Supplementary material
Methods used for mutation analysis by the panel labs
Extraction of DNA from formalin-fixed paraffin-embedded tissue
Two 10 m thick sections were cut from each tissue block and either collected in 1.5 ml
reaction tubes or mounted on slides. Paraffin was removed by xylene treatment followed by
washing the slides with ethanol. Depending on the lab, this procedure was repeated up to
three times. After drying tissue lysis was performed by proteinase K digestion. The kits used
for further DNA extraction and purification differed between the panel labs and are listed in
Table S1. All kits were used according to the manufacturers’ instructions.
The amount and integrity of extracted DNA were estimated semi-quantitatively by agarosegelelectrophoresis.
PCR amplification and purification of PCR products prior to cycle sequencing
In order to obtain enough material for cycle sequencing, the relevant exons were amplified by
PCR. The panel labs used different primer-sets which are specified for KIT exon 9 and exon
11 and PDGFRA exon 18 in Table S2. Concerning PCR approaches and cycling profiles the
PCR protocols of all panel labs followed standard conditions. Parameters as annealing
temperatures, MgCl2 concentration and the polymerases used for PCR amplification differed
and are also detailed in Table S2. To rule out contaminations a negative control with distilled
water instead of DNA was included in each PCR run. Prior to cycle sequencing unbound
primers were removed using different methods or kits (Table S3).
Cycle sequencing and precipitation of sequencing products
In all panel labs except Lab B employing an external core facility, bidirectional sequencing
was carried out in-house using the dideoxy chain termination method of Sanger. The
different fluorescence labelled dye-terminators and the devices used for electrophoresis are
listed Table S4. For the bidirectional cycle sequencing reactions the same primers as for the
primary PCR amplification were used in all laboratories except Lab B. The annealing
temperatures and cycle numbers differed from the primary PCR protocol and are also
specified in Table S4. In all panel labs except Lab E who applies CentriSep Spin Columns
(empBiotech, Berlin, Germany), the cycling products were precipitated with standard sodiumacetate/ethanol precipitation. All kits were used according to the manufacturers’ instructions.
1
Proposed standard operation procedure for testing and reporting of common KIT and
PDGFRA mutations in GIST
In general, as PCR amplification is a highly sensitive method and susceptible to carry over
contaminations, the following fundamental rules should be the gold standard in every
diagnostic molecular pathology laboratory. The working area should be divided in pre- and
post-PCR sections, optimally three independent rooms for extracting DNA, preparing the
PCR and performing the post-PCR analysis. Each section should have assigned equipments
and reagents. Plugged pipette tips have to be used throughout and gloves to be changed
between the working sections.
Evaluation of the tissue area to be analyzed
An experienced pathologist has to evaluate H&E and immunohistochemical staining and to
define the most appropriate tissue block to be tested. Furthermore he has to mark on the
H&E stained section the tumor area for DNA extraction to allow manual microdissection for
reducing the amount of non-tumorous tissue.
Sectioning of formalin-fixed, paraffin-embedded tissue blocks
For cutting the paraffin blocks by microtome, some rules have to be observed to avoid
contamination between blocks:
- blocks that are proposed for the same analysis should not be cut consecutively
- the microtome and the working area should be cleaned from paraffin material after each
block
- depending on the type of microtome, the knives should be removed or relocated after each
block
- if the sections are mounted on slides for manual microdissection, the water-bath should be
cleaned regularly with filter-paper and the water should be changed as often as possible
Two different methods can be used to perform manual microdissection: The marking of the
H&E stained slide is transferred to the tissue block before cutting one to six 10 m thick
slides in a reaction tube. This method impairs the quality of tissue blocks for further
investigations. The alternative protocol is to prepare tissue sections first and to perform the
manual microdissection on glass slides after deparaffinization. The slides have to be
incubated for at least 30 min at 60°C before deparaffinization.
The number of slides needed for DNA extraction varies depending on the tissue area marked
by the pathologist.
2
DNA extraction and quantification
Depending on the method chosen for manual microdissection two different protocols for
deparaffinization are possible:
Deparaffinization of tissue sections prior to macrodissection
- incubate slides twice for 10 minutes in xylol and ethanol, respectively, followed by
rehydration in 100%, 96%, 80%, and 70% ethanol for 5 minutes each
- scrape areas marked on the H&E stained section from the deparaffinized slides and
transfer into a reaction tube
- centrifuge for 5 minutes at 13000 rpm; discard supernatant
- resuspend pellet in the appropriate buffer and continue with purification protocol
Deparaffinization of tissue sections cut into an reaction tube
- add 1 ml of xylol to the reaction tube, vortex and incubate at room temperature for 10
minutes
- centrifuge for 5 minutes at 13000 rpm; discard supernatant
- repeat the first two steps
- resuspend pellet in 1 ml ethanol, vortex and incubate at room temperature for 10 minutes
- centrifuge for 5 minutes at 13000 rpm; discard supernatant
- repeat the last two steps
- dry pellet in the open reaction tube for 10 minutes at 37°C, resuspend pellet in suitable
buffer and continue with purification protocol
The deparaffinization is followed by tissue lysis with the appropriate buffers and proteinase K.
After proteolysis, DNA purification is indispensable. A number of DNA purification methods
has been described to date mostly being offered commercially by several companies. The
kits which can be recommended due to the own experience of the panel labs are listed in
Table S1.
For the assessment of DNA amount and the extent of degradation we highly recommend to
use agarose gelelectrophoresis. Because the PCR result depends on the number of
amplifiable fragments it should be avoided to use standard DNA amounts as PCR template.
It is strongly recommended not to analyze samples with bad DNA quality. In such cases,
additional material (e.g. fresh frozen tissue, if available, or another paraffin block) should be
used.
3
PCR for amplification of KIT exon 9 and 11 and PDGFRA exon 18
Each laboratory offering the mutational analysis of GIST should at least perform sequencing
of KIT exon 9 and 11 and PDGFRA exon 18. From the results of our trials, we decided to
recommend two different primer sets for each exon:
KIT exon 9:
A-9 forward
agtgcattcaagcacaatgg
A-9 reverse
gacagagcctaaacatcccc
B-9 forward
cagggcttttgttttcttcc
B-9 reverse
atcatgactgatatggtagacagagc
KIT exon11:
A/B-11 forward
gtgctctaatgactgagac
A/B-11 reverse
tacccaaaaaggtgacatgg
C/D-11 forward
ccagagtgctctaatgactg
C/D-11 reverse
agcccctgtttcatactgac
PDGFRA exon 18:
A-18 forward
catggatcagccagtcttgc
A-18 reverse
tgaaggaggagtagcctgac
B-18 forward
cagctacagatggcttgatc
B-18 reverse
gaaggaggatgagcctgac
A standard PCR protocol for amplification with all recommended primer pairs is as follows:
PCR set-up
template:
1-20 µl (according to gelelectrophoresis)
nucleotide:
100 M
reaction buffer:
5 l
forward primer:
0,4 M
reverse primer:
0,4 M
polymerase enzyme:
1U
distilled water:
up to 50 µl
4
PCR program
first denaturation
94°C
3 min
denaturation
94°C
40 sec
annealing
s. o.
40 sec
extension
72°C
40 sec
final extension
72°C
5 min
40 cycles
For each amplification experiment, positive and negative controls should be carried along. A
sample with water instead of DNA serves as negative control; a positive control may be DNA
extracted from FFPE tissue which was amplified successfully in a previous approach. The
control reactions should be checked by agarose gelelectrophoresis. The number of PCR
cycles should not exceed 40 cycles.
If amplification failed twice, the analysis should be stopped at this point. The analysis can be
re-tried with another paraffin block.
Purification of PCR fragments
Unbound primers and an excess of nucleotides have to be separated from the PCR
fragments prior to cycle sequencing. This can be either done by polyethylenglycol (PEG)
precipitation or with commercially available DNA purification columns. An overview over the
methods used within the panel is given in Table S3. The protocol for the PEG purification is
as follows:
Purification of PCR fragments by polyethylen glycol (PEG) precipitation
- mix equal volumina of PCR product and PEG mix
- vortex and incubate for 10 minutes at room temperature
- centrifuge for 10 minutes at 13.000 rpm
- discard supernatant
- add 100 l 100% ethanol, DON’T VORTEX
- centrifuge for 10 minutes at 13.000 rpm
- discard supernatant
- dry pellet at room temperature
- resuspend pellet in distilled water
5
- PEG-mix:
52,4 g PEG 8000
40 ml
3M NaOAc pH5,2
1,32 ml
1M MgCl2
add distilled water up to 200 ml
Cycle sequencing and precipitation
The cycle sequencing should always be performed as bidirectional sequencing and may be
done with different sequencing kits. Some examples are listed in Table S4. The cycle
sequencing products may be either precipitated or narrowed using spin columns.
Exemplarily, the cycle sequencing protocol for working with the BigDye Terminator Cycle
Sequencing Ready Reaction Kit (Applied Biosystems, Darmstadt, Germany) as well as the
protocol for precipitation of cycling products is given:
Standard PCR protocol for cycle sequencing
Cycle sequencing set-up
template:
1-8 µl (according to gelelectrophoresis)
primer (forward OR reverse):
10 pmol
terminator ready reaction mix:
1 µl
buffer
2 l
distilled water:
ad 20 µl
PCR program for cycle-sequencing
first denaturation
1 min
96°C
denaturation
10 sec
96°C
annealing
5 sec
55/60°C*
extension
4 min
60°C
bis zur Fällung
4°C
cooling down
25 Zyklen
*KIT exon 9 and 11: 55°C; PDGFRA exon 18: 60°C
6
Protocol for precipitation of cycle sequencing products
- mix the following components in a 1,5 ml reaction tube and vortex
80 µl distilled water HPLC grade
10 µl 3 M sodium acetate, pH4,6
250 µl 100% ethanol
product of sequencing reaction
- centrifuge at room temperature for 15 minutes at 13.000 rpm, discard supernatant
- add 250 l ethanol, vortex
- centrifuge at room temperature for 5 minutes at 13.000 rpm, discard supernatant
- dry pellet for 30 minutes
- freeze at -20°C or use directly for electrophoresis
If necessary, the purified cycling products are diluted prior to electrophoresis. Most
conveniently, the separation of products is done by capillary electrophoresis, but depending
on the available laboratory equipment it is also possible to perform polyacrylamide
gelelectrophoresis. Generally these methods should be performed according to the
manufacturers’ instructions.
Description of sequence data
For diagnostic mutation analysis DNA changes should be related to the cDNA sequence.
The first nucleotide refers to the “A” from the ATG initiator methionine codon. The DNA
sequence change is preceded by “c.” standing for coding cDNA sequence. Substitutions at
DNA level are designated by “>” (e.g.: c.1669T>A denotes a missense substitution at
nucleotide 1669 in the coding DNA sequence where a Thymidine was changed to an
Adenine). Deletions are designated by “del” after the deleted amino acid or a deleted region
followed by the deleted nucleotides (e.g.: c.1669_1683del15 denotes a deletion of the 15
nucleotides 1669 – 1683 in the coding DNA sequence). Accordingly, insertions are
designated by “ins”.
Amino acid changes are described using the single letter amino acid code. The amino acid is
preceded by “p.”. For example, a substitution of Valin 559 by Aspartic acid is designated as
“p.V559D”. Deletions are designated by “del”, the “_” symbol should be used to separate the
first from the last affected amino acid (e.g.: “p.W557_E561del” denotes a deletion of the five
amino acids 557 – 561).
7
Table S1 Kits used for DNA extraction
Lab
Kit for DNA extraction
A
BioRobot M48/ QIAmp DNA Mini-Kit (Qiagen, Hilden, Germany)
B
QIAmp DNA Mini-Kit (Qiagen, Hilden, Germany)
C
QIAmp DNA Mini-Kit (Qiagen, Hilden, Germany)
D
Pure Gene Tissue Kit (Qiagen, Hilden, Germany)
E
Nucleospin Tissue Kit (Macherey & Nagel, Düren, Germany)
F
Magna Pure (Roche, Mannheim, Germany)
8
Table S2 Primersets and conditions for PCR amplification of KIT exon 9 and 11 and
PDGFRA exon18
Lab
Exon forward primer (5’-3’)
PCR enzyme
reverse primer (5’-3)
A
9
11
annealing
no. of length
temp. [°C] cycle in bp
agtgcattcaagcacaatgg
HotStar Taq
gacagagcctaaacatcccc
(Qiagen, Hilden,
gtgctctaatgactgagac
Germany)
57
40
146
57
40
232
60
40
256
57
45
266
57
45
232
57
40
213
54
50
284
54
50
215
tacccaaaaaggtgacatgg
18
catggatcagccagtcttgc
tgaaggaggagtagcctgac
B
9
11
18
cagggcttttgttttcttcc
Platinum Taq
atcatgactgatatggtagacagagc
(Invitrogen,
gtgctctaatgactgagac
Karlsruhe,
tacccaaaaaggtgacatgg
Germany)
cagctacagatggcttgatc
gaaggaggatgagcctgac
C
9
11
18
tcctagagtagcagggctt
AmpliTaq Gold
tggtagacagagctaacatcc
(Applied
ccagagtgctctaatgactg
Biosystems,
agcccctgtttcatactgac
Darmstadt,
cagggtgatgctattcagc
Germany)
54
50
238
gccacatcccaagtgttttatg
Fideliti Taq (USB,
55
40
310
gagcctaaacatccccttaaattg
Cleveland, USA)
60
40
215
55
40
265
64
35
270
64
35
199
64
35
214
294
gattaaagtgaagaggatgagc
D
9
11
ccagagtgctctaatgactg
agcccctgtttcatactgac
18
tcttgcaggggtgatgctat
agaagcaacacctgactttagagatta
E
9
cta gag taa gcc agg gct ttt gtt
Hot Goldstar
cct aaa cat ccc ctt aaa ttg gat t (Eurogentec, Köln,
11
aaaggtgatctatttttccctttctc
Germany)
ccaaaaaggtgacatggaaagc
18
cag ggg tga tgc tat atc agc
gtc cag tgt ggg aag tgt gga c
F
9
11
18
tcctagagtaagccagggctt
Fermentas Taq
65-55
10
tggtagacagagcctaaacatcc
(Fermentas,
55
30
gatgattctgacctacaaat
St.Leon-Roth,
65-55
10
aggaagccactggagttcctt
Germany)
55
30
accatggatcagccagtctt
65-55
10
tgaaggaggatgagcctgacc
55
30
342
264
9
Table S3 Purification of PCR products prior to cycle sequencing
Lab
Purification
Purification Kit
Method
A
polyethylenglycol
-
precipitation
B
-
Wizard columns (Promega, Madison, USA)
C
-
Exo Sap-IT (USB, Cleveland, USA)
D
-
High Pure PCR purification kit (Roche, Mannheim, Germany)
E
-
Mini Elute PC purification kit (Qiagen, Hilden, Germany)
F
-
QIAquick PCR purification kit (Qiagen, Hilden, Germany)
10
Table S4 Kits, devices and conditions used for cycle sequencing
Lab
Kit
Capillary/Gel
Primer
Cycling program
Electrophoresis
A
Annealing
Cycles
Big Dye Terminator 1.1 ABI 3130 Genetic
as
Exon 9: 55°C
25
(Applied Biosystems)
above
Exon 11: 55°C
25
Exon 18: 60°C
25
Analyzer (Applied
Biosystems)
B
Sequencing is done in an external lab (SeqLab, Göttingen, Germany)
C
Big Dye Terminator 1.1 ABI 3130 Genetic
as
Exon 9: 54°C
25
(Applied Biosystems)
above
Exon 11: 54°C
25
Exon 18:58°C
25
Exon 9: 50°C
25
Analyzer (Applied
Biosystems)
D
DYEnamic ET
ABI 377 DNA
as
Terminator Cycle
Sequencer (Applied above
Exon 11: 50°C
25
Sequencing Kit (GE
Biosystems)
Exon 18: 50°C
25
Healthcare)
E
Big Dye Terminator 1.1 ABI 3100 Genetic
as
Exon 9: 50°C
25
(Applied Biosystems)
above
Exon 11: 50°C
25
Exon 18: 55°C
25
Analyzer (Applied
Biosystems)
F
Big Dye Terminator 1.1 ABI 310 Genetic
as
Exon 9: 52°C
25
(Applied Biosystems)
above
Exon 11: 52°C
25
Exon 18:52 °C
25
Analyzer (Applied
Biosystems)
11