination (F+)
    • 51. Methods  DNA sequencing  Southern Blot  PCR  RT-PCR  RealTime PCR  Methylation-Specific PCR  In-situ PCR  ProteinTruncationTest  Transcription-Mediated Amplification  Strand Displacement Amplification  Nucleic Acid Sequence- Based Amplification  Signal amplification  Branching DNA  Hybrid Capture  Invader  FISH  DNA arrays and chips
    • 52. Branched DNA makes the complicated matters simple  The technology uses variety of branched DNA ( bDNA ) probes and signal amplification reporter molecules  And generate Chemiluminescent signal.  The signal correlates with target nucleic acid  Doctortvrao’s ‘e’ learning series
    • 53. Capture Extenders (CEs), Label Extenders (LEs), and Blocking Probes (BLs)
    • 54. Branched DNA applications  Detection HIV, HBV, and HCV  Measures viral loads  Less sensitive than PCR  Doctortvrao’s ‘e’ learning series
    • 55. Hybrid Capture  Qiagen  Signal amplification technique  Denaturated DNA gets hybridized to complimentary unlabeled RNA sequences (if DNA sequence is present)  Antibody bound to the well is attracted to RNA:DNA hybrids  A second conjugated anti RNA:DNA hybrid antibody is added  Chemiluminescent signal is generated in proportion of target DNA present
    • 56. Product Overview – Update  CervistaTM HPV HR is an FDA approved test that screens for the presence of 14 high-risk HPV types  100% detection of CIN3+ and 99.1% NPV for CIN2+  Only FDA approved HPV screening test with an internal control  Reduces patient call backs  Limits QNS (only 2 ml sample volume required)  <1% indeterminate rate  No equivocal zone for interpretation 75 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 57. HPV HR Indications Indications for use: • To screen patients with atypical squamous cells of undetermined significance (ASC-US) cervical cytology results to determine the need for referral to colposcopy • In women 30 years and older the CervistaTM HPV HR test can be used with cervical cytology to adjunctively screen to assess the presence or absence of high-risk HPV types. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management 76 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 58. Product Design & Chemistry
    • 59. Invader® Chemistry  A technology protected by 128 issued U.S. patents  Structure-specific recognition and cleavage with Cleavase® enzyme  Signal amplification  Isothermal reactions: no thermal cycling needed  Fluorescence detection Probe Repeating Process Amplifies Signal Cleavase® Enzyme 78 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 60. CervistaTM HPV HR Test Design  Developed from phylogenetic tree of A-superfamily HPV strains, based on L1 region  Cervista HPV is specific for high-risk types selected from the A5/A6, A7 and A9 virus groups: 51, 56, 66A5/A6 A7 18, 36, 45, 59, 68 A9 16, 31, 33, 35, 52, 58 A5 A7 A6 A9 79 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 61. Invader® Chemistry Overview - Summary Primary reaction Secondaryreaction (Simultaneous) Signal amplification is typically ~107 per molecule of target sequence. 80 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 62. Invader® HPV Biplex Reaction Format F1 F2 C G A A C C Invader® Oligo Probe Probe FRET Cassette 1 FRET Cassette 2 Released 5´ Flap Cleavage Site Human DNA-specific target HPV-specific targets Cleavage Site Invader® Oligo Released 5´ Flap Cleavage Site Cleavage Site A T F1 F2 Q 81 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA Q
    • 63. Cervista™ HPV HR Test Workflow Pellet Cells Genfind Extraction IncubationRead & Analyze Reaction Setup MAGNET 82 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 64. Cervista™ HPV HR Test Process Full 4-hour walk-away time enhances productivity. 83 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 65. Interpretation of Results User-friendly  Intuitive user interface  Screen-by-screen walk- through of process steps Flexible  Multiple reporting options Data Analysis Software 84 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 66. Clinical Performance
    • 67. Cervista™ HPV Clinical Trial Overview  Screened >50,000 women; enrolled approximately 4,000  Samples collected from 89 sites in 23 states, providing diversity  Included both ASC-US and normal cytology arms  ASC-US: (>1,300 women)  Primarily intended to show that women with abnormal Pap test results and negative HPV results have < 1% probability of cervical disease (CIN2/3), or conversely >99% NPV  Also intended to show a sensitivity ≥ 90% for detecting cervical disease  Normal cytology (NILM) arm: (>2,000 women)  Intended to establish that HPV-positive subjects are more likely than HPV- negative subjects to develop cervical disease (CIN2+) over a 3-year period  All clinical objectives achieved 86 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 68. Clinical Performance CIN3+ detection:  Cervista™ HPV HR versus Colposcopy/Consensus Histology results (CIN3+) among women with ASC-US cytology Cervista™ HPV HR Colposcopy/Consensus Histology Positive Negative‡ Total Positive 22 747 769 Negative 0 563 563 Total 22 1310 1332 100% detection ‡No CIN, CIN1 or CIN2 by Central Histology or Colposcopy without Central Histology. 87 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 69. Clinical Performance CIN2+ detection:  Cervista™ HPV HR versus Colposcopy/Consensus Histology results (CIN2+) among women with ASC-US cytology Cervista™ HPV HR Colposcopy/Histology Positive Negative‡ Total Positive 64 705 769 Negative 5 558 563 Total 69 1263 1332 93% detection ‡ No CIN or CIN1 by Central Histology or Colposcopy without Central Histology 88 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 70. Clinical Performance  ASC-US/LSIL Triage Study (ALTS)  Organized and funded by the NCI  Included over 5,000 patients  Benchmark ASC-US triage study a Cervista™ HPV HR multicenter clinical trial, 2006–2008. Clinical and analytical data on file, Hologic, Inc. b Immediate colposcopy arm of ALTS. c Number of subjects with known disease status and Cervista™ HPV HR results. d Referral rate for women 30 years of age and older was 43%. 89 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA Comparison of Cervista™ HPV HR clinical trial andALTS
    • 71. CervistaTM HPV HR Benefits
    • 72. Confidence of an Internal Control  Confirms the presence of adequate cellular material for testing  Confirms that no inhibitory substances are present  Minimizes false-negatives due to insufficient sample cellularity © 2009 Hologic, Inc. All right reserved.91 B0043-0309 RevA Test Contains an Internal Control CervistaTM HPV HR:The only FDA-approved HPV test with an internal control Example Invader Call ReporterTM Output Only FDA approved HPV screening test with an internal control
    • 73. Minimizes False Positives  Cross-reactivity to these common low-risk HPV types causes false-positive results, which can lead to unnecessary colposcopies 1Hybrid Capture® 2 High-Risk HPV DNA Test® package insert #L00665, Rev. 2, 2007 2Castle PE, Solomon D., et al. A Comparison of Two Methods to Determine the Presence of High-Risk HPV Cervical Infections. Am J Clin Pathol 2008;130:401-408. 92 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 74. Substantially Reducing Patient Call Backs  Requires only half the sample volume of other HPV tests (2 ml vs. 4 ml)  Increases the likelihood of useful sample volume remaining for additional testing  Providing clear results without an equivocal (gray) zone  Reduces the indeterminate rate to <1%, compared with 4% or more for other HPV tests1 1Solomon et al, JNCI, 2001. Minimum Sample amount Required to Perform Test1 hc2 2 ml 4 ml hc2 4.7% <1% Indeterminate Rate 93 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 75. Cervista™ HPV vs. hc2 ® and RCS ® © 2009 Hologic, Inc. All right reserved.94 B0043-0309 RevA
    • 76. Summary Key Factor CervistaTM HPV HR hc2® Internal control Yes Limits false negatives None Minimum TPPT sample vol. required 2 ml Limits QNS 4 ml More QNS Cross-reacts to common low-risk HPV types? No Yes Types 6,11,42,43,44,53 Equivocal zone for interpretation? No Not required Yes (1.0 to 2.5 RLUs/CO) Maximum walk-away time (manual process) 4 hours 1 hour 95 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
    • 77. Methods  DNA sequencing  Southern Blot  PCR  RT-PCR  RealTime PCR  Methylation-Specific PCR  In-situ PCR  ProteinTruncationTest  Transcription-Mediated Amplification  Strand Displacement Amplification  Nucleic Acid Sequence- Based Amplification  Signal amplification  Branching DNA  Hybrid Capture  Invader  FISH  DNA arrays and chips
    • 78. In Situ Hybridization  Probe types:  Centromeric or CEP (chromosome enumeration probe)  Whole chromosome probes or paints [metaphase only]  Locus specific probe or identifier (LSI)  Section pretreatment.  The labeled probe is first denatured (by heating or under alkaline conditions) into single DNA strands  Hybridized to the target DNA (~Southern blotting) or RNA (~northern blotting) immobilized on a membrane (blotting) or in situ.  Metaphase and Interphase cells
    • 79. ISH Advantages  Interphase nuclei  Archive material  Can detect anomalies hidden to other methods  Detects polysomy, losses, amplifications, translocations Disadvantages  Only provides information of the specific target  Work together with classic cytogenetics  Minimal Residual Disease  Do not provide allele- specific information  Do not detect small changes
    • 80. In Situ Hybridization  More in use Chromosomal translocations  Useful in CMV, HSV,VZV  Sub types of papilloma virus  Useful in Mycobacteria, fungi and parasites  Helicobacter pylori from gastric biopsies  Legionella pneumophila  Pneumocystis jiroveci  Tests done on paraffin embedded specimen  Need applications in Infectious diseases  Doctortvrao’s ‘e’ learning series
    • 81. ISH - PNA probes  Traditionally either cloned probes or synthesized oligonucleotide probes have been used for hybridization.  Peptide nucleic acid (PNA) probe, a nucleotide analogue capable of binding to DNA/RNA in a sequence-specific manner obeying the Watson-Crick base pairing rules.  In PNA, the sugar phosphate backbone of DNA/RNA has been replaced by a synthetic peptide backbone keeping the distances between bases exactly the same as in DNA/RNA.  Further, the PNAs are very stable molecules. Experiments have shown virtually no degradation by DNases, RNases, proteinases or peptidases.  The PNA probes are labeled with fluorescein and detected using a sensitive PNA ISH Detection Kit (colorimetric).
    • 82. Clinical Uses for ISH
    • 83. ISH - Examples  Genotyping of Neoplasms  Polysomy and other gains  Trisomy 12 in B-CLL  Losses  del 1p / del 19q  Amplification  HER2/neu  Translocations  t(9,22)(q34;11) BCR/ABL in CML
    • 84. ISH - Examples  Constitutional Molecular Genetics  Sex chromosome enumeration  Gender  Polysomy and other gains  Trisomy 21 in Down’s  Losses  del(22q11.2) in DiGeorge syndrome
    • 85. ISH - PNA probes  EBER  EBV lytic  Kappa and Lambda
    • 86. ISH Urinary Cytopathology
    • 87. Urovision™  >60,000 new cases of bladder cancer  Recurrence of Urothelial carcinomas 50-80%  Follow up cytoscopy and urine cytology (sensitivity ~48%)  Aneuploidy 3, 7, 17 and loss of 9p21 (CDKN2A p16)  Sensitivity 96% in HG UCs  Interpretation: 35 abnormal cells  ≥4 cells with aneuploidy in ≥2 chromosomes  Loss of 9p21 in ≥12 cells
    • 88. ISH SolidTumors
    • 89. 1p / 19q - Oligodendroglioma
    • 90. Dual FusionTranslocation Probe Break ApartTranslocation Probe
    • 91. ISH – SolidTumors 1p36/19q13 – Oligodendroglioma panelIncludes:1p36/1q25 (1p36 deletion)19q13/19p13 (19q13 deletion) Oligodendrogliomas, mixed oligoastrocytomas EGFR/CEP7 Colorectal, breast and non-small cell lung carcinomas, and glioblastoma multiforme. EGFR gene amplification by FISH may identify tumors predicting responsiveness to EGFR-targeted therapies EWSR1 (22q12) translocations (Breakapart) Clear cell sarcoma, Extraskeletal myxoid chondrosarcoma, PNET/Ewing sarcoma, Desmoplastic small round cell tumor
    • 92. ISH – SolidTumors HER2/CEP17 Identifies the subset of breast carcinoma patients eligible for Herceptin™ (trastuzumab) therapy. MDM-2/SE12 Well-differentiated liposarcoma, dedifferentiated liposarcoma, atypical lipomatous tumor, and pleomorphic lipoma SS18 (SYT) translocations (Breakapart) Synovial Sarcoma TOP2A / CEP17 A predictive biomarker in a subset of breast carcinomas.TOP2A gene amplification may predict response to anthracycline-containing breast chemotherapy.
    • 93. ISH - Lymphomas MALT1 (18q21) translocations (Breakapart) Translocations involving the MALT1 gene have been detected in approximately 20-30% of patients with extranodal low grade marginal zone B- cell lymphomas of MALT type (i.e., MALT lymphomas). Patients with t(11;18)(q21;q21)-positive gastric MALT lymphomas do not respond to Helicobacter pylori eradication therapy, are associated with more advance stage disease, and usually do not show transformation to large cell lymphoma. t(14;18) IGH/MALT1 Subset of MALT lymphomas (Marginal zone B cell lymphoma) t(11;18), MALT1/API2 Subset of MALT lymphomas (Marginal zone B cell lymphoma)
    • 94. ISH - Lymphomas MYC (8q24) translocations (Breakapart) Burkitt lymphoma; MYC translocations (MYC/IGH, MYC/kappa, MYC/lambda); t(8;14), t(2;8), t(8;22) t(11;14) CCND1/IGH Identifies mantle cell lymphoma and subset of plasma cell neoplasms. Patients with multiple myeloma that have a t(11;14)(q13;q32) have been reported to have a neutral to slightly improved clinical course. FISH-based assays provide the most sensitive and specific methodology for detecting the t(11;14)(q13;q32).
    • 95. ISH - Lymphomas t(14;18) IGH/BCL2 Identifies follicular lymphoma and subset of DLBCL with the t(14;18)(q32;q21), which results in constitutive overexpression of the BCL- 2 protein leading to alterations in programmed cell death (i.e., apoptosis) and tumor cell proliferation. FISH- based assays provide the most sensitive and specific methodology for detecting the t(14;18)(q32;q21).
    • 96. ISH - Leukemia t(9;22) BCR/ABL CML and subset of ALL. CML has been traditionally diagnosed by detection of a Philadelphia chromosome (Ph) which has become the hallmark of this disease and is the result of a reciprocal translocation between the BCR gene on chromosome 22 and the ABL gene on chromosome 9. Detection of the Philadelphia chromosome by FISH (or other techniques) helps to confirm and/or monitor patients with CML or other myeloproliferative disorder. An alternate translocation involving BCR and ABL, which is also detected by this assay, can be seen in acute lymphoblastic leukemia (ALL).