Transcript 7 mutations - BIORON Diagnostics

MOLECULAR DIAGNOSTICS IN ONCOLOGY
Dr. Sergey Kovalenko
What is the benefit of molecular diagnostics in cancer?
• Early detection of the disease for hereditary cancer
• Optimal therapy selection
EGFR-initiated signal transduction pathways
Anti-EGFR Monoclonal antibodies-based drugs
• Cetuximab (Erbitux) – Merck Serono
• Vectibix (Panitumumab) - Amgen
Activation mutations in KRAS gene
• Mutations can be found in 40% cases
• Anti-EGFR therapy is not effective if KRAS mutations appear
• Package inserts to Cetuximab and Vectibix clearly specify the need to
analyze KRAS mutation before drugs prescriptions
EGFR-initiated signal transduction pathways
Thyrosine kinase domain is the target
Activation mutations in EGFR gene
• Mutations in TK domain of EGFR can be found in 10-40% cases
• TK inhibitors (Iressa, Tarceva) are effective only for patients with
mutations
• Package inserts to Iressa and Tarceva clearly specify the need to
analyze EGFR mutations before drugs prescriptions
PF Survival of lung cancer patients under
Iressa treatment
Cetuximab and Vectibix
• The medications are effective only for CRC patients without
KRAS mutations in tumor cells
• PF and Overall Survival under Cetuximab and Vectibix is
much better in comparison with Best Available Treatment
The need to analyze KRAS mutations
in CRC tumors
Iressa and Tarceva
Iressa and Tarceva
• The medications are effective only for Lung Cancer patients with
EGFR mutations in tumor cells
• PF and OS under Iressa and Tarceva is much better in
comparison with Best Available Treatment
The need to analyze EGFR mutations
in lung cancer tumors
Zelboraf (Vemurafenib)
• Melanoma treatment
• highly effective new drug Vemurafenib
(Roche).
• The drug is effective only for patients
with V600 mutations in BRAF gene
The need to analyze BRAF mutations
in melanoma patients
Problems in mutations analysis in tumor samples
• The sample contains a mixture of malignant and normal
cells – sequencing is problematic
• Just a few cells are suitable for analysis
• The sample is fixed in paraffin
Percentage of malignant cells in 313 lung cancer
samples (our lab results)
160
151
140
120
100
71
80
60
48
43
40
20
0
<10%
10-25%
25-50%
>50%
Principle of allele-specific RealTime PCR analysis
A. Control PCR
TaqMan- probe
Forward
primer
Mutant
DNA
Wild-type
DNA
Mutation
Reverse
primer
Wild-type
B. Allele-specific PCR
Mutation-specific
primer
Mutant
DNA
Mutation
Wild-type
DNA
Wild-type
TaqMan-probe
Reverse
primer
Results of mutations detection by allele-specific
Real-time PCR
RealLine BRAF V600 PCR test
DNA Standards
Skin melanoma
WT
V600E2
1% V600E
V600E
Control PCR
Mutation-specific PCR
Wild-type blocking PCR
Forward
primer
Oligonucleotide
Blocker
Wild-type
DNA
Wild-type
Mutant
DNA
Mutation
Reverse
primer
Enrichment of PCR fragments with mutations
Mutations to be analyzed
Mutations of BRAF-V600 in skin melanoma
Codon
Number of
tumors, (%)*
WT
V600E
V600K
GTG
GAG
AAG
146 (33.7%)
236 (54.5%)
36 (8.3%)
V600R
V600E2
V600D1
AGG
GAA
GAT
7 (1.6%)
7 (1.6%)
0 (0.0%)
V600D2
GAC
1 (0.2%)
Aminoacid
Total
*Sequencing by Sanger and 454 Roche
433 (100.0%)
Anderson et al (2012) Arch Pathol Lab Med. Feb 14.
Bioron Dia vs competitor (R)- BRAF
Parameter
Bioron Diagnostics
Competitor
Mutation
spectrum
V600E/K/D
V600E/K
Sensitivity
V600E/D -1%
V600K – 5%
5%
Price
<15 Euro/test
Up to 300
Euro/test
Sensitivity of commercial tests for mutations of
BRAF V600
Analytical sensitivity, % mutant allele
Cobas 4800
BRAF V600
(Roche)
THxID BRAF
(bioMerieux)
RealLine
BRAF V600
(BIORON)
V600E
5
5
1
V600E2
65
5
1
V600D
10
5
1
V600K
35
5
10
V600R
nd*
nd
nd
Mutation
*nd – not detected
K-Ras Mutations
K-Ras
Mutation
Codon
Sequence
12 13
wt ---GGT GGC---
G12S ---AGT GGC--G12R ---CGT GGC--G12C ---TGT GGC---
codon 12
G12D ---GAT GGC--G12A ---GCT GGC--G12V ---GTT GGC--G13D ---GGT GAC---
codon 13
Bioron Dia vs competitor (R)- KRAS
Parameter
Bioron
Diagnostics
Competitor
Mutation
spectrum
7 mutations (1213 codons)
7 mutations (1213 codons)
Sensitivity
1-5%
5% - 30%
Price
<15 Euro/test
Up to 300
Euro/test
EGFR Mutations
Bioron Dia vs competitor EGFR
Parameter
Bioron Diagnostics
Competitor
Mutation
spectrum
77 mutations mutations
del19 (70 variants),
L858R, L861Q,
G719A/C/S, S768I,
T790M
29 mutations
Sensitivity
1-5%
5% - 30%
Price
<25 Euro/test
Up to 300
Euro/test
Sensitivity of EGFR-7R and Therascreen EGFR kits
(Qiagen)
PCR Mix
Exon
Mutation
Del19
19
Various deletions (b)
1
1-17
L858R
L861Q
21
21
p.L858R
p.L861Q
G719X
18
p.G719A
1
1
5
6
9
33
p.G719C
5
NA (c)
p.G719S
5
NA
p.S768I
p.T790M
1
5
8
18
S768I
T790M
20
20
Ins 20
20
Sensitivity, % (a)
EGFR-7R
Therascreen
p.H773_V774insH
ND (d)
p.D770_N771insG
ND
(a) % of mutant allele; (b) 70 variants of del19 in EGFR-7R and
19 variants of del19 in Therascreen; (c) NA – not available; (d) ND – not detected;
4
20
Products for sequencing analysis
• KRAS 12-13 codons Mut-sequencing enrichment
• BRAF V600 Mut sequencing enrichment
• EGFR del746-750 Mut sequencing enrichment
KRAS G13D sequencing
1%
-blocker
+blocker
5%
20%
50%
CONCLUSIONS
(1) Bioron tests for mutations analysis cover
comprehensive spectrum of mutations
(2) The sensitivity of tests is superior due to the use of
“enrichment technique”
(3) The prices of the tests are strongly competitive
(4) Enrichment technique can be used in sequencing labs
on the basis of BIORON reagents
Thank you for your attention !
BIORON Diagnostics GmbH
Rheinhorststrasse 18 - 67071 Ludwigshafen - Germany
www.bioron.de – [email protected]