br onset of symptoms prompting

133 onset of symptoms prompting clinical intervention suggests the need for a screening test for
134 earlier detection of endometrial cancer in asymptomatic patients. The increasing incidence and
135 mortality of endometrial cancer over the past 20 years further suggests the need for this approach
136 particularly among our underserved populations at higher risk for poor cancer related outcome.
137 We conducted a nested case-control study using prediagnostic serum from
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138 postmenopausal women enrolled in the screening arm of the Prostate, Lung, Colorectal and
139 Ovarian (PLCO) Cancer Screening Trial. The primary objective was to identify serum proteins
140 able to distinguish endometrial cancer cases from controls ≤ 2 years of diagnosis, and to
141 determine whether there was a temporal relationship in abundance of these LNicotine among cases
142 as time of cancer diagnosis approached.
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161 Materials and Methods
162 We performed a nested case-control study within the screening arm of the PLCO Cancer
164 recruited from 10 screening centers across the United States. Participants were randomized to
165 either a screening or non-screening arm; participants randomized to the screening arm provided
166 serum samples upon enrollment and at five subsequent medical examinations. Incident cancers
167 were determined by participant self-report and confirmed by review of the participant’s medical
168 record and pathology report. All participants provided written informed consent and the PLCO
169 Cancer Screening Trial was approved by the institutional review board of the National Cancer
171 We evaluated postmenopausal women randomized to the screening arm of the PLCO
172 Cancer Screening Trial that met the following inclusion criteria: intact uterus, available serum
173 sample, no previous cancer diagnosis, and completion of intake questionnaire. Participants also
174 had to provide written consent allowing biochemical study of their serum. Endometrial cancer
175 cases were defined as having a primary diagnosis of an invasive epithelial tumor of the uterus
176 between the initial screening visit and January 1, 2010. Participants with a preexisting
177 malignancy at enrollment or a mesenchymal uterine tumor were excluded.
178 Controls were matched 1:1 based on age, race, study site, year of blood draw, and year of
179 randomization. Controls were restricted to women with no history of a hysterectomy and were
180 required to be alive at time of diagnosis of their matched case.
181 Serum samples from 552 participants were provided by the PLCO central bank and were
182 stored at -80˚C until use. No more than two freeze- thaw cycles were allowed for each sample.
183 Samples were blinded and randomized for processing and analysis. Serum samples were thawed
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184 on ice and highly abundant proteins were immunodepleted using Multiple Affinity Removal
185 Human-14 Spin Cartridge (Agilent Technologies) according to manufacturer’s instructions. The
186 unbound protein fraction was eluted and buffer-exchanged with 100 µL x 6 of 100 mM
187 triethylammonium bicarbonate (TEAB) in a Microcon-10 (Millipore) spin-column filter. A total
189 were then transferred to 0.5 mL MicroTubes (Pressure Biosciences) and incubated at 99 ⁰C for
190 30 min, after which they were cooled to ambient temperature and 1 µL of SMART Digest
191 Trypsin (Thermo Scientific) was added to each. The MicroTubes were capped with MicroPestles
192 (Pressure Biosciences) and digestion was performed in a Barocycler 2320EXT (Pressure
193 BioSciences) by cycling between 45 kpsi for 50 s and atmospheric pressure for 10 s for 60 cycles
194 at 50 ⁰C. Peptide digests were dried by vacuum centrifugation, resuspended in 100 mM TEAB,
195 and quantified using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific).
196 Equivalent amounts of peptide were labeled with tandem-mass tag (TMT) isobaric labels 197 (TMT11plex™ Isobaric Label Reagent Set, ThermoFishe r Scientific) as per manufacturer’s
198 recommendations. TMT-labeled plexes were fractionated using high pH reversed-phase liquid
199 chromatography (1260 Infinity II, Agilent) into 96 fractions through development of a linear
200 gradient of acetonitrile (0.69% min). The 96 fractions were concatenated into 12 in a serpentine
202 TMT-labeled peptide digest fractions were analyzed by nanoflow liquid chromatography-