Emerging Technologies
In
Cancer Molecular Pathology
2011
2010 Metropolitan Health Service of the Year
Cancer Molecular Pathology
Inherited Cancer Predispositions
– Tumour Suppressor Genes – loss of function
• Breast Cancer
• Colorectal Cancer
• Rare familial cancer syndromes
– Endocrine
– Gastric Cancer
– AIP (Pituitary Adenoma)
– Skin Cancer
Looking for Heterozygote DNA variation:
– Point mutations
– Large scale rearrangements
2010 Metropolitan Health Service of the Year
Somatic Markers of Cancer
– Oncogenes & Tumour Suppressor Genes – loss & Gain of
function
Catalogue of Somatic Mutations in Cancer - COSMIC database
Tumours
548 399
Samples
551 325
Mutated Samples
138 836
Mutations
143 772
Unique Mutations
25 079
Papers Curated
18 647
Fusions
5 050
Structural Variants
2 306
Looking for low frequency mutations in mixed samples
2010 Metropolitan Health Service of the Year
COSMIC V49 – 29th Sept 2010
Reasons for Current Somatic
Testing
Gene
Target Regions
Drug
LUNG CANCER
EGFR
Exons 18,19,20 & 21
Gefitinib
Erlotinib
BRAF
Exon 15, codon 600
Vemurafinib
KIT
Exons 9,11 & 13
Imatinib
Exons 9,11,13 & 17
18
Imatinib
Imatinib
MELANOMA
GIST
KIT
PDGFRD
CRC
KRAS
2010 Metropolitan Health Service of the Year
Exon 2, codons 12&13 Cetuximab
Panitumumab
OR
||||||||||
10M000
Smith , j
Routine Work Flow for Somatic Mutation
Analysis
+
PATHOLOGY
REPORT
Path Review
Macro-dissection
DNA extraction
Pre-screen
D enn
(HRM)
Sequencing
D
NEXT
GEN
D
D
-VE
E
Report
2010 Metropolitan Health Service of the Year
Duplicate
PCR
Cancer Pathways
2010 Metropolitan Health Service of the Year
Current Requirements
•
Assay Single or small # targets for individual samples
– Melanoma
• BRAF,KIT
•
Short time frames for actionable & clinically relevant testing
– BRAF, KRAS, EGFR, BRCA1/2 (PARP inhibitors)
•
Interpret genetic variants for drug sensitivity and resistance
•
Analyse a larger number of individual genes contributing moderate or
low risk of cancer predisposition
•
Introduce more target assays as therapies increase
– Melanoma
• GNAQ,GNA11,PI3K,NRAS
•
Provide high sensitivity & reproducible assays at low cost
2010 Metropolitan Health Service of the Year
NGS
(Next Gen Sequencing)
•
•
Advantages
Parallel sequencing of multiple targets •
Disadvantages
Requires pre-selection of target DNA
Sample multiplexing to increase
throughput & reduce cost
•
Potentially complex analysis
•
Identification of normal variation on a
larger scale - interpretation
•
Sensitivity increase over standard
sequencing
•
Digital read out of sequence
2010 Metropolitan Health Service of the Year
Choice of Strategy
•
Just sequence it all
– Genome
– Exome
– Kineome
•
•
•
•
Shallow depth of coverage
Unwanted information
IT issues, data storage
Validation
•
Hybe capture
•
Amplicon capture
– Long Range PCR
– Multiplex PCR
– Micro Fluidics
•
•
•
•
•
•
Uneven capture (GC regions)
Template quantity high (FFPE)
Library prep required
Specificity
Routine workflow
Suitable for poor & small
samples
Tedious for numerous targets
2010 Metropolitan Health Service of the Year
•
Targeting Method Comparisons
PCR
MIP
Array Capture
Solution Capture
Cost
High
<10 samples; high
>10 samples; low
Medium
<10 samples; medium
>10 samples; low
Ease
Low
High
Medium
High
DNA input
~8ug; 1Mb
200ng
10-15ug;30Mb
3ug;30Mb
Sensitivity
>99.5%
>98% with
stringent design
98.6% of CTR
>99.5% of CTR
Specificity
93% for HapMap
72% for whole
genome amplified
>98%
Up to 70%
Up to 80%
Uniformity
80% within 2 fold
range
58% within 10 fold
88% within 100
fold
60% within 1-5 fold
61% within 1.5 fold
Reproducibility
Up to 100%
0.92 rank order
>95%
>96%
2010 Metropolitan Health Service of the Year
Mamanova et al 2010 Nature Methods
PCR Targeting Chosen
•Similar to current workflow
•Suitable for degraded DNA
•Micro fluidic forms of PCR require low sample DNA input
•Amenable to automation – conventional or micro fluidic
•Specificity controlled by primer selection
•Redundant coverage of targets important for FFPE samples
•PCR incorporated adaptors & barcodes – no library prep
•Unlikely to produce incidental findings
2010 Metropolitan Health Service of the Year
Specificity
Significant homology between genes & pseudo genes exists for a number of
targets
•BRAF
•PMS2
BRAF chromosome 7
Precise control of specificity possible with primer selection compared to
hybridisation
BRAF pseudo gene (BRAFP1) X chromosome
2010 Metropolitan Health Service of the Year
Initial Proof of Principle
Path Review
Sample 1
Macro-dissection DNA extraction
Path Review
Sample 2
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MID1
PCR:
KRAS ex2
NRAS ex3
BRAF ex15
KIT ex9,11&13
PDGFRa ex18
with addition of
MID’s & 454
adaptors A & B
MID2
AmPure
Equimolar
Mix
emPCR
&
454 sequencing
8 Channels on a
single run
Reproducibility
2010 Metropolitan Health Service of the Year
BRAF c.[1801A>G]+[=]:p,Lys601Glu
G: 21.84% Coverage 5784
2010 Metropolitan Health Service of the Year
BRAF c.[1801A>G]+[=]:p,Lys601Glu
G: 21.67% Coverage 5884
Ability to Detect different
Mutation Types
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KRAS c.38G>A:p.Gly13Asp
Coverage 4859
Allele 42.4%
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KIT Exon 11 c.1657_1673delinsGA:p.Tyr553_Lys558
Coverage 8769
delins96%
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KIT Exon 9 c.1504_1509dup: p.Ala502_Tyr503dup
Coverage 9729
Ins 33%
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Sensitivity of NEXT GEN
NCI-H1650 adenocarcinoma
c.2235_2249del15:
p.Glu746_Ala750
2 fold
dilutions
With normal
DNA
NCI-H1975 adenocarcinoma
c.2369C>T:p.Thr790Met
c.2573T>G:p.Leu858Arg
2010 Metropolitan Health Service of the Year
MID1
PCR EGFR
Ex19,20 & 21
with addition
of MIDs &
454 adaptors
MID2
AmPure
Equimolar
Mix
emPCR
H1975 2% EGFR
c.2573T>G:p.Leu858Arg
G allele 2.10%
Coverage 20240
2010 Metropolitan Health Service of the Year
H1975 1% EGFR
c.2573T>G:p.Leu858Arg
G allele 0.98%
Coverage 15972
2010 Metropolitan Health Service of the Year
Automation of Amplicon Targeting
Initial PoP promising however, multiple targets and
multiplexed samples would require many PCR’s and
increase assay difficulty & cost
Two examples of Micro fluidics
– Raindance
– Fluidigm
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RainStorm™ Droplet-Based
Microfluidics
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RainStorm™ Droplet-Based
Microfluidics
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RainDance
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RainDance
2010 Metropolitan Health Service of the Year
Fluidigm
Access-Array
2010 Metropolitan Health Service of the Year
48 - ~480 targets for 48 samples
Can include primers with
Specific bar codes
Samples
(1ul of 50ng/ul)
Reagents
Load-PCR-Recover
Single or Multi - plex
Pool Samples
(1 ul each)
Ampure Clean
Quantify
~300 template
molecules
/reaction
NGS
2010 Metropolitan Health Service of the Year
Array Matrix
Detection
Samples
BRCA1: c.1175_1214del
MID22
BRCA1: c.1687C>T: p.Gln563X
MID36
BRCA1: c.1823_1826delAGAA
MID16
BRCA1: c.2369C>T:p.Thr790Ile
MID5
BRCA1: c.2641G>T p.Glu881X
MID15
BRCA1: c.3481_3491del:p.Glu1161PhefsX3
MID43
BRCA1: c.3662A>C:p.Asn1121Thr and
c.5357T>C:Leu1786Pro
MID14
BRCA1: c.3756_3759delGTCT:p.Ser1253ArgfsX10
MID46
BRCA1: c.4364T>G:p.Leu1455X
MID8
BRCA1: c.5030_5033delCTAA:p.Thr1677IlefsX2
MID34
BRCA1: c.5030_5033delCTAA:p.Thr1677IlefsX2
MID50
BRCA1: c.5266dupC
MID30
BRCA1: c.5425G>T: p.Val1809Phe
MID33
BRCA1: c.566A>G: p.Asp189Gly
MID20
BRCA1: c.68_69delAG:p.Glu23ValfsX17
MID18
BRCA1: c.68_69delAG:p.Glu23ValfsX17
MID48
BRCA1:c.4065_4068delTCAA:p.Asn1355LysfsX10
MID37
BRCA2: c.1310_1313delAAGA
MID7
BRCA2: c.1310_1313delAAGA
MID47
BRCA2: c.2175delA
MID3
BRCA2: c.2880delG
MID27
BRCA2: c.3975_3978dupTGCT
MID1
BRCA2: c.5279C>G
MID28
BRCA2: c.5357delG
MID31
BRCA2: c.5576_5579delTTAA
MID23
BRCA2: c.5722_5723delCT
MID13
BRCA2: c.6275_6276delTT
MID6
BRCA2: c.6405_6409del
MID42
BRCA2: c.68-7T>A
MID19
BRCA2: c.755_758delACAG
MID21
BRCA2: c.771_775delTCAAA
BRCA2: c.778_779delGA
MID35
MID45
BRCA2: c.8172_8175dupGTGG
MID25
BRCA2: c.8172_8175dupGTGG
MID49
BRCA2: c.8575delC
MID11
BRCA2: c.9097dupA
MID41
BRCA2: c.9414_9417dupATTT
MID32
BRCA2:c.2808_2811delACAA
MID44
BRCA2:c.3847_3848delGT
MID24
BRCA2:c.5073dupA
MID10
BRCA2:c.787G>A
MID38
BRCA2:c.8904delC
MID40
BRCA2:c.9117G>A
MID39
NEGATIVE
MID2
NEGATIVE
Single-plex BRCA1
Primers
Samples
MID17
2010 Metropolitan Health Service of the Year
Primers
External primers
to include
Sample specific
MIDs
Exon
2
5
6
8
11
11
11
11
11
11
11
11
11
12
13
14
15
16
18
19
21
22
24
Amplicon
BRCA1_600_1887836_1_2
BRCA1_600_1945886_1
BRCA1_600_1900448_2
BRCA1_600_1840043_1
BRCA1_600_36588_184
BRCA1_600_36588_137
BRCA1_600_36588_124
BRCA1_600_36588_123
BRCA1_600_36588_217
BRCA1_600_36588_89
BRCA1_600_36588_88
BRCA1_600_1936588_4
BRCA1_600_1936588_1
BRCA1_600_0729436_1
BRCA1_600_0371140_1
BRCA1_600_1816775_1
BRCA1_600_0865524_1
BRCA1_600_0865521_1
BRCA1_600_1368002_2
BRCA1_600_1895889_2
BRCA1_600_0865503_1
BRCA1_600_0865496_1
BRCA1_600_1831829_1
NM_007294.3 position
c.-19-249_80+32
c.135-179_212+88
c.213-171_301+140
c.442-141_547+182
c.671-46_1116
c.1031_1570
c.1413_1998
c.1744_2341
c.2031_2627
c.2548_3081
c.2942_3528
c.3448_3944
c.3893_4096+296
c.4097-154_4185+170
c.4186-111_4357+131
c.4358-168_4484+110
c.4485-133_4675+171
c.4676-56_4986+71
c.5075-98_5153-200
c.5152+223_5193+131
c.5278_189_5332+156
C.5333-199_5406+129
C.5468-76_*220
0
Sample
Samples
2010 Metropolitan Health Service of the Year
Sample
Samples
11M0542_DUP
09M3771
11M0542
10M3715
10M4833
10M2895
10M3767_DUP
10M0410
10M5156
10M3767
10M4624
10M3065
10M4833_DUP
10M0494
10M5164
09M2536
10M4296
10M3079
10M3065_DUP
10M0584
10M5382
10M3986
10M4478
10M3172
09M2454
10M0617
10M5579
10M3992
10M4603
10M3654
09M4051
10M0628
10M5781
10M4618
10M2102
BRCA1 & Tp53
10M3222
09M4059
10M6071
10M5907
10M4806
10M2123
10M3331
09M449
11M0429
2
10M5983
3
Conc.ng/ul
5
10M4817
4
ng/ul
6
10M2679
Conc.ng/
7
10M3418
11M0542_DUP
09M3771
11M0542
10M3715
10M4833
10M2895
10M3767_DUP
10M0410
10M5156
10M3767
10M4624
10M3065
10M4833_DUP
10M0494
10M5164
09M2536
10M4296
10M3079
10M3065_DUP
10M0584
10M5382
10M3986
10M4478
10M3172
09M2454
10M0617
10M5579
10M3992
10M4603
10M3654
09M4051
10M0628
10M5781
10M4618
10M2102
10M3222
09M4059
10M6071
10M5907
10M4806
10M2123
10M3331
09M449
11M0429
10M5983
10M4817
10M2679
10M3418
ng/ul
Single-Plex BRCA1/2 & Tp53
• Simultaneous analysis of 3 genes for 48 samples
• Single-plex PCR using 2 access arrays for 73 amplicons
Bioanalyser Traces
10
BRCA2
9
8
7
6
5
4
1
3
2
1
0
Individual Sample Bioanalyser traces
Access Array 1
BRCA1/Tp53
31 Amplicons
Access Array 2
BRCA2
43 Amplicons
2010 Metropolitan Health Service of the Year
BRCA1 Coverage – All Samples
2
3
5 67
2010 Metropolitan Health Service of the Year
10 11
12
8 9
13
1415 16 17 1819
20
22 24
21 23
BRCA 2 Coverage All Samples
2
3
5,6 7
4 8 910
11
2010 Metropolitan Health Service of the Year
12 13
15
18
14 16 17
20
19
22,23,24
21
26
25 27
BRCA1 Exon 11 Coverage
3 amplicons
45 bp
2010 Metropolitan Health Service of the Year
BRCA2 Exon 11 Coverage
2010 Metropolitan Health Service of the Year
BRCA1 Results from Fluidigm
&
Roche 454 Junior
Number of variants
19
Number of variants covered by primer design
14
Number of variants not detected due to failed amplicons
3
Number of variants covered but not detected
1
2010 Metropolitan Health Service of the Year
RESULTS – BRCA1
Capillary Sequencing
Exon
Theoretical
c.514C>T p.Gln172X
MID26
8
Yes
Yes
BRCA1_600_1840043_1
NM_007294.3
c.442141_547+182
c.1175_1214del:p.Leu392GlnfsX5
MID22
11
Yes
No
BRCA1_600_36588_137
c.1031_1570
No
c.1687C>T: p.Gln563X
MID36
11
Yes
No
BRCA1_600_36588_124
c.1413_1998
No
c.1823_1826delAGAA:p.Lys608IlefsX3
MID16
11
Yes
No
BRCA1_600_36588_123
c.1744_2341
No
c.2369C>T:p.Thr790Ile
MID5
11
Yes
Yes
BRCA1_600_36588_217
c.2031_2627
Yes
26
c.2641G>T p.Glu881X
MID15
11
Yes
Yes
BRCA1_600_36588_89
c.2548_3081
Yes
29
c.3481_3491del:p.Glu1161PhefsX3
MID43
11
Yes
Yes
BRCA1_600_1936588_4
c.3448_3944
No*
48
c.3662A>C:p.Asn1121Thr
MID14
11
Yes
Yes
BRCA1_600_1936588_4
c.3448_3944
Yes
53
c.5357T>C:Leu1786Pro
MID14
11
Yes
Yes
BRCA1_600_1936588_4
c.3448_3944
Yes
77
c.3756_3759delGTCT:p.Ser1253ArgfsX10
MID46
11
Yes
Yes
BRCA1_600_1936588_4
Yes
58
c.4364T>G:p.Leu1455X
MID8
14
Yes
Yes
BRCA1_600_1816775_1
c.3448_3944
c.4358168_4484+110
Yes
30
c.5030_5033delCTAA:p.Thr1677IlefsX2
MID34
17
No
No
Not Covered
c.5030_5033delCTAA:p.Thr1677IlefsX2
MID50
17
No
No
Not Covered
c.5266dupC:p.Gln1756ProfsX74
MID30
20
No
No
Not Covered
c.5425G>T: p.Val1809Phe
MID33
23
No
No
Not Covered
c.566A>G: p.Asp189Gly
MID20
9
No
No
Not Covered
c.68_69delAG:p.Glu23ValfsX17
MID18
2
Yes
Yes
BRCA1_600_1887836_1_2 c.-19-249_80+32
Yes
41
c.68_69delAG:p.Glu23ValfsX17
MID48
2
Yes
Yes
BRCA1_600_1887836_1_2 c.-19-249_80+32
Yes
43
BRCA1:c.4065_4068delTCAA:p.Asn1355LysfsX10
MID37
11
Yes
Yes
BRCA1_600_1936588_1
Yes
71
2010 Metropolitan Health Service of the Year
*Allele drop out ?
Experimental Fluidigm Amplicon
Mutation Coverage
MID
c.3893_4096+296
Yes
39
Forward Primer
Reverse Primer
2010 Metropolitan Health Service of the Year
BRCA1 c.4065_4068delTCAA
coverage 71
37% del
2010 Metropolitan Health Service of the Year
Artefact OR Variant in homo polymers
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All 48 Samples
2010 Metropolitan Health Service of the Year
BRCA2 5073DupA in Homopolymer
Missed
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Separate Assay for ins & dels in
homopolymers
When Using Pyrosequencing based
NGS
Simple Fragment Analysis
2010 Metropolitan Health Service of the Year
0
Sample
Samples
2010 Metropolitan Health Service of the Year
11M0542_DUP
09M3771
11M0542
10M3715
10M4833
10M2895
10M3767_DUP
10M0410
10M5156
10M3767
10M4624
10M3065
10M4833_DUP
10M0494
10M5164
09M2536
10M4296
10M3079
10M3065_DUP
10M0584
10M5382
10M3986
10M4478
10M3172
09M2454
10M0617
10M5579
10M3992
10M4603
10M3654
09M4051
10M0628
10M5781
10M4618
10M2102
10M3222
09M4059
10M6071
10M5907
10M4806
10M2123
10M3331
09M449
11M0429
10M5983
10M4817
10M2679
10M3418
Conc.ng/
ng/ul
Multi-Plex
BRCA1/2;Tp53;APC;KIT;EGFR
• Simultaneous analysis of 6 genes for 48 samples
• Multi-plex PCR using 1 access array for 177 amplicons
Bioanalyser Traces
7
6
5
4
3
2
1
Multiplex
Samples
(1ul of 50ng/ul)
~300 template
molecules
/reaction
Peform 2nd PCR to add adaptors
and sample MIDs
Pool Samples
(1 ul each)
Ampure Clean
Quantify
2010 Metropolitan Health Service of the Year
NGS
Reagents
~4 plex
FFPE – Fluidigm - NGS
• Objective to achieve 1% sensitivity (~5000 depth)
Assess Degradation
(qPCR)
Samples Primers
no
Degraded
yes
Pre-amplification
Primer Pool
2nd PCR to add
adaptors and sample
MIDs
NGS
2010 Metropolitan Health Service of the Year
Is this All Feasible for Somatic
Testing?
•
Assumptions:
– Average Output from Small NGS- ~60Mb data / run
– 1% sensitivity requires ~ 5000 depth of coverage
– 200 bp amplicons
– $1000/small NGS run
1%
5%
NoAmplicons
MaxSamples
cost/sample
NoAmplicons
5
12
83
5
60
17
10
6
167
10
30
33
15
4
250
15
20
50
20
3
333
20
15
67
25
2
417
25
12
83
30
2
500
30
10
100
2010 Metropolitan Health Service of the Year
MaxSamples cost/sample
Small NGS Instruments
• Roche 454 Junior
~50Mb
400bp reads
30h total analysis time
• Ion Torrent
~10Mb 100Mb 1Gb
100bp – 200bp reads
8h total analysis time
• MiSeq
~1Gb
150bp reads
27h total analysis time
2010 Metropolitan Health Service of the Year
Single Molecule Sequencing
• Oxford Nanopore
• Two technologies
•Exonuclease sequencing
• Strand sequencing
• Pacific Biosciences
– Read lengths ~1,000bp
• More accurate assembly
• Detect large rearrangements
2010 Metropolitan Health Service of the Year
Overall Germ line Strategy
Blood
OR
FFPE
Sample
FFPE
Somatic
Strategy
Blood
Micro Fluidic
PCR
(48+ samples)
NGS
(48 samples)
Complimentary Assays
GeneScan – Homopolymers
MLPA - Rearrangements
2010 Metropolitan Health Service of the Year
Report
(48 samples)
Overall Somatic Strategy
FFPE
OR
CTC/Plasma
DNA
Sample
CTC/Plasma DNA
CTC Chip/EpCAM
Plasma DNA isolation
FFPE
EGFR Mutations In Plasma DNA
Tumour
Stream
Panels
Complimentary Assays
Targeted Assays
Gene Expression Panels
Micro RNA assays
Translocation Assays
2010 Metropolitan Health Service of the Year
Micro Fluidic
PCR
(48+ samples)
NGS
(48 samples)
Report
(48 samples)
Validation of New Technologies
• NPAAC/NATA
“Requirements for the development and use of in-house in vitro diagnostic
devices (IVDs) (2007 Edition)
– High individual risk/moderate public health risk Class 3
– Section 4 Particular requirements - technical validation
2010 Metropolitan Health Service of the Year
BRCA1/2 Validation Strategy
•Primer design
•All the old design issues of SNP’s & allele drop out etc
•Homopolymer regions with 454 and ion torrent sequencing
•Retrospective comparisons
•Targeted/Blinded
•New Platform comparisons
•Software validation
Cross validation of software packages & comparison to in house
pipelines
2010 Metropolitan Health Service of the Year
Summary
• Technology is enabling extensive variant detection
• Now Need Better Interpretation tools
• Emerging sampling methods likely to improve & extend
tumour profiling
• There are many other emerging technologies &
understanding all their quirks will be difficult but
interesting
2010 Metropolitan Health Service of the Year
Diagnostic
Cliff Meldrum
Victoria Beshay
Michelle McBean
Hazel Phillimore
Stephen
McCaskill
Dong Li Guo
Wasanthi De
Silva
Timmy Chan
Su Ping Chang
Ravi Vedururu
Heidi Williams
Erin McKay
Ayiguli Ha
Danilo Acosta
Kateh Namdarian
Harshana Vashi
Susan Andric
Megan Rehfisch
2010 Metropolitan Health Service of the Year
Nidhi Bhatt
Trent Bosma
Director
Stephen Fox
Research
Alex Dobrovic
Angela Tan
Chelsee Hewitt
Giada Zapparoli
Hongdo Do
Renato Salemi
Elena Takano
Katie Huang
Ida Candiloro
Heather Hondow
Toni-Marie
Rogers
Stephen Wong
Pathologists
Bill Murray
Max Yan
Stephen Lade
Catherine
Mitchell
Sarah Swain
Sid Deb
2010 Metropolitan Health Service of the Year
RainDance
2010 Metropolitan Health Service of the Year
Alternative Sampling for Somatic
Testing
Alternative methods for sampling tumour cells
– Difficult biopsy
– Monitoring
– Non-invasive “liquid biopsy” for cancer
• Circulating Tumour Cells (CTC’s)
• Plasma DNA
• Couple with NGS profiling may lead to new
diagnostics
2010 Metropolitan Health Service of the Year
PDGFRa c.2525A>T:p.Asp842Val
Coverage 6665
2010 Metropolitan Health Service of the Year
c.4065_4068delTCAA 71 fold coverage
2010 Metropolitan Health Service of the Year
EGFR Mutations In Plasma DNA
2010 Metropolitan Health Service of the Year
Automated CTC Isolation
• CTC Chip
• Nano-velcro chip
•Antibodies
•Physical properties of cells
2010 Metropolitan Health Service of the Year
Sample Preparation
• Blood to DNA
• FFPE tissue to DNA
– Isolate tumour DNA (laser micro dissection)
– Fixatives
• Plasma/CTC DNA
2010 Metropolitan Health Service of the Year
How Do We Deliver Current & Future Requirements
• Methods Directed at known mutations
Advantages
Disadvantages
Sensitive
Specific for targeted mutation only
Specific for targeted mutation only
Relatively simple -inexpensive
• Methods Directed at unknown mutations
Advantages
Disadvantages
Specific for unknown mutations
Generally less sensitive
Sequencing more expensive
Pre-Screening methods need
sequencing characterisation
2010 Metropolitan Health Service of the Year
Ion TorrentsSemi conductor Sequencing
2010 Metropolitan Health Service of the Year
Ion TorrentsSemi conductor Sequencing
•
Simplicity
– The Chip is the Machine™
– Natural nucleotides,
no enzymatic cascade,
no fluorescence,
no chemiluminescence,
no optics
•
Speed
– Detection is real time
– Run time - Hours
•
Scalability
– 1 Trillion $ investments in
Silicon Semiconductor
industry
– Any CMOS chip Production
facilities
2010 Metropolitan Health Service of the Year
Ion TorrentsSemi conductor Sequencing
2010 Metropolitan Health Service of the Year
Diagnostic Situation
• Sample Heterogeneity
– Normal & tumour tissue
– Clonal tumour cell populations
• Variable quality
• FFPE with little control over processing conditions
– Fixation conditions
» Induced sequence artefacts
– Age of block
Require high sensitivity of detection
&
independent confirmation of mutations
2010 Metropolitan Health Service of the Year
What to test
~20% will NOT have primary tumour available
94%-100%
concordance
Veronese J Clin Oncol 4217-8 2008
Clin Cancer Res 2004: 4830
2010 Metropolitan Health Service of the Year
What to test
• primary
Resection
Post chemo-radiation
metastasis
Biopsy
2010 Metropolitan Health Service of the Year
Cytology
Biopsy
Sequencing of positives: G12D
2010 Metropolitan Health Service of the Year
12 13
GGT GGC
A
Gly Gly
Asp
KRAS codon 12 & 13 mutations
10%
G12D
G12V
G13D
G12A
G12C
G12R
Others
32%
7%
8%
21%
5%
17%
2010 Metropolitan Health Service of the Year
All KRAS mutations may not be the same?
Patients
with
tumours
harbouring
the
p.Gly13Asp
mutation were associated with
longer progression-free survival
than patients whose tumours
carried other KRAS mutations.
2010 Metropolitan Health Service of the Year
How much mutant KRAS is important?
•
What is the threshold for non-response to anti EGFR treatment ?
• Sensitive techniques can detect 1-5%
2010 Metropolitan Health Service of the Year
Problems with Current Technologies
• Compromise between sensitivity & mutation coverage
• Independent analysis required to distinguish signal from noise
2010 Metropolitan Health Service of the Year
• Parallel Analysis of multiple targets
• Parallel analysis of multiple samples
• High Sensitivity potential (high depth of
coverage) – possibly ~ 2%
2010 Metropolitan Health Service of the Year
•
Redundant coverage of each target to provide replicate PCR’s from
FFPE tissue
•
Tumour stream specific assay’s
– including multiple targets for Melanoma example:
•
•
•
•
•
•
•
•
BRAF
NRAS
PI3K
PTEN
KIT
MEK
ERBB4
Refine amplicon approach to maximise template DNA availability &
efficiency
– Multiplexing
– Fluidigm
– Raindance
2010 Metropolitan Health Service of the Year
2010 Metropolitan Health Service of the Year
Future Tumour Profiling in
Melanoma
• MAPK is a key pathogenic pathway in melanoma
• BRAFV600E inhibitors suppress the MAPK cascade in
melanoma
• BRAF inhibitors can activate the MAPK pathway in
BRAF wild type cells particularly those with activated
NRAS
• Future tumour profiling in melanoma is likely to require
the analysis of multiple targets
2010 Metropolitan Health Service of the Year
MAPK Pathway Inhibitors
2010 Metropolitan Health Service of the Year
Romano et al Lancet 2011
Mutation testing at Peter Mac
2010 Metropolitan Health Service of the Year
Mutation testing at Peter Mac
ASCO
2009
ASCO
2008
2010
10
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Met
Metropolitan
etro
et
ropo
ro
poli
po
lita
li
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ta
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Heal
Health
alth
al
th Service
Ser
Ser
ervi
vice
vi
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off the
the Year
Year
Sequenom
PCR then Single Base Extension
2010 Metropolitan Health Service of the Year
Recent Example
• Turner et al Nature Methods 2009
– Modified protocol overcomes representational and
allelic bias
– Simultaneous amplification of 50,000 exons followed
by accurate sequencing
– Direct sequencing of MIP amplicons with 99%
concordance to HapMap sequence
2010 Metropolitan Health Service of the Year
Modified Protocol
Hybridisation
Polymerisation
Ligation
Multi-template inverse PCR
Append Illumina Adaptors
Next Gen Sequencing
2010 Metropolitan Health Service of the Year
Modified from Turner et al Nature Methods 2009
Programmable Microarray
100 mer
Precursor MIPs
Single strand digestion
70mer MIPs
Nt.AlwI +Nb.BsrDI
Amplification
6% PAGE –urea gel
ExoI + ExoIII
Gap fill + ligase
Hybridisation
2X Inverse PCR
End Repair +T4 Ligase
Concatenation
2010 Metropolitan Health Service of the Year
Library Prep
Sequence
NRAS c.182A>G:p.Glu61Arg
Coverage 3982
2010 Metropolitan Health Service of the Year
Common Methods for Tumour Profiling
Specific
Probes
Capillary “Sanger”
Sequencing
HRM
(High Resolution
Melt)
Pyrosequencing
Low-Med
High
Sensitivity
High
<5%
Low
10-20%
Specificity
Low - Med
High
Med
(follow-up Seq)
Med
High
Cost
Low-Med
High
Low
Low-Med (High
-instrument)
High
Fast
Slow
Fast
Med
Med
Simple
Moderately Simple
Simple
Difficult
Mod-Difficult
Detect Unknown
Mutations
No
Yes
Yes
No
Yes
Requires followup Analysis
No
No
Yes
Yes
No
Speed
Complexity
2010 Metropolitan Health Service of the Year
High
Sequenom