molecular pathology testing of nucleic acids within a clinical context helpful hereditary disorders...
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Introduction to Molecular Pathology
Luis E. Ferrer Torres, MD FCAP
Molecular PathologyTesting of nucleic acids within a clinical context
Helpful Hereditary disorders Oncology Infectious diseases
Molecular PathologySpecific purposes
Diagnosis Prognosis Prenatal testing Pharmacotherapy
PharmacogeneticsPharmacogenomics
Watson and CrickThe 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
Watson and Crick discovers DNA / Feb 28th 1953
Watson and Crick Builds a Model DNA 7th March 1953
First Document on DNA published in Nature 25th April 1953
Watson and Crick - Awarded Nobel Prize in 1962
DNAA molecule contains two polynucleotide strands
that form an an antiparallel double helix.
Nucleotides: Nitrogenous base (AT GC,U) Deoxyribose Phosphate
DNA - StructureThe 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).
DNAA purine always links
with a pyrimidine base to maintain the structure of DNA.
Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.
Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.
Chemical structure of DNA
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.
DNAExample
First strand GGGTTTAAACCC
Second strand CCCAAATTTGGG
Central Dogma of Molecular Biology
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.
DNA ReplicationDNA 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 ?
TranscriptionRNA polymerase II mediates transcription and
generates a precursor ss-mRNA identical to the sense (coding) stand except for U for T.
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
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
DNA – RNA – DNAa 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
Translationm-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
RNA = Ribonucleic acid. RNA is similar to
DNA except:
It has one strand instead of two strands. Has uracil instead of thymine3.Has Ribose instead of Deoxyribose
Gene ExpressionDNA level expression control
Transcriptional Post-Transcriptional
Epigenetics DNA methylation Histone modification
Gene ExpressionDNA level expression control
TranscriptionalHouse keeping genes
Always on
Transcription factors Usually lie upstream in the promoter region Enhancer and silencer elements
Gene Expression Post transcriptional
Export of mRNA out of nucleusAlternative splicingmRNA stabilizationmRNA degradationRNA interference or silencing
miRNA and siRNA
Gene ExpressionDNA level expression control
Transcriptional Post-Transcriptional
Epigenetics DNA methylation Histone modification
What is GeneThe 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.
Modes of inheritanceMutations 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
Modes of inheritanceGenomic imprinting
Different expression of alleles depending on parent origin
Uniparental Disomy Both copies inherited from one parent
Environmental influence Chronic diseases
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.
Single Nucleotide Polymorhisms and HaplotypesSNPs 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
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
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…
Methods DNA sequencing
Southern Blot
PCR RT-PCR Real Time PCR
Methylation-Specific PCR
In-situ PCR
Protein Truncation Test
Transcription-Mediated Amplification
Strand Displacement Amplification
Nucleic Acid Sequence-Based Amplification
Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
Gene sequencingDetermining 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)
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
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]
Methods DNA sequencing
Southern Blot
PCR RT-PCR Real Time PCR
Methylation-Specific PCR
In-situ PCR
Protein Truncation Test
Transcription-Mediated Amplification
Strand Displacement Amplification
Nucleic Acid Sequence-Based Amplification
Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
Southern BlotEdwin 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
Southern Blotworking protocol
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
Methods DNA sequencing
Southern Blot
PCR RT-PCR Real Time PCR
Methylation-Specific PCR
In-situ PCR
Protein Truncation Test
Transcription-Mediated Amplification
Strand Displacement Amplification
Nucleic Acid Sequence-Based Amplification
Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
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
PCRDenaturation
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
RT-PCRTo detect or quantify RNA transcripts or viral
RNA
RNA is converted to DNA
Reverse transcriptase (Avian Myeloblastosis Virus and Moloney Murine Leukemia virus)
Isothermal reaction with primers: oligo dT, random hexamer primers, or target specific primers
One step vs. two steps
PCR or RT-PCR Product analysis / detection
Real Time Hybridization
Membrane boundReverse line blotsLiquid Bead Array with Flow Cytometry
ElectrophoresisAgaroseCapillary
Cycle sequencer
Multiplexed – PCR and ELISA
Protein Expression ProfilingCancer MarkersCardiac MarkersCellular SignalingCytokines, Chemokines, and Growth FactorsEndocrineIsotypingMatrix MetalloproteinasesMetabolic MarkersNeurobiologyTranscription Factors/Nuclear Receptors
Genomic ResearchFlexmiR® v2 Custom microRNA AssayFlexmiR microRNA PanelsGene Expression ProfilingGenotyping
Genetic DiseaseCystic FibrosisCytochrome p450
ImmunodiagnosticsAllergy TestingAutoimmune DiseaseHLA TestingInfectious DiseaseVaccine TestingNewborn Screening
Biodefense/Environmental
Real Time - PCRAmplifies 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
Real Time - PCRThe 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
PCRAdvantages
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+)
Methods DNA sequencing
Southern Blot
PCR RT-PCR Real Time PCR
Methylation-Specific PCR
In-situ PCR
Protein Truncation Test
Transcription-Mediated Amplification
Strand Displacement Amplification
Nucleic Acid Sequence-Based Amplification
Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
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
Capture Extenders (CEs), Label Extenders (LEs), and Blocking Probes (BLs)
Branched DNA applications
Detection HIV, HBV, and HCV
Measures viral loads
Less sensitive than PCR
Doctortvrao’s ‘e’ learning series
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
InvaderFRET / Cleavase
Signal amplification
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
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
Product Design & Chemistry
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 ProcessAmplifies Signal
Cleavase® Enzyme
78 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
CervistaTM HPV HR Test Design
Developed from phylogenetictree 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
A6A9
79 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
Invader® Chemistry Overview - Summary
Pri
mary
re
acti
on
Secon
dary
re
acti
on
(S
imu
ltan
eou
s)
Signal amplification is typically ~107 per molecule of target sequence.
80 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
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
CleavageSite
CleavageSite
A
T
F1F2 Q
81 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
Q
Cervista™ HPV HR Test Workflow
Pellet Cells Genfind Extraction
IncubationRead & Analyze Reaction Setup
MAGNET
82 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
Cervista™ HPV HR Test Process
Full 4-hour walk-away time enhances productivity.
83 © 2009 Hologic, Inc. All right reserved. B0043-0309 RevA
Interpretation of ResultsUser-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
Clinical Performance
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
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
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
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 and ALTS
CervistaTM HPV HR Benefits
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
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, 20072Castle 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
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
Cervista™ HPV vs. hc2 ® and RCS
®
© 2009 Hologic, Inc. All right reserved.94 B0043-0309 RevA
SummaryKey Factor CervistaTM HPV HR hc2®
Internal controlYes
Limits false negativesNone
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
Methods DNA sequencing
Southern Blot
PCR RT-PCR Real Time PCR
Methylation-Specific PCR
In-situ PCR
Protein Truncation Test
Transcription-Mediated Amplification
Strand Displacement Amplification
Nucleic Acid Sequence-Based Amplification
Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
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
ISHAdvantages
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
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
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).
Clinical Uses for ISH
ISH - ExamplesGenotyping of Neoplasms
Polysomy and other gainsTrisomy 12 in B-CLL
Lossesdel 1p / del 19q
AmplificationHER2/neu
Translocationst(9,22)(q34;11) BCR/ABL in CML
ISH - ExamplesConstitutional Molecular Genetics
Sex chromosome enumerationGender
Polysomy and other gainsTrisomy 21 in Down’s
Lossesdel(22q11.2) in DiGeorge syndrome
ISH - PNA probesEBER
EBV lytic
Kappa and Lambda
ISHUrinary
Cytopathology
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
ISHSolid Tumors
1p / 19q - Oligodendroglioma
Dual Fusion Translocation Probe
Break Apart Translocation Probe
ISH – Solid Tumors
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
ISH – Solid Tumors
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.
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)
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).
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).
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).