Kinase activity profiles distinguish papillary thyroid cancers with
AACR 2015# 4322
and without BRAF V600E mutations
1
Hilhorst ,
1
Berg ,
2
Wezel ,
3
Kievits ,
Riet
Adriënne van den
Tom van
Tim
Rik de
Rob
, Hans
1PamGene International BV, 's-Hertogenbosch, Netherlands; 2Leiden University Medical Center , Leiden, Netherlands, 3Vitromics Holding BV, 's-Hertogenbosch, Netherlands
Background
1
Wijn ,
1
,
Ruijtenbeek Wim
2
Corver
2
Morreau .
Results
Serine/threonine kinase activity in malignant thyroid tumors, notably ATC’s and PTC’s, is in many instances
higher than in benign tumors (Fig. 2). The V600E mutation results in many cases in increased activity. To
predict whether a sample is benign or malignant, a model was built with Partial Least Squares Discriminant
Analysis (PLS-DA) with validation by Leave One Out Cross Validation (Fig. 3).
We investigated whether inhibition profiles with the inhibitors dabrafenib, regorafenib or sorafenib could
discriminate BRAF WT and V600E PTC thyroid tumors (Fig. 4).
11
0
V600E
WT
malignant
VE
PTC
Basal kinase activity profiles of PTC’s were not able
to distinguish BRAF V600E and BRAF WT samples
due to biological heterogeneity.
Tumor subgroup
benign
Tumor properties
Muation status
BRAF V600E
0
Not determined
10
Tumor subgroup
ATC BRAF WT
BRAF V600E
FTC BRAF WT
PTC BRAF WT
BRAF V600E
*
one sample: ATC from PTC
Malignant
35
WT
FTC
WT
7
2
10
7*
9*
4x Mean
Mean of row
0.25x Mean
PamChip® Cell-Based Kinase Assay
Kinase activity profiles without inhibitor.
B
C
Tumor lysates
D
Kinase activity
Profiles
C1 C2
C
Inhibition
profile
low
1
0.5
Inhibition profiles with sorafenib
BRAF V600E
PKA signaling
BRAF WT
Cytoskeleton
reorganisation
1
0.5
Ratio inhibited/non inhibited
2
Peptides
1
Ratio inhibited/non inhibited
Ratio inhibited/non inhibited
C2
signal intensity
Inhibition by drug
2 log
Dynamic
incubation
drug
Drug added
ex vivo
BRAF WT
2
C1
high
20-50 µl
D
 Serine/threonine kinase activity profiling appears
to be able to differentiate benign and malignant
thyroid tumors.
 Ex vivo spiking in of kinase inhibitors shows the
ability to differentiate BRAF V600E mutants only
for dabrafenib.
P
array
96 PamChip® arrays
Inhibition profiles with regorafenib
BRAF V600E
Peptides
Lysate,
no drug
Phosphosite
in peptide
E
signal intensity
18
BRAF WT
Ratio inhibited/non inhibited
BRAF V600E
Kinase inhibition profiles of PTC’s with dabrafenib, an
inhibitor directed against BRAF V600E, overcame
the interpatient heterogeneity. 34/144 peptides were
significantly different (p<0.05) between the BRAF
V600E and BRAF WT groups.
The RAF inhibitors sorafenib and regorafenib
showed very similar inhibition profiles that did not
discriminate BRAF V600E from BRAF WT samples.
Conclusions
cAMP signaling
Inhibition profiles with dabrafenib
Peptides
BRAF WT
Peptides
BRAF V600E
B
2 log
A
0.0
A
Fig. 3. Prediction scores for benign/malignant for 57
thyroid tumors. Prediction scores are coloured by
clinical assessment and by tumor subgroup. Green
stars indicate a BRAF mutation.
Fig. 2. Normalised kinase activity profiles of 57 lysates of thyroid
tumors. Signals are expressed with respect to the mean per peptide
(row). ND: BRAF mutation status not determined. VE: BRAF V600E.
Tissue cryosections from fresh frozen thyroid tumors
were lysed. Tumor specimens were analyzed for BRAF
mutations. Serine/threonine kinase (STK) activity
profiles of the lysates were generated on PamChip®
peptide microarrays, comprising peptide sequences
from known human phosphorylation sites. The ex vivo
effect of BRAF inhibitors sorafenib, regorafenib and
dabrafenib on kinase activity profiles of 14 PTC’s was
determined as well. Data were analysed with
Bionavigator software.
A classifier built on the STK kinase activity profiles of
57 thyroid cancer samples was able to classify
malignant and benign tumors with a limited error rate.
Leave One Out Cross Validation classified 17/22 of
benign and 26/35 of malignant samples correctly
(PPV = 0.65, NPV = 0.84). Misclassification was not
associated with tumor subgroup or BRAF mutation
status.
BRAFV600E
malignant
Benign
22
ND
ATC
WT
Clinical assessment
Prediction score
Table 1. Subgroup and
mutation status of 57
samples.
Malignant
benign
Benign
Peptides
Most differentiated non medullary thyroid cancers
(DTC) are curatively treated by surgery and radio-active
iodine ablation therapy. A subset of patients shows
recurrence due to a loss of iodine transport. Two main
subgroups of recurrent DTC are seen: papillary thyroid
cancers (PTC) with somatic BRAF mutations (V600E )
and oncocytic follicular cancer. Recurrent DTC are
clinically treated by multi-kinase inhibitors such as
sorafenib, with a low affinity for BRAF V600E.
Vemurafenib and dabrafenib were specifically designed
against this mutant.
The aim of this study was twofold:
- Can benign and malignant DTC be classified based
on kinase activity profiles?
- Can dabrafenib (or sorafenib or regorafenib) inhibition
profiles differentiate BRAF WT and mutant PTC
tumors?
Discussion
2
Potentially, an industrial prediction platform can be
envisioned for testing of novel drugs in tumor tissue.
Whether individual patient responses against
registered kinase inhibitors can be predicted must be
investigated.
1
0.5
0.1
Fig. 1. Kinase activity profiling of lysates of tumor tissue on serine/threonine
PamChip® peptide microarrays (A) without and with inhibitor (B, C). Signal
intensities for samples were converted to activity profiles (D) and to inhibition
profiles (E) by calculation of the ratio of signals in the presence and absence of
inhibitor. 1 implies no change in activity, 0.1 implies 10 % remaining activity.
Fig. 4. Kinase activity profiles of lysates of 14 PTC thyroid tumors without inhibitor (A) and inhibition
profiles obtained by addition of 10 µM dabrafenib (B), 50 µM regorafenib (C) or 10 µM sorafenib (D) in
the assay. The inhibition profiles show ratios compared to the incubations without inhibitor. Peptides
are presented in the same order in heatmaps A-D.
Fig. 5. cAMP and PKA signalling are differentially
affected by dabrafenib in BRAF V600E and BRAF
WT samples.
Info: rhilhorst@pamgene.com