10/29/2015

1

MOLECULAR DIAGNOSTICS PRIMER Part 3 of 3

Cecilia CS Yeung, MDFred Hutchinson Cancer Research Center Nilesh Dharajiya, MD

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

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.

2

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

3

10/29/2015

2

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

4

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

5

Branched DNA signal amplification

6

10/29/2015

3

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

10/29/2015

4

• 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)

10

• Isothermal amplification, no need for denaturation

step

• Addition of Revere Transcriptase will amplify RNA

(RT-LAMP)

• Amplification efficiency is very high

LAMP video

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

LAMP

12

Key features1. Expensive equipment not required2. Very sensitive and specific.3. Isothermic.4. High tolerance for inhibitors.5. Visual Detection using dye 

10/29/2015

5

K562 RNA Dilution

SYBR Green 

100 ng 10 ng 1 ng 100 pg 10 pg 1 pg Neg Neg

13

HNB

100 ng 10 ng 1 ng 100 pg 10 pg 1 pg Neg Neg

Nanostring

14

Sample processing

15

10/29/2015

6

Sample processing

16

Data Collection

17

Digital PCR

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

10/29/2015

7

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”

21

Fluidigm 192 chip

10/29/2015

8

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

22

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.

23

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

24

10/29/2015

9

What is happening here? Why is the HHV6 persistently high?

Patient persists to be very sick, in spite of

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

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?

26

Next step‐ perform digital droplet PCR for CI‐HHV6

27

10/29/2015

10

QA/QC check of droplets read/sample

28

Positive control/negative control

29

Patient sample with positive result

30

10/29/2015

11

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

31

• disseminated Candida krusei infection, • persistent AML (unable to be treated due), • myocardial infarction

Sample to result Molecular Diagnostic Devices

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

GeneXpert

33

10/29/2015

12

Cepheid cartridge

34

Filmarray

35

Oxford Nanotechnology ‐ Nanopore sequencing

36

10/29/2015

13

POC

37quantumdx.com

POC

38

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

39

clinical molecular lab

• Point of care molecular devices offers

innovative clinical applications

10/29/2015

14

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

40

Thank you for your kind attention!

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

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!

42

10/29/2015

15

Basic molecular questions

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

Who first described the double helix structure of DNA?

A. Gregor MendelB. Phoebus LevineC. Franklin and GoslingD. Watson and Crick

44

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

45

10/29/2015

16

How many histones are needed to form a nucleosome? 

A. 2B. 4C. 6D. 8E. 12

46

D. 8 histones form a nucleosome bead

47

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

48

10/29/2015

17

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

49

Answer: A. Look for strand with the most GC

50

What are the acceptor and donor sequences of a splice site

A. AU…….AGB. GU…....ACC. GG…….AAD. GU…….AG

51

10/29/2015

18

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

53

54http://carolguze.com/text/442-1-humangenome.shtml

10/29/2015

19

Which is not a post translational modification?

A. GlycosylationB. PhosphorylationC. Vitamin C or K mediated modificationD. Alternative splicingE. Selenium addition

55

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.

56

Thi k P i Sh

What are the differences between DNA and RNA? 

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

Think Pair Share

10/29/2015

20

Answer: 

1. Single versus double strands2. Uracil versus Thymine3. Ribose versus Deoxyribose

58

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

60

10/29/2015

21

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

61

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

62

Find the contaminant

in the lab

Expertise that advances patient care through education, innovation, and advocacy.

www.amp.org

in the lab

10/29/2015

22

D

AB

C

A

B

CD

A

C

B

10/29/2015

23

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%

67

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

69

• Other relatives with developmental delay

Patient with global developmental delay

2 maternal cousins with Fragile X

• Other relatives with undefined “syndrome” (cannot live alone)

10/29/2015

24

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

70

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

71

< 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

72

29

32

29

Normal male with repeat size 29

Note AGG interruptions which create “dips” in the stutter

10/29/2015

25

Testing Results

• 3 yo proband

73

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

74

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

75

10/29/2015

26

Expanded Genetic Alphabet

iC:iG Base Pair• Pairs with novel complement only• Recognized by nature’s enzymes• Bayer, Promega

G

C

76

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

77

Testing for Influenza A/B

78

10/29/2015

27

Testing for Influenza A/B

79

Different years –Possibly different strains –Possibly different Tms

Testing for Influenza A/B

80

Subtyping Influenza A

H1N1H1N1

H3N2H3N2

81Stone et al., J. Virol Methods 117:103‐112, 2004

NTCNTC

10/29/2015

28

Subtyping Influenza A

H3N2H3N2

PatientPatient

H1N1H1N1

82

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

83

state lab for further testing

CA and TX confirms trip to Mexico

H1N1, not H3N2, probably swine flu

Specimen sent to CDC 

for confirmation