Selecting the right drugs for the right patients
(pitfalls of diagnostic testing)
SStephen
tephen Fox
Fox
Department
D
epar tment of
of Pathology
Pathology
Peter
MacCallum
Cancer
Centre
P
eter M
acCallum C
ancer C
entre
Wednesday, 22 June 2011
Rationale for molecular testing
Selecting the right
drugs for the right
patients
Selecting the right
patients for the right
drugs
•identify treatment to
•The ability to select
those most likely to
benefit
•Avoid toxic harmful
side effects
•enable personalised
medicine
the right patients for
the right drug to hit
target
•Improves outcomes,
quality of life benefits,
etc
Wednesday, 22 June 2011
RCPA Structured reporting of lung cancer
G4.01 Molecular testing of NSCLC should be
considered when it will influence treatment.
G4.02 Molecular pathology testing result(s)
should be recorded if performed.
Wednesday, 22 June 2011
Molecular classification of lung carcinoma
vs
Travis et al (2011) J Thor Oncol 244-285
Wednesday, 22 June 2011
KRAS
EGFR
BRAF
PI3KCA
FGFR4
EML-ALK
HER2
Others
Diagnosis of lung carcinoma
• CK5/6
• MUC-1
• TRIM29
• CEACAM
• SLC7A5
Wednesday, 22 June 2011
Molecular testing in tumour pathology
•
•
•
Diagnosis
Prognosis
Treatment
A+B+C
Omit D
Wednesday, 22 June 2011
Selecting the right drugs for the right patients
in the diagnostic environment
• laboratory framework issues
• lesson on how not to perform a molecular test
• laboratory workflow for somatic testing
• pre-analytical variables
• fixation
• test selection...
• KRAS and EGFR
• start of the journey....
Wednesday, 22 June 2011
Logistics
• incidence 100K in Australia
• lung carcinoma
• prevalence 700K
• patient access
• suite of tests on several platforms
• manpower?
• expertise?
• funding?
Wednesday, 22 June 2011
Mutation testing by sequencing at Peter Mac
Number of tests
3000
Total External
2500
Total Internal
2000
1500
1000
500
0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Year
Wednesday, 22 June 2011
Mutation testing by sequencing at Peter Mac
Number of tests
600
ASCO
2009
BRAF
EGFR
500
KIT
ASCO
2008
KRAS
400
PDGFRa
300
200
100
0
2000
2001
2002
2003
2004
Year
Wednesday, 22 June 2011
2005
2006
2007
2008
2009
Selecting the right drugs for the right
patients in a diagnostic laboratory
•
•
diagnostic laboratory framework
accredited facility
•
RCPA, NATA, NPAAC, TGA
•
•
•
•
quality assurance program
specimen tracking system (LIS)
accredited personnel
•
•
Wednesday, 22 June 2011
maintenance program
pathologists
scientists
A lesson in diagnostic testing
‘0’ (negative)
‘1+’ (negative)
‘2+’ (equivocal)
‘3+’ (positive)
http://www.medicareaustralia.gov.au/provider/patients/late-breast-cancer.shtml
Wednesday, 22 June 2011
Analyses of IHC 3+ FISH amplification rates
No. 3+
IHC
% FISH AMPLIFIED
Sydney data; last 1000 FISH cases
118
69% [outside IHC]
nover
Dolann & SSnover
ath 2005;1
123:766
Am J Clin Path
2005;123:766
113
3
338%
8% [outside
[outside IHC]
Paik et al [NSABP]
JNCI 2002;94:852
104
79% [outside IHC]
94% [in house IHC]
Roche et al [Intergroup]
JNCI 2002;94:855
110
66% [outside IHC]
Tubbs et al
J Clin Oncol 2001;19:2714
48
79%
20-35% false
positives
31
Hecep
pte study]
97% [Aust Heceptest
5-10%
false
negatives
wsett
Dowsett
ett al
270
2
70
94%
9
4% [[outside
outside IHC]
Bilous et aall [HER2000]
ast 2003;12:92
The Breast
2003;199:418
003 19
99 418
J Pathol 20
Wednesday, 22 June 2011
Despite these data.......
• RCPA QAP IHC module 2010 audit of 100 cases
• 123 of the 171 labs (72%) submitted data
• 7667 tests were reported, an average of 62 per lab
• 24% were significantly outside the expected range
of HER2 positive
• further 12% were marginally outside the range of
expected HER2 positive results
36% potentially incorrect!
Wednesday, 22 June 2011
Her-2 Testing algorithm for early breast cancer
Breast Cancer Sample
C/SISH
NON-AMP
Not eligible
under PBS
Wednesday, 22 June 2011
AMPLIFIED
Herceptin
Therapy
History repeating itself...gastric cancer
Basolateral
Wednesday, 22 June 2011
Circumferential
Diagnostic laboratory: variation
•
•
•
Wednesday, 22 June 2011
pre-analytical
amount of material
•
• fixation
• front end preparation
analytical
test
•
post-analytical
interpretation
reporting
•
•
Amount of material for testing
KRAS
EGFR
10%
0.98%
39.50%
MUTATION
30%
WILD TYPE
INSUFFICIENT TUMOUR
TUMOUR
59.52%
KRAS mutant
n=337 (41%)
60%
w type
KRAS wild
No Mutation Identified
n=234
(59%)
Mutation
Identified
No Result
Wednesday, 22 June 2011
Effect of specimen handling on nucleic
acids
• type of fixative
• delayed, short and prolonged fixation leads
to shorter fragment size
• temperature storage significantly alters
fragment size
• type of paraffin
• storage conditions
• PCR artefact
Wednesday, 22 June 2011
Fixation
Limited material
Manuel Salto-Tellez
Wednesday, 22 June 2011
PCR artefact in EGFR sequencing?
Weber et al Br J Cancer 2005
Wednesday, 22 June 2011
Effect of fixation on SISH HER2
1 hour fixation
22 hour
4 hour fixation
52 hour
Adrienne Morey
Wednesday, 22 June 2011
Which method of somatic testing?
“Horses for courses”
Virchows Arch (2008) 453:417–431
Wednesday, 22 June 2011
Choice of assay
Sanger
sequencing
20%
Pyrosequencing
HRM+seq
5%
PCR-based
<1%
All
mutations
All
mutations
Defined
mutations
only
100%
10%
$$
Wednesday, 22 June 2011
$
$$$
Which assay to use?
(total=155; 80 frozen, 65 FFPE, 10 biopsies )
Tib-MolBiol kit
Allele specific PCR
HRM
DNA sequencing
Pyrosequencing
SSCP
t
HRM
Journal of Molecular Diagnostics 2009
Wednesday, 22 June 2011
Comparability of tests
Sensitivity vs specificity
Wednesday, 22 June 2011
What is the gold standard?
Sequencing
(Amgen)
Allele-specific
PCR
(DxS/Histogenex)
Sequencing
(Gentris)
0.90
[n=40]
0.75
[n=32]
0.13
0.94
[n=27]
[n=35]
0.75
-0.04
0.89
[n=32]
[n=27]
[n=35]
0.75
-0.09
0.74
[n=32]
[n=21]
[n=31]
Allele-specific
PCR (DxS/
Histogenex)
Direct Sequencing
(Gentris)
Allele-specific
Allele-specific PCR
hybridization (Invitek) extensions (Genzyme)
Allele-specific
-0.04
-0.09
-0.08
Hybridization
(Invitek)
[n=27]
[n=21]
[n=25]
0.89
0.74
-0.08
[n=35]
[n=31]
[n=25]
Allele-specific PCR
extensions
(Genzyme)
AACR 2008
Wednesday, 22 June 2011
EGFR testing
Drug
Antibodies
trastuzumab/pertuzumab
cetuximab/panitumamab
Small molecule inhibitors
imatinib
gefitinib/erlotinib
lapatinib
sorafenib/sunitinib
Vemurefenib
Critzotinib
Wednesday, 22 June 2011
Target
Tumour
HER2
EGFR
breast, stomach
CRC
KIT
EGFR
HER2
TKs
BRAF
EML-ALK
GIST, melanoma
lung ca
breast ca
HCC, RCC
Melanoma
lung ca
Method does matter
“Method in the madness” Hamlet
Wednesday, 22 June 2011
Antibody detection of mutant protein
Brevet et al J Mol Diagnostics, 2010
Wednesday, 22 June 2011
%$ #' $ " $ (
(%+)),
Wednesday, 22 June 2011
Selection of assays
• CLIA
• accuracy, precision, reportable range, reference
range, analytical sensitivity, and analytical specificity
• ESP
• laboratories have SOPs, report sensitivity, specificity,
method validation, analysis success rate and cost
• NATA/TGA
• register IVD, demonstrate test is fit for purpose,
SOPs etc
Wednesday, 22 June 2011
Is the future already here?
Current
practice
Macrodissection
DNA extraction
(automation)
Sequencing
PCR
2015
NGS
Sequenom
Oncomap
20-30 cases
Wednesday, 22 June 2011
Is the future already here?
Sequenom
#*()
n=10
10% hit rate
Wednesday, 22 June 2011
Continual evolution of Pathology
Macro Micro
IHC
FISH
C/SISH PCR
Arrays, PCR,
sequencing etc
Molecular merges with conventional pathology
Wednesday, 22 June 2011