TP53 Mutation Status of Tubo-ovarian and Peritoneal High-grade Serous Carcinoma with a Wild-type p53 Immunostaining Pattern

Materials and Methods

Case selection. This study (2017-1479-001) was reviewed and approved by the Institutional Review Board at Severance Hospital (Seoul, Republic of Korea). We selected 153 patients diagnosed with tubo-ovarian or peritoneal HGSC during the period from July 2015 to June 2016. Clinicopathological information was obtained from the electronic medical record system and pathology reports. Clinical and pathological details that were reviewed included age of patient at initial diagnosis, origin of tumor, association with serous tubal intraepithelial carcinoma (STIC), tubo-ovarian surface extension, presence of malignant cells in ascites or intraoperative peritoneal washings, pelvic or extrapelvic peritoneal metastasis, lymph node metastasis, lymphovascular invasion, and International Federation of Gynecology and Obstetrics (FIGO) stage.

Pathological examination. The tissues resected by gynecological oncology surgeons at Severance Hospital (Seoul, Republic of Korea) were initially examined by pathologists before fixation in 10% neutral-buffered formalin. After fixation for 12-24 h, the tissues were thoroughly examined macroscopically and sectioned. After processing with an automatic tissue processor (Peloris II; Leica Microsystems, Newcastle Upon Tyne, UK), the sections were embedded in paraffin blocks. Four micrometer-thick slices were cut from each formalin-fixed, paraffin-embedded tissue block using a rotary microtome (RM2245, Leica Microsystems) and stained with hematoxylin and eosin using an automatic staining instrument (Ventana Symphony System; Ventana Medical Systems, Tucson, AZ, USA). After staining, the slides were covered with a glass coverslip and sent to two Board-certified pathologists specialized in gynecological oncology (K.N. and H.-S.K.). They examined all available hematoxylin and eosin-stained slides by light microscopy (BX43 System Microscope; Olympus, Tokyo, Japan) and made definitive pathological diagnoses. In addition, the most representative slide for each case was chosen for immunohistochemical staining.

Immunohistochemical staining. Four micrometer-thick slices obtained from the formalin-fixed, paraffin-embedded tissue blocks were placed on Superfrost Plus glass slides (Thermo Fisher Scientific, Waltham, MA, USA). These slices were deparaffinized in xylenes and rehydrated through graded alcohols. Immunohistochemical staining was performed using automatic immunostaining instruments, either a Ventana Benchmark XT automated staining system (Ventana Medical Systems) or Dako Omnis system (Dako, Agilent Technologies, Carpinteria, CA, USA), according to the manufacturer's recommendations (13-17). Antigen retrieval was performed using Cell Conditioning Solution (CC1; Ventana Medical Systems) or EnVision FLEX Target Retrieval Solution, High pH (Dako, Agilent Technologies). The slices were incubated with primary antibody against paired box 8 (PAX8; 1:50, polyclonal; Cell Marque, Rocklin, CA, USA), Wilms tumor 1 (WT1; 1:200, clone 6F-H2; Cell Marque), p16 (prediluted, clone E6H4a; Ventana Medical Systems), p53 (1:300, clone DO-7; Novocastra, Leica Biosystems, Newcastle Upon Tyne, UK), estrogen receptor (ER; 1:150, clone 6F11; Novocastra), progesterone receptor (PR; 1:100; clone 16; Novocastra), hepatocyte nuclear factor 1β (HNF1β; 1:400, polyclonal; Sigma-Aldrich, St. Louis, MO, USA), and AT-rich interaction domain 1A (ARID1A; 1:400, polyclonal; Sigma-Aldrich). After chromogenic visualization using an ultraView Universal DAB Detection Kit (Ventana Medical Systems) or EnVision FLEX /HRP Kit (Dako, Agilent Technologies), the slices were counterstained with hematoxylin, dehydrated in graded alcohols and xylene, and then embedded in mounting solution. Appropriate positive and negative controls were concurrently stained to validate the staining method. For negative controls, non-specific activity was assessed by omitting the primary antibodies. The p53 immunostaining pattern was interpreted as a missense mutation, nonsense mutation, or wild-type pattern when p53 expression was diffuse and strong (>60% of tumor cell nuclei), completely absent (0%), or focal and weakly positive, respectively.

Direct sequencing. Five cases of tubo-ovarian and peritoneal HGSC with a wild-type p53 immunostaining pattern were analyzed for TP53 mutational status, as previously described (16). Briefly, histopathological confirmation for the presence of tumor tissue was performed by the two Board-certified pathologists. They marked the most dense tumor area on the hematoxylin and eosin-stained slides, avoiding necrotic and hemorrhagic areas as much as possible. Ten-micrometer-thick sections were cut from the formalin-fixed, paraffin-embedded tissue blocks. The areas that corresponded to the marked ones on the hematoxylin and eosin-stained slides were macrodissected with a scalpel and placed into 1.5-ml tubes for tissue digestion. Extraction of genomic deoxyribonucleic acid (DNA) was performed using a QIAamp DNA FFPE Mini Kit (Qiagen, Valencia, CA, USA). DNA concentrations of extracted DNA were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and adjusted to a concentration of 50 ng/μl. Exons 1-11 of TP53 were amplified by polymerase chain reaction, using a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences used are shown in Table I. Briefly, 50 ng of DNA was amplified with a thermal cycling profile of 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, with a final extension at 72°C for 7 min. Amplimers were purified using a Multiscreen Filter Plate (EMD Millipore, Billerica, MA, USA) and resolved by agarose gel electrophoresis to confirm successful amplification. Bidirectional Sanger sequencing of all polymerase chain reaction products was performed using a BigDye Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed using an Applied Biosystems 3730xl Genetic Analyzer (Applied Biosystems) and Variant Reporter Software Version 1.1 (Applied Biosystems).