Supplemental Methods
Tissue specimens and matched normal blood were obtained at the time of surgery,
snap frozen and stored at -70 ºC. DNAs were prepared using standard procedures. For all
tumors, H & E stained sections were evaluated to identify tissues will neoplastic
cellularity of >66% that were then used for DNA preparation. Normal (non-neoplastic
DNA) was prepared from peripheral blood leukocytes for the majority of cases, or when
blood was not available, from normal uninvolved myometrium.
Microsatellite analysis was performed as previously described (Kowalski et al.
1997) using five National Cancer Institute consensus microsatellite markers (BAT25,
BAT26, D2S123, D5S346 and D17S250) (Boland et al. 1998). Tumors were designated
as having high-level MSI (MSI+) if novel PCR bands were identified in at least two of
the five consensus panel markers. Tumors were considered microsatellite stable if there
was no evidence of MSI in any marker.
PCR primer sequences were M13 tailed and sequences are available by request
from the author. PCR reactions were purified after thermocycling using solid phase
reversible immobilization (SPRI)-based technology (AMPure®; Agencourt Biosciences
Corp., Beverly, MA), resulting in the removal of unincorporated dNTPs, primers, and
salts. PCR products were eluted in 30l distilled H2O. Both strands of each PCR
product were sequenced as follows: Sequencing reactions were performed using 3l
(approximately 25ng) of purified PCR product in a 6l reaction containing 0.33l
BigDye Terminator v3.1 premix, 3.2 pmol of either M13 forward
(TGTAAAACGACGGCCAGT) or M13 reverse (CAGGAAACAGCTATGACC)
primer, and 1.03l 5X BigDye sequencing buffer. Cycle-sequencing was performed for
35 cycles following the manufacturers recommendations on GeneAmp 9700 PCR
machines (Applied Biosystems, Foster City, CA). Sequencing reactions were purified
using CleanSEQ® (Agencourt Biosciences Corp., Beverly, MA) to remove
unincorporated dye-terminators, and analyzed on 3730xl DNA analyzers (Applied
Biosystems, Foster City, CA). Sequences were analyzed using Sequencher (Gene Codes).
All mutations were absent in the corresponding normal DNA demonstrating they arose
somatically and all mutations were confirmed in a second independent PCR.
It should be noted that the A315T reported in ESS-1 derived from an endometrial
stromal sarcoma occurred in the mesenchymally expressed isoform (FGFR2c) and all
carcinosarcomas (tumors with both malignant epithelial and stromal elements) were also
screened for mutations in exon 9 (NM_000141).
1. Kowalski LD, Mutch DG, Herzog TJ, et al: Mutational analysis of MLH1 and MSH2
in 25 prospectively-acquired RER+ endometrial cancers. Genes Chromosomes
Cancer 18:219-227, 1997
2. Boland CR, Thibodeau SN, Hamilton SR, et al: A National Cancer Institute
Workshop on Microsatellite Instability for cancer detection and familial
predisposition: development of international criteria for the determination of
microsatellite instability in colorectal cancer. Cancer Res 58:5248-5257, 1998