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Supplemental Digital Content
Detailed information on p53 immunohistochemistry and KRAS mutational analysis.
p53 immunohistochemistry
From each patient one representative formalin-fixed, paraffin-embedded tissue block was selected
and sections at 3.5 µm were cut.
DAKO EnVision FLEX+, HRP, Mouse, High pH (Link) (DAKO kit K8002, DAKO, Glostrup,
Denmark) was used for the immunohistochemical staining and additional reagents (Mouse LINKER
K2021and DAKO REAL antibody diluents K8005 DAKO, Glostrup, Denmark) were obtained for
the pre-treatment of the slides and the detection of the p53 antibody (clone DO-7, dilution 1:12000,
M7001, DAKO, Glostrup, Denmark).
The stainings were performed on the Autostainer link 48 (AS480, DAKO, Glostrup, Denmark).
Here the slides were submitted to blocking of endogen peroxidase and incubated with the primary
antibody for 30 minutes. A Mouse LINKER was then added for 20 minutes to enhance the signal
and after washing in wash buffer the polymer-hrp complex EnVision Flex+ was added for 30
minutes. The visualization of the immunoreaction was done by adding DAB for 12 minutes. The
signal was then enhanced by 0.5% Cobber sulphate in TBS (pH 7.6) for 10 minutes and after rinse
in deionised water the slides were counterstained in Mayer’s sour haematoxylin. Finally, the slides
were dehydrated and cleared in xylene before mounted in pertex.
The p53 antibody used in the study is well validated, also for routine use. As positive control a slide
containing small pieces of normal thyroid tissue, normal placental tissue, small cell carcinoma of
the lung and serous adenocarcinoma of the ovary was included in each run. As negative control a
similar slide was incubated with diluents but without the primary antibody.
The immunohistochemical reaction was evaluated by an experienced gynecopathologist using a
semiquantitative score combining the intensity of the reaction with the number of positive nuclear
staining of tumor cells. The results of p53 immunohistochemistry were reported categorically as
either negative or positive based on intensity of staining and percentage of positive tumor cells. p53
immunohistochemical staining was considered positive, when more than 50% of the tumor cells
showed moderate or strong nuclear staining. This cut-off is also used in other studies (14;15).
KRAS mutational analysis
KRAS analyses were performed using a DxS TheraScreen KRAS Mutation kit (Qiagen) and/or an inhouse KRAS assay developed during this study. All 512 samples were assessed by the in-house
KRAS assay and 294 samples were in addition determined by the DxS TheraScreen KRAS Mutation
kit. Both assays utilize an Amplification Refractory Mutation System-Quantitatine PCR (ARMSqPCR) methodology and both detect 6 mutations in KRAS codon 12 (Gly12Ala, Gly12Arg,
Gly12Asp, Gly12Cys, Gly12Ser, Gly12Val) and one in codon 13 (Gly13Asp).
The DxS TheraScreen KRAS Mutation kit was used to analyze 294 samples in single reactions on an
ABI7900HT (Applied Biosystems, Foster City, CA) qPCR instrument according to the
manufacturer's instructions
Primers and probes for the in-house KRAS assay were developed with the use of the OLIGO 7
software (Molecular Biology Insights Inc, Cascade, CO). A number of refractory primers were
tested on DNA from KRAS mutated colorectal cancer samples as well as normal donor DNA. In
order to increase the specificity of the qPCR reactions a wild-type blocking oligo was added in
some reactions. The blocking oligos were oligos modified by including HyNA nucleotides
(Pentabase Aps, Soendersoe, Denmark), which increased the melting temperature and blocked
extension. As positive control and reference an in-house cyclophilin (gCYC) qPCR was included.
The specificities of the in house KRAS mutation assays varied between 0.1% and better than 0.01%.
The final primer mixtures are shown in Table 1. All qPCR reactions were performed in a volume of
25 µl in duplicates on an ABI Prism7900HT using ABI Universal Mastermix with UNG. In all
assay series a mixture of patient sample DNA representing all 7 KRAS mutations was included as
positive controls as well as water controls (negative control) and wild-type donor DNA controls.
The qPCR reaction conditions were: 2 min at 50C and 10 min at 95C, followed by 50 cycles of 15
s at 95C and 60 s at 60C.
The in-house KRAS mutation assay was validated by comparing the test results with the results of
the 294 samples analyzed by the DxS TheraScreen kit .Of these samples 19 were not evaluable due
to poor DNA quality. Of the remaining 275 samples there was a 100% concordance between the
TheraScreen kit and the in-house KRAS assay. The remaining samples were analyzed using the inhouse KRAS assay.