Adrenocortical carcinoma liver metastasis
Supplemental data
Supplemental data 1: Genomic DNA isolation, PCR amplification and sequencing, and
Mutation detection
Genomic DNA was extracted from five 10 m slices (“curls”) of formalin-fixed, paraffin
embedded tissue using the Formapure kit (Beckman Coulter Genomics, Beverly, MA), in a 96-well
format, using a modified version of the manufacturers’ method, in a semi-automated fashion.
Briefly, 400 µl of FormaPure Lyse Buffer were added per well manually, followed by 1 h incubation at
65°C and 15 minutes at 95°C, addition of 20 µl of 40 mg/ml proteinase K, shaken at 1400 rpm for 2
minutes and then incubated for 72 h at 65°C. This extended heating incubation allows for reversal of
the DNA-protein crosslinking from formalin fixation1. For frozen specimens, DNA was extracted from
five curls of OCT material using the GeneFind kit (Beckman Coulter Genomics). All subsequent steps
of solid phase reverse immobilization (SPRI) bead binding, isopropanol and ethanol washes, and
elution were automated on a Beckman Coulter Biomek® NX, following the manufacturers’
instructions. DNA was eluted in 65 µl of nuclease-free water for the FFPE specimens and in 100 µl for
the frozen specimens. Double stranded DNA was quantified by Quant-iT™ PicoGreen ® reagent
(Invitrogen, Carlsbad, CA).
The exonic regions of interest, exon 3 of CTNNB1 gene and the whole TP53 gene (NCBI
Human Genome Build 36.1) were broken into amplicons of 350 bp or less, and specific primers were
designed using Primer 3, to cover the exonic regions plus at least 50 bp of intronic sequences on
both sides of intron-exon junctions2. M13 tails were added to facilitate Sanger sequencing. PCR
reactions were carried out in 384 well plates, in a Duncan DT-24 water bath thermal cycler, with 10
ng of template DNA (Repli-G Midi, Qiagen, Valencia, CA) as template, using a touchdown PCR
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Adrenocortical carcinoma liver metastasis
protocol with Kapa2G Fast HotStart Taq (Kapa Biosystems, Cape Town, South Africa). The touchdown
PCR method consisted of: 1 cycle at 95°C for 5 min; 3 cycles at 95°C for 30 sec, 64°C for 15 sec, 72°C
for 30 sec; 3 cycles at 95°C for 30 sec, 62°C for 15 sec, 72°C for 30 sec; 3 cycles at 95°C for 30 sec,
60°C for 15 sec, 72°C for 30 sec; 37 cycles at 95°C for 30 sec, 58°C for 15 sec, 72°C for 30 sec; 1 cycle
at 70°C for 5 min. Templates were purified using AMPure (Beckman Coulter Genomics, Beverly, MA).
The purified PCR reactions were divided and sequenced bidirectionally with M13 forward and
reverse primer and Big Dye Terminator Kit v.3.1 (Applied Biosystems, Foster City, CA), at Beckman
Coulter Genomics.
Dye terminators were removed using the CleanSEQ kit (Beckman Coulter
Genomics), and sequence reactions were run on ABI PRISM 3730xl sequencing apparatus (Applied
Biosystems, Foster City, CA).
Mutations were detected using an automated detection pipeline at the MSKCC
Bioinformatics Core.
Bi-directional reads and mapping tables (to link read names to sample
identifiers, gene names, read direction, and amplicon) were subjected to a QC filter which excludes
reads that have an average phred score of < 10 for bases 100-200. Passing reads were assembled
against the reference sequences for each gene, containing all coding and UTR exons including 5Kb
upstream and downstream of the gene, using command line Consed 16.0 (PMID: 9521923).
Assemblies were passed on to Polyphred 6.02b (PMID: 9207020), which generated a list of putative
candidate mutations, and to Polyscan 3.0 (PMID: 17416743), which generated a second list of
putative mutations. The lists were merged into a combined report, and the putative mutation calls
were normalized to ‘+’ genomic coordinates and annotated using the Genomic Mutation
Consequence Calculator (PMID: 17599934). The resulting list of annotated putative mutations was
loaded into a Postgres database along with select assembly details for each mutation call (assembly
position, coverage, and methods supporting mutation call). To reduce the number of false positives
generated by the mutation detection software packages, only point mutations supported by at least
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one bi-directional read pair and at least one sample mutation called by Polyphred were considered,
and only the putative mutations annotated as having non-synonymous coding effects, occuring
within 11 bp of an exon boundary or with a conservation score >0.699 (http://genome.ucsc.edu/cgibin/hgTrackUi?hgsid=108554407&g=multiz17way) were included in the final candidate list. Indel
mutations called by any method were manually reviewed and included in the candidate list if found
within an exon. All putative mutations were confirmed by a second PCR and sequencing reaction, in
parallel with amplification and sequencing of matched normal tissue DNA. All mutation calls were
manually reviewed.
1.
Gilbert MT, Haselkorn T, Bunce M, et al. The isolation of nucleic acids from fixed, paraffinembedded tissues-which methods are useful when? PLoS One 2007; 2(6):e537.
2.
Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.
In: Krawetz S, Misener S, eds. Bioinformatics Methods and Protocols: Methods in Molecular
Biology. Totowa, NJ: Humana Press; 2000:pp. 365-386.
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Supplemental data 2: Patients, tumors characteristics and details of procedures
n (%) / median (IQ)
Patient characteristics
Age (years)
45 (36-63)
Sex (female)
20 (71%)
Race (white)*
22 (88%)
Primary tumor characteristics
Primary tumor ENSAT stage
I
1 (4%)
II
4 (14%)
III
12 (43%)
IV
11 (39%)
Side of the initial lesion (right)
12 (43%)
Functional tumors
15 (54%)
Synchronous liver metastases
11 (39%)
Details of procedures
Major hepatectomy
17 (61%)
Extrahepatic metastasis at/prior hepatectomy
3 (11%)
Operative time (min)
Blood loss (ml)
Postoperative mortality
240 (210-320)
1000 (300-2400)
0 (0%)
Postoperative morbidity*
55 (12%)
Redo hepatectomy
4 (14%)
Surgical treatment of recurrence
11 (39%)
*based on patients with accurate available data
IQ: interquartile
ENSAT: European Network for the Study of Adrenal Tumors
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Supplemental table 3: Description of CTNNB1 and P53 mutations (n=15 patients)
Supplemental table 3: Description of CTNNB1 and P53 mutations (n=15 patients)
Type of mutation
n
CTNNB1 mutations
Exon 3
c. /p.Ilr35Thr heterozygous
1
Exon 3
c. /p.Thr41Ala heterozygous
2
Exon 3
c. /p.Gln76Glu heterozygous
1
P53 mutations
Exon 5
c. /p.Gln144X heterozygous
1
Exon 5
c. /p.ins GG 151 heterozygous
1
Exon 7
c. /p.del TGC 255 heterozygous
1
Exon 8
c. /p.Pro278Leu homozygous
1
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