Supplementary Methods
Immunohistochemistry
Immunohistochemistry was performed using a panel of antibodies specified in supplementary
table 1, according to the previously published techniques1. Detection was carried out on either
a Ventana Benchmark automated immunostainer using UltraView detection, or a Dako
Autostainer Plus using either Envision+ or Flex detection systems. Detailed protocols are
available on request.
Fluorescence-in-situ-hybridization
Interphase fluorescence in-situ hybridization (FISH) studies were performed on 5 microns
Formalin-Fixed-Paraffin-Embedded (FFPE) tumor sections using standard protocols2. For all
cases, FISH was performed using the D7S522 (7q31)/CEP7 probe (Abbot Molecular, Illinois,
U.S.A.). Some cases were also tested for loss of chromosome 7p copy number using
7p11.2/7q31 probe mix: (7p11.2, BAC clones RP11-56P1, RP11-371M7 in Spectrum Green,
Empire Genomics)/ (7q31, Spectrum Orange, Empire Genomics). Hybridizations were
performed according to standard protocols using a ThermoBrite denaturation/hybridization
system (Abbot Molecular, Illinois, U.S.A.). Image acquisition and analysis were performed on
the BioView Duet-3 fluorescent scanning system using 63X-oil objective and
DAPI/FITC/Rhodamine single-band pass filters (Semrock, Rochester, NY). At least 100
interphase cells were analyzed for each patient.
T-cell receptor gamma-chain gene rearrangement studies
DNA from each case was extracted using the Qiagen QIAamp DNA FFPE Tissue Kit on a QIAcube
robotic system, according to the instructions of the manufacturer. PCR was performed using
consensus primers to the T-cell receptor variable and joining regions. The primers used
interrogate primers all known Vγ family members and the Jg1/2, JP1/2, and JP joining
segments. The products were analyzed by either acrylamide gel electrophoresis or by capillary
electrophoresis on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA).
Mutational Analysis of STAT3 and STAT5B
Targeted analysis for STAT3 mutations located between codons 614 through 661 and for
STAT5B mutation located between codons 642 through 665 were performed using
pyrosequencing on a PyroMark Q24 instrument (Qiagen, Alameda, CA). The pyrosequencing
assay was designed to target all hotspot mutations identified in recent publications3-6 using
PyroMark Assay Design v2.0 (Qiagen). PCR amplification primers and sequencing primers are as
follows:
STAT3
codons
640-665
assay
(118
bp
amplicon),
STAT3-FW
5'-
TAAGACCCAGATCCAGTCCG, STAT3-REV 5'-biotin-CCAGTGGAGACACCAGGATATTG, STAT3-S1 5'CCAGTCCGTGGAACC, STAT3-S2 5'-TGAAATCATCATGGGC; STAT3 codons 614-618 assay (102 bp
amplicon),
STAT3-FW2
5'-CGGGCCATCTTGAGCACTAA,
STAT3-REV2
5'-biotin-
GATGTCCTTCTCCACCCAAGTG, STAT3-S3 5'-GCTAAGATTCAGTGAAAGC; STAT5B codons 642-665
assay (122 bp amplicon), STAT5b-FW 5'-TTGATCTAGAGGAAAGAATGTTTTGG, STAT5B-REV5'biotin CCGATCAGGAAACACGTAGATAA, STAT5b-S 5'CTAGAGGAAAGAATGTTTTG. PCR reactions
were conducted in a total volume of 25 ul containing genomic DNA template, 200 nM of each
forward and reverse primers, and 12.5 ul 2x HotStarTaq Master Mix (Qiagen) with conditions at
95ºC 15 min, 40x (95ºC 20 sec, 60ºC 30 sec, 72ºC 20 sec), 72ºC 10 min, 15ºC hold. For the
pyrosequencing reactions, 10 ul of PCR product was immobilized on streptavidin-coated
Sepharose beads (GE Healthcare) and processed according to the manufacturer’s instructions.
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3.
Jerez A, Clemente MJ, Makishima H, Koskela H, Leblanc F, Peng Ng K, et al. STAT3
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