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MOLECULAR DIAGNOSTICS PRIMER Part 3 of 3
Cecilia CS Yeung, MDFred Hutchinson Cancer Research Center Nilesh Dharajiya, MD
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Fred Hutchinson Cancer Research Center University of Washington
Seattle, WA
October 28, 2015ASCP Annual Meeting Session Number: 9001
j y ,Sequenom Laboratories
San Diego, CA
Disclosure(s)
Cecilia Yeung: In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation.
Nilesh Dharajiya MD is a full time employee of Sequenom LaboratoriesNilesh Dharajiya, MD is a full-time employee of Sequenom Laboratories and as such receive salary and equity compensation.
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Objectives
At the end of this course you will be able to:
1. Describe current technologies used in molecular diagnostics2. Discuss clinical applications of molecular diagnostics3. Differentiate between diagnostic, prognostic and targeted therapy applications4. Identify cost-efficient ways of doing molecular testing
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OUTLINE
1. Brief Review of Molecular Biology2. Molecular Diagnostics tools:
A. Target Amplification: Polymerase Chain Reaction (PCR) and related methodsB. DNA sequencing – Sanger to Next gen and beyondC. Microarrays - Gene expression, SNP/CNA
3. Upcoming/other technologies of interest:A. Isothermal PCRB. Signal Amplification methodologiesC. PCR alternatives – non amplification methodsD. Sample to answer – point of care
4. Summary
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Amplification Methodologies
• Signal amplification assays:– Branched‐chain DNA (bDNA)– Invader Technology– Hybrid Capture
• Isothermal PCR:– Nucleic Acid Sequence Based Amplification (NASBA)– Strand Displacement Amplification (NASBA)– Loop Mediated Amplification (LAMP)– Helicase‐Dependent Amplification
• PCR alternative non‐target amplification assays:– Nanostring
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Branched DNA signal amplification
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Benign Lymphoid hyperplasia shows mixed kappa and lambda signal
Bright field in situ hybridization (BRISH) for detection of restricted clonal expression of low-abundance immunoglobulin light chain mRNA in B-cell lymphoproliferative disorders.
Tubbs RR, Am J Clin Pathol. 2013 Nov;140(5):736‐46.
Case 5: What is your differential diagnosis?
35 year old man with no prior illness presents with 4 month history of cervical adenopathy.
DDX: Benign follicular hyperplasia vs. Follicular
lymphoma
Excisional biopsy of a lymph node shows
Kappa (black) and Lambda (red) Bright-field in situ hybridization (BRISH): How would you interpret this?
Tubbs RR, Am J Clin Pathol. 2013 Nov;140(5):736‐46.
BRISH shows restricted lambda signal
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• The assay uses Bst DNA Polymerase with Strand
Displacement Activity at 63° C
I th l lifi ti d f d t ti
Loop‐mediated Isothermal Amplification (LAMP)
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• Isothermal amplification, no need for denaturation
step
• Addition of Revere Transcriptase will amplify RNA
(RT-LAMP)
• Amplification efficiency is very high
LAMP video
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LAMP
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Key features1. Expensive equipment not required2. Very sensitive and specific.3. Isothermic.4. High tolerance for inhibitors.5. Visual Detection using dye
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K562 RNA Dilution
SYBR Green
100 ng 10 ng 1 ng 100 pg 10 pg 1 pg Neg Neg
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HNB
100 ng 10 ng 1 ng 100 pg 10 pg 1 pg Neg Neg
Nanostring
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Sample processing
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Sample processing
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Data Collection
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Digital PCR
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Digital droplet PCR
SLIPCHIP
66–267
2
20
Lab Ch
ip. 2
010 Oct 21; 10(20
): 26
Newer generation digital PCR chips
Variety of Chips
Multiple assays can be performed on lots of psamples on a single chip.
Load both samples and assay reagents into chip – “semi-closed systems”
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Fluidigm 192 chip
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Power of digital PCR
• Absolute quantitation compared to real time
PCR
• “single cell” or single target analysis
• Massive data in a very short time
• Low cost (relative) if you have the volume
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Case 6
37 year old man with high risk MDS, but rapidly transformed to AML. Significant social history: Heroin addictionTransferred for fever, tachycardia and tachypnea following episode of bacterial meningitis, and pseudomonas pneumonia.
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Workup showed high plasma quantitative HHV6 levels and blood culture was positive for Serratia marcescensAfter appropriate antibiotic and antiviral therapy, HHV6 levels remained high but fluctuated
Clinical progress
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What is happening here? Why is the HHV6 persistently high?
Patient persists to be very sick, in spite of
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Patient persists to be very sick, in spite of treatment.
Does he really have HHV6 infection?
Could this be chromosomally integrated HHV6? What does this mean?
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Next step‐ perform digital droplet PCR for CI‐HHV6
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QA/QC check of droplets read/sample
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Positive control/negative control
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Patient sample with positive result
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Follow‐up
Determined high fluctuating HHV6 titers due to chromosomal integration and not true infection.
Focus on other infections and problems.Patient eventually died from:
disseminated Candida krusei infection
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• disseminated Candida krusei infection, • persistent AML (unable to be treated due), • myocardial infarction
Sample to result Molecular Diagnostic Devices
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GeneXpert
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Cepheid cartridge
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Filmarray
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Oxford Nanotechnology ‐ Nanopore sequencing
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POC
37quantumdx.com
POC
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Genie III (LAMP)BioHelix (HDA)
CorisBio (NASBA)
Summary
• Molecular testing is area of fastest growth in
Pathology
• PCR and NGS are prevalent test methods in
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clinical molecular lab
• Point of care molecular devices offers
innovative clinical applications
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Acknowledgments
• Ted E. Schutzbank, PhD, D(ABMM)• Giovanni Insuasti‐Beltran, MD (FCAP, FASCP)• Association for Molecular Pathology – Melvin Limson, PhD
and Kathleen Carmody• Linda Cook and Greg Levin –Molecular Virology Laboratory, g gy y,
Virology Division, Laboratory Medicine and the Infectious Diseases Division, FHCRC
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Thank you for your kind attention!
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What is your Status?
A. ZzzzzzzzB. What did you just say?C. Meh…D. Ready for moreE. Molecular is so awesome, applying for MGP tonight!
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Basic molecular questions
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Who first described the double helix structure of DNA?
A. Gregor MendelB. Phoebus LevineC. Franklin and GoslingD. Watson and Crick
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Answer: D
Gregor Mendel: described inheritance in 1866Phoebus Levine: 1919 defines the nucleotide unit = base sugar and phosphateFranklin, Wilkins, and Gosling: 1952 took picture of DNA by X-ray diffraction imageWatson and Crick: 1953 proposed double helix structure of DNA, shared Nobel with Wilkins
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How many histones are needed to form a nucleosome?
A. 2B. 4C. 6D. 8E. 12
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D. 8 histones form a nucleosome bead
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Pritchard; Medical genetics at a glance 2008
Why is a nucleosome important to surg path?
A nucleosome has ~ 200 bpsFormalin stabilizes histone/DNA bondsWhile the rest of the genome is fragmented in processingDNA packed onto a nucleosome is generally "protected" during FFPE extraction
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Which strand of DNA has a higher melting temperature?
A. G-C-G-G-CB. A-T-T-A-G-C-TC. T-T-T-G-G-G-D. C-T-G-T-C-A-A-AE. G-T-A-C-G-G-A
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Answer: A. Look for strand with the most GC
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What are the acceptor and donor sequences of a splice site
A. AU…….AGB. GU…....ACC. GG…….AAD. GU…….AG
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Splicing of introns
Splicesome – multiunit protein, binds to the acceptor and donor sequence as well as a intro into A to cleave out intron, which will leave as a lariat structure to be degraded
52http://en.wikipedia.org/wiki/RNA_splicing
Which part of the gene gets translated?
A. Poly A tailB. IntronC. PromoterD. TrailerE. Exon
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54http://carolguze.com/text/442-1-humangenome.shtml
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Which is not a post translational modification?
A. GlycosylationB. PhosphorylationC. Vitamin C or K mediated modificationD. Alternative splicingE. Selenium addition
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Answer D
Splicing occurs before translationPost translational modifications increased the function of the polypeptide through additional enzymatic changes
Example: some studies looking at Alzheimers show hyperphosphorylation or glycosylation of the tau protein thus leading to plaque development.
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Thi k P i Sh
What are the differences between DNA and RNA?
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Think Pair Share
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Answer:
1. Single versus double strands2. Uracil versus Thymine3. Ribose versus Deoxyribose
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Make a molecular block
20 x 15 x 3mmIdeally: one tumor block, one normal blockFor the tumor block:
> 60% viable tumor, the higher the betterthe better
<20% necrotic tissue
59https://commons.wikimedia.org/wiki/File:Lung_cancer.jpg
What is more abundant in a human cell?
A. DNAB. RNA
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Answer: B, but trick questionApproximately 1% of a cells weight is DNA vs 5-10% RNADNA is highly stable, especially in FFPE due to additional cross-linking with packing proteins induced by formalinmRNA levels can change dramatically depending on the microenvironment
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Degradation of your RNA and DNA samples
TemperatureIce RNA during use, -80 for
storageDNA room temp is ok
during use, -20 at least for storagefor storage
RNAses – very stable, and ubiquitous DNAses – not so stable but still can destroy your sample
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Find the contaminant
in the lab
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in the lab
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D
AB
C
A
B
CD
A
C
B
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O_A_A_I F_A_M_N_S
A. L D K I C K WB. L V L V G B OC. G K Z R E K TD. Q X A S V B P
25%25%25%
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25%
Read from 3’ to 5’Synthesize 5’ to 3’*add NT to 3’-OH end
Case 7
3 yo male with global developmental delay - failing 9 of 11 developmental milestonesPMH: normal vaginal term delivery, no medications
68Case with slides courtesy of A. Kim
Family History
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• Other relatives with developmental delay
Patient with global developmental delay
2 maternal cousins with Fragile X
• Other relatives with undefined “syndrome” (cannot live alone)
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Fragile X
• Fragile X: most common inherited cause of mental retardation after Down’s Syndrome
Incidence: 1 of 6000-7000 males, 1 of 8000 to 9000 females“fragile” site at Xq27.3 where the FMR1 gene is locatedFMR1 is inactivated by additional CGG trinucleotide repeats
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Fragile X
Molecular tool needed: testing to examine the number of CGG repeats
Molecular tool used: Sizing PCR ‐ Requires special polymerase to transcribe through Cs and Gs
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< 44 repeats = normal45‐54 repeats = grey zone 55‐199 repeats = premutation, may be associated with
premature ovarian failure200 repeats = affected
Molecular Testing
Normal female with repeat sizes 29 and 32
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29
32
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Normal male with repeat size 29
Note AGG interruptions which create “dips” in the stutter
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Testing Results
• 3 yo proband
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Boy has a full mutation with >200 repeats and is therefore affected by Fragile X
Molecular Testing Results
Mother: Why should she be tested?Further reproductive counselingRisk of premature ovarian failure
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Mother is mosaic with a normal allele, a premutation, and a full mutation30 140 >200
Case 8: Influenza A/B
Molecular detection of influenza viruses has become routine
Quickly identifying the type and subtype has been used to indicate appropriate antiviral therapy
Beginning with the 2008-2009 season, most season H1N1 infections were resistant to oseltamivir (Tamiflu) whereas most H3N2 infections were resistant to adamantanes (MMWR December 12 2008 / 57(49):1329-resistant to adamantanes (MMWR December 12, 2008 / 57(49):13291332 )
2-step real-time PCR approach:Detection of influenza A and B was performed on the Roche LightCycler
using an ASR-based assay with MultiCode-RTx chemistry (Eragen, Madison, WI)
Samples positive for influenza A were reflexed to a 2nd real-time PCR assay targeting the matrix protein gene (Stone et al., J. Virol. Methods 117:103–112, 2004) to determine subtype based on differential melting of FRET probes
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Expanded Genetic Alphabet
iC:iG Base Pair• Pairs with novel complement only• Recognized by nature’s enzymes• Bayer, Promega
G
C
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Boost Performance with iC:iG Base Pair• One easy to use platform technology• Simplified multiplexing• Standardized protocols• Fast, accurate results• Complete software solutions• Covers >95% of nucleic acid tests
iG
iC
Loss of Signal = Upside‐down Curves
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Testing for Influenza A/B
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Testing for Influenza A/B
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Different years –Possibly different strains –Possibly different Tms
Testing for Influenza A/B
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Subtyping Influenza A
H1N1H1N1
H3N2H3N2
81Stone et al., J. Virol Methods 117:103‐112, 2004
NTCNTC
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Subtyping Influenza A
H3N2H3N2
PatientPatient
H1N1H1N1
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Result: Influenza A positive – unable to subtype
Stone et al. J. Virol Methods 117:103‐112, 2004
H1N1H1N1
NTCNTC
Timeline Review
Date 04/22 04/23 04/25 04/27 05/01
Event Specimen collection and testing
Influenza B neg, A positive ‐ unable to subtype. Sent to state lab for
News of pandemic flu in Mexico with confirmed cases in CA and TX
Patient contacted by physician, confirms trip
State lab report: B neg, A pos ‐ not H1N1, not
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state lab for further testing
CA and TX confirms trip to Mexico
H1N1, not H3N2, probably swine flu
Specimen sent to CDC
for confirmation
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