molecular pathology testing of nucleic acids within a clinical context helpful hereditary disorders...

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

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

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

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

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

• 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]

Southern Blot

In-situ PCR

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

Southern Blot

In-situ PCR

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 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

Luminex

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

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+)

Southern Blot

In-situ PCR

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

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

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

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

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

Repeating ProcessAmplifies Signal

Cleavase® Enzyme

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

Invader® Chemistry Overview - Summary

Signal amplification is typically ~107 per molecule of target sequence.

Invader® HPV Biplex Reaction Format

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

F1F2 Q

Cervista™ HPV HR Test Workflow

Pellet Cells Genfind Extraction

IncubationRead & Analyze Reaction Setup

MAGNET

Cervista™ HPV HR Test Process

Full 4-hour walk-away time enhances productivity.

Interpretation of ResultsUser-friendly

Intuitive user interface

Screen-by-screen walk- through of process steps

Flexible

Multiple reporting options

Data Analysis Software

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

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.

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

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%.

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

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.

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

Indeterminate Rate

Cervista™ HPV vs. hc2 ® and RCS

SummaryKey Factor CervistaTM HPV HR hc2®

Internal controlYes

Limits false negativesNone

Minimum TPPT sample vol. required

Limits QNS

More QNS

Cross-reacts to common low-risk HPV types? No

Types 6,11,42,43,44,53

Equivocal zone for interpretation?No

Not required

(1.0 to 2.5 RLUs/CO)

Maximum walk-away time (manual process) 4 hours 1 hour

Southern Blot

In-situ PCR

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).

molecular pathology testing of nucleic acids within a clinical context helpful hereditary disorders...

dna structure

nucleus slide

dna example

model dna

dna replicates

strand cccaaatttggg

dna double helix

functional translations

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