Use of Cervical Mucus to Screen for Gynecological Malignancies

A Pilot Study

Ihab Lamzabi; Lela Buckingham; Mezgebe Gebrekiristos; Richa Jain; Paolo Gattuso; Vijaya Reddy; Alfred Guirguis; Summer Dewdney; Jacob Rotmensch; Pincas Bitterman

Disclosures

Mod Pathol. 2013;26(11):1508-1513. 

In This Article

Materials and Methods

Case Selection

With approval of the Rush Institutional Review Board, hysterectomy specimens received fresh for frozen section with suspected gynecological malignancies and a group of presumed benign cases received for routine surgical pathology were randomly selected from April 2012 to July 2012. The uteri from which samples were taken had to be intact with no grossly visible cervical or serosal tumor involvement to minimize direct contamination of the mucus.

Histologic Examination

Histologic sections stained with hematoxylin and eosin were examined by board certified pathologists. The pathologists were blinded and did not know which cases were included in the study.

Immunohistochemistry

Immunostain for p53 protein was performed using p53 monoclonal antibody (DAKO (M7001) clone DO-7) run at 1:1000 dilution with Leica's pretreatment of ER2 for 20 min, on Leica Bond III instrument. All fallopian tube sections and a sample from the tumors or precancerous lesions that were prospectively identified on histologic examination were stained. Only strong (3+) positivity that was continuous and diffuse was considered positive by the authors (PB and IL).

Mucus Sample Collection

Mucus was collected by aspiration using a 10 cc syringe and then labeled with the patients identification and pathology number, snap frozen, and stored at −86°C.

DNA Extraction

DNA was extracted from 100–200 μl of mucus using the 5 PRIME cell/tissue extraction buffers (5 PRIME, Inc., Gaithersburg, MD). The mucus was rinsed from the needle-less syringe tip with 600 μl lysis buffer in a 35 × 10 mm Petri Dish. The mixture was transferred to a 2 ml microcentrifuge tube, using a transfer pipet, and incubated at 55°C for 4 h or until the solution cleared. Samples were placed on ice and cooled before adding 200 μl protein precipitation buffer. After vigorous vortexing for 20 s, the mixture was centrifuged at 12 000 g for 5 min. The supernatant was added to 500 μl isopropanol in a 1.5 ml microcentrifuge tube and mixed by inversion at least 50 times. The precipitated DNA was pelleted by centrifugation at 15 000 g for 10 min. The resulting pelleted DNA was rinsed one time by adding 500 μl 70% ethanol and centrifuging at 15 000 g for 5 min. The tubes were inverted on absorbent paper for 10 min and then resuspended in 100–250 μl rehydration buffer, depending on the size of the pellet.

TP53 Exons 5–9 Amplification

TP53 exons 5–9 were amplified for each specimen using HotStarTaq (Qiagen, Valencia, CA). Each 25 μl reaction mix contained 1X HotStarTaq buffer brought to 2 mM MgCl2, 0.2 mM dNTP mix, 0.1 μM each forward and reverse primers (Table 1) and 1 unit HotStarTaq enzyme. Five μl of template was added to each reaction mix and amplified in an ABI 9700 thermal cycler (Applied Biosystems, Foster City, CA) with the following program: 95°C 15 min, 40 cycles of (94°C 1 min, 58°C 50 s, 72°C for 1 min), 72°C 5 min. Amplified samples were held at 4°C. Amplification was confirmed by agarose gel electrophoresis of 5 μl of each sample.

Single Strand Conformation Polymorphism

Nine μl of PCR product was mixed with 1 μl denaturation solution (0.2M NaOH, 0.04M EDTA, pH8). After incubation at 56 °C for 5 min, 2 μl formamide loading buffer (0.05% bromphenol blue in formamide) was added. Five μl of each sample mix was resolved on 12.5% polyacrylamide at 5 °C. Band patterns were visualized using the DNA Silver Staining Kit (GE Healthcare Life Sciences, Pittsburgh, PA) according to manufacturer's directions.

TP53 DNA Sequencing

Cases that showed TP53 mutation band pattern by SSCP were subject to Sanger sequencing. For sequencing of selected exons, PCR was repeated using a 50 μl reaction mix volume. The entire reaction mix was cleaned using S-400 spin columns (GE Healthcare), followed by ethanol precipitation at −20°C for one hour. Pellets were rinsed once in 70% ethanol and resuspended in 10 μl nuclease-free water. Two microliters of template DNA was mixed with 0.5 μl forward or reverse primer (5 μM); and 4 μl Terminator Read y Mix (Applied Biosystems). Cycle sequencing was performed in the ABI 9700 thermal cycler. The program was comprised of 96°C one minute followed by 25 cycles of (96°C 10 s, 50°C 5 s and 60°C 4 min). Unincorporated nucleotides were removed using S-200 spin columns (GE Healthcare) followed by ethanol precipitation. The resulting pellets were resuspended in 12 μl formamide and resolved by capillary electrophoresis.

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