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We developed a monoclonal mouse antibody capable of detecting mutant IDH1 of the R132H type on paraffin sections. This mutation is frequent in specific subsets of gliomas and its detection by immunohistochemistry proved to be a valuable an inexpensive diagnostic tool now widely employed in neuropathological diagnostics. The mutational status of IDH1 has also a strong effect on overall survival in astrocytoma and glioblastoma patients and will be essential for future classification and grading of malignant gliomas with subsequent implications for therapy. Encouraged by this we continued by generating a monoclonal mouse antibody capable of detecting mutant BRAF V600E (VE1) on paraffin sections. We tested this antibody for its potential of rapid BRAFV600E testing in a diagnostic setting. The specificity and sensitivity of VE1 proved very high in different tumor types. With the availability of small molecule drugs targeting the BRAFV600E mutant protein, wide spread screening with VE1 might be a powerful tool to identify patients suffering from tumors with low BRAF V600E incidences who might be eligible for such targeted therapy. In addition the detection of this mutation has diagnostic potential for various special tumor entities such as Lynch syndrome, sessile serrated adenoma, hairy cell leukemia or Langerhans cell histiocytosis and, among brain tumors, ganglioglioma and pleomorphic xanthoastrocytom.
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Development and application of mutation
specific antibodies: from IDH1-R132H to BRAF-V600E
Andreas von Deimling
Under a licensing agreement between DIANOVA GmbH, Hamburg,
Germany and the German Cancer Research Center,
and
under a licensing agreement between Roche, Tucson, USA and the
German Cancer Research Center,
Dr. von Deimling is entitled to a share of royalties received by the
German Cancer Research Center on sales of H09 and VE1 antibody.
The terms of this arrangement are being managed by the German
Cancer Research Center in accordance with its conflict of interest
policies.
Disclosures
Early molecular pathologists
isocitrate dehydrogenases
IDH1 cytosolic NADP+ specific
IDH2 mitochondrial NADP+ specific
IDH3A / IDH3B / IDH3G mitochondrial NAD+ specific
Gene Nucleotide change
Amino acid change
N (%)
IDH1 (716) G395A R132H 664 (92.7%)
C394T R132C 29 (4.2%)
C394A R132S 11 (1.5%)
C394G R132G 10 (1.4%)
G395T R132L 2 (0.2%)
IDH2 (31) G515A R172K 20 (64.5%)
G515T R172M 6 (19.3%)
A514T R172W 5 (16.2%)
IDH1 IDH2 1010 diffuse gliomas
IDH1-R132H mutation
Enzyme activity of Isocitrat-dehydrogenase (IDH1)
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
0.450
0.500
0 5 10 15 20 25 30 35 40 45
untr.
wt
H
E
Q
S
C
V
L
G
GFP
C OH H
NADP+ NADPH
H
CO2
COO-
CH2
COO-
C COO- H
C O
COO-
CH2
COO-
C COO- H
COO-
CH2
COO-
C H H
C O
isocitrate oxalosuccinate a-ketoglutarate (2-oxoglutarate)
wt IDH
2-hydroxyglutarate
COO-
CH2
COO-
C OH
CH2
H
a-ketoglutarate (2-oxoglutarate)
NADPH NADP+ COO-
CH2
COO-
C O
CH2
mut
0
10
20
30
40
50
60
70
80
90
100
IDH1 wt IDH1R132H
IDH1R132C
IDH1R132G
IDH1R132S
IDH1R132L
IDH2 wt IDH2R172K
frequ
ency
1
10
100
1000
10000
nmol
2-H
G/m
g to
tal p
rote
in
% of total IDH mutations in glioma2-HG concentration in cell pellet
IDH1 mutants
2-HG concentrations inversely correlate with the frequency of IDH mutation occurrence
David
mIDH1R132H H09
rIDH1
55 kD
36 kD
72 kD
1 3 2 4 5 3 2 4 5 1
Mouse monoclonal H09 Antibody (IDH1R132H)
Pilocytic astrocytoma
Oligodendroglioma III Clear cell ependymoma Central neurocytoma
DNT Oligodendroglioma II
GBM – A III cohort and prognostic parameters
Months 0 12 24 36 48 60 72 84 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
p<0.0001 Months 0 12 24 36 48 60 72 84 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
p<0.0001
A III (n=145) GBM (n=237) IDH wt (n=278)
IDH mut (n=104)
Months 0 12 24 36 48 60 72 84 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
p<0.0001 Months 0 12 24 36 48 60 72 84 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
p<0.0001
MGMT not meth (n=170) MGMT meth (n=168) Age <= 60 (n=225)
Age > 60 (n=157)
Histology IDH1 status
Age MGMT status
382 patients NOA04 and GGN overall survival
GBM – A III and IDH1 status
Months
0 12 24 36 48 60 72 84 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
p<0.0001
A III & IDH mut
(n=58) GBM & IDH mut (n=17) A III & IDH wt
GBM & IDH wt (n=220)
(n=87)
overall survival
GBM – A III and IDH1 status
Age > 60
Months
0 12 24 36 48 60 72 84
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
p=0.457
(n=22) A III & IDH wt
GBM & IDH wt (n=124)
overall survival
0 12 24 36 48 60 72 84 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
p<0.0001
A III & IDH mut
(n=58) GBM & IDH mut (n=17) A III & IDH wt
GBM & IDH wt (n=220)
(n=87)
Months
GBM – A III and IDH1 status
progression free survival
Structure of IDH1
NADP+
Isocitrate
Homodimer(E.coli) Mesecar et al.(1997)
Mesecar et al. (1997)
http://fightcolorectalcancer.org
Pilocytic astrocytoma
Green = BRAF Red = KIAA1549
BRAF/KIAA1549 - duplication and fusion in pilocytic astrocytoma
BRAF
Michaloglou et al. 2008
T599 and S602 phosphorylation activate BRAF BRAF V600E mutation mimicks phosphorylation and leads to constitutive activity
BRAF V600 mutations in human tumors
In up to 5-7% of all human tumors ~95% BRAF V600E Carcinomas
• thyroid cancer (~30-50%) (papillary and anaplastic) • colorectal cancer (~5-10%) • ovarian carcinoma (~30% in serous forms)
Skin tumors
• malignant melanoma (~60%) Hematologic tumors
• Hairy cell leukemia (>90%) • Langerhans cell histiocytosis (~50%)
Primary brain tumors
• pleomorphic xanthoastrocytoma (~60%) • ganglioglioma (~20%) • pilocytic astrocytoma (~5-10%) (mostly BRAF-KIAA1549 fusion)
BRAF V600E inhibitors
Selective small - molecule BRAFV600E inhibitors
Vemurafenib (Zelboraf™) Flaherty NEJM 2010, Chapman NEJM 2011 FDA approval for advanced melanoma (08/2011)
GSK2118436 Long, ESMO 2010
BRAF V600E antibody (VE1)
Antibody confirmation by sequencing of 85 tumors
Match in 97%
Papillary Thyroid Carcinoma
Koperek et al.
N
VE1 pos
VE1 neg
total 144 76 (53%) 68 (47%)
Papillary micro-adenoma <1cm 72 42 30
Papillary macro-adenoma >1cm 72 34 38
BRAF V600E
BRAF wt
verified by sequencing
39 VE1 pos
39 0
Bullock et al.
N VE1 pos VE1 neg
total 96 68 (71%) 28 (29%)
BRAF V600E BRAF wt
verfied by sequencing
96 56 (61%) 37 (39%)
Koperek et al. Am J. Surg Pathol 2012 Bullock et al. Endocr Relat Cancer 2012
Papillary Thyroid Carcinoma
Koperek et al. Am J. Surg Pathol 2012 Bullock et al. Endocr Relat Cancer 2012
Papillary Thyroid Carcinoma
Ovarian Carcinoma Bösmüller et al.
N VE1 pos VE1 neg
total 179 23 (13%) 156 (87%)
Serous ovarian borderline tumor 31 22 (71%) 9 (29%)
Serous ovarian carcinoma G1 7 1 6
Serous ovarian carcinoma G3 141 0 141 (100%)
BRAF V600E
BRAF wt
verified by sequencing
23 VE1 pos
21 2 (low tumor
fraction)
Preusser et al.
N VE1 pos VE1 neg
total 144 7 (5%) 137 (95%)
Serous ovarian borderline tumor
31 4 27
Serous ovarian carcinoma G1 15 3 12
Serous ovarian carcinoma G3 44 0 44
BRAF V600E BRAF wt
verfied by sequencing
6 VE1 pos
6 0
Bösmüller et al. Hum Pathol 2013 Preusser et al. Appl Immunhistochem Mol Morphol 2013
Ovarian Carcinoma
Bösmüller et al. Hum Pathol 2013 Preusser et al. Appl Immunhistochem Mol Morphol 2013
Borderline tumor grade 3
Serous ovarian carcinoma grade 1
Serous ovarian carcinoma grade 1
Borderline tumor grade 3
Histiocytoses Sahm et al.
N
VE1 pos
VE1 neg
Langerhans Cell Histiocytosis
89 34 (38%) 55 (62%)
BRAF V600E
BRAF wt
verified by sequencing
46 select
ed
Concordance of IHC and
sequencing was 96%
Haroche et al.
N BRAF V600E BRAF wt
total 127 7 (5%) 137 (95%) Langerhans Cell Histiocytosis
29 11 (38%) 18 (62)
Erdheim Chester disease 24 13 (54%) 11 (46%)
Juvenile Xanthogranuloma 12 0 23
Rosai Dorfman disease 23 0 23
VE1 pos
VE1 neg
verfied by sequencing
7 select
ed
Concordance of IHC and sequencing was 100%
Sahm et al. Blood 2012 Haroche et al. Blood 2013
Histiocytoses
Sahm et al. Blood 2012 Haroche et al. Blood 2013
Langerhans Cell Histiocytosis VE1
Erdheim Chester Disease VE1
Erdheim Chester Disease VE1
Langerhans Cell Histiocytosis S100
BRAF V600E
S100
CD1a
CD207
Eosinophilic Granuloma
Hairy Cell Leukemia
Andrulis et al. N
VE1 pos
VE1 neg
total 52 76 (53%) 68 (47%)
Hairy cell leukemia 32 32 (100%) 0
Spleenic lymphoma/leukemia unclassifiable 6 0 6
Splenic marginal zone lymphoma 8 0 8
BRAF V600E
BRAF wt
verified by sequencing
30 VE1 pos 28 2
Andrulis et al. Am J. Surg Pathol 2012
UltraView OptiView
Hairy Cell Leukemia
VE1 (BRAF V600E)
Gastro-esophageal cancer
Preusser et al.
N VE1 pos
total 534 0
esophageal squamous cell carcinoma 119 0
esophageal adenocarcinoma 72 0
GEJ / cardia 63 0
gastric cancer (corpus / antrum) 199 0
gastric GIST 81 0
Preusser et al. Appl Immunohistochem 2013
Lung adenocarcinoma Ilie et al.
N VE1 pos
VE1 neg
Total
450 (wt for
EGFR, KRAS, Pi3Ka, Her 2, ALK-EML4)
19 (4%) 231 (96%)
acinar 285 5 (*) 280
papillary 142 5 (*) 137
micropapillary 10 9 1
other 13 0 13
BRAF V600E
BRAF wt
verified by sequencing
450 21 429
Ilie et al. Ann Oncol 2013
lung acinar adeno-carcinoma
VE1 staining absent
Melanoma
Long et al.
N VE1 pos
VE1 neg
melanoma 97 38 59
VE1 missed 11 other BRAF mutations
5 discrepant cases between VE1 and V600E sequencing data in 3 cases initial testing was false positive in 1 case initial testing was false negative in 1 case VE1 was false negative
BRAF V600E
BRAF wt
verified by sequencing
97 38 (*+11) 48
Busam et al.
N VE1 pos
VE1 neg
total 44 7 (5%) 137 (95%) metastatic melanomas with BRAF V600E (DNA sequencing)
22 22 0
metastatic melanomas without BRAF V600E (DNA sequencing)
22 0 22
Inhomogenous in some staining raises the question of response to targeting drugs
Long et al. Am J Surg Pathol 2013 Preusser et al. Appl Immunhistochem Mol Morphol 2013
Melanoma at primary site
Melanoma metastasis to lymph node (repeatedly sequenced BRAF wt)
Melanoma
Wilmott et al.
VE1 intensity
melanomas with BRAF V600E (DNA sequencing)
Treated with BRAF inhibitors
36 strong
20 moderate
2 weak
Inhomogenous in some staining raises the question of response to targeting drugs
Wilmott et al. B J Cancer 2013
Serrated lesions and polyps of the colon
Mesteri et al.
N
VE1 pos
total 115 79
Sessile serrated adenoma 38 38 (100%)
Traditional serrated adenoma 16 15 (93%)
Microvesicular hyperplastic polyp
44 26 (59%)
Goblet-cell rich hyperplastic polyp 17 0
Mesteri et al. submitted Sessile serrated adenoma
Microvesicular hyperplastic polyp
Colon carcinoma
Lynch Syndrome in colorectal cancer
Toon et al. in press Capper et al. submitted
Recognition of Lynch syndrome requires detection of high level microsatellite instability (MSI-H).
Most tumors with MSH-I are sporadic. Therefore germline mutation analysis is required BRAFV600E in MSI CRC excludes Lynch Syndrome
Pleomorphic xanthoastrocytoma
Pleomorphic xanthoastrocytoma
VE1 (BRAF V600E)
Pan- BRAF
glioblastoma with BRAF V600E mutation
VE1
GBM BRAF wt
GBM BRAF V600E
VE1
Ganglioglioma
VE1 (BRAF V600E)
Ganglioglioma
BRAF V600E in 874 brain metastases
Melanoma 42/76 (55.3%)
Ovarian cancer 1/15 (6.7%)
Lung cancer 1/355 (0.3%)
Thyroid cancer 2/6
Colorectal cancer 4/72 (5.5%)
Chorioncarcinoma 1/2
BRAF V600E
melanoma
Lynch syndrome
papillary thyroid
carcinoma
Colon carcinoma
Serrated adenoma
hairy cell leukemia
Langerhans cell
histiocytosis
Erdheim Chester disease
Ovarian carcinoma
PXA
GG
Glio-blastoma
Antibodies targeting IDH1 and BRAF Mutations
- tumor specific - diagnostic relevance
- stratify tumor patients for targeted therapy
Jörg Balss David Capper Andreas von Deimling Antje Habel Christian Hartmann Christian Kölsche Andrey Korshunov Jochen Meyer Stefan Pusch Felix Sahm Genevieve Schindler
Peter Birner Matthias Preusser Ildiko Mesteri Mindaugas Andrulis Julien Haroche Hans Bösmüller Oskar Koperek Georgina Long Christopher Toon Marcel Ilie Klaus Busam Jams Wilmott Alexander Sorokhod Boris Bastian
Hanswalter Zentgraf
Group and Cooperation Partners
David Capper
For the German Glioma Net: Michael Weller
Thank you !
