Immunohistochemical stain
CK, ER, PR, AR, P16, and Ki67 were performed using an automated system (Benchmark XT, Roche Ventana, Tucson, AZ, US). Other antibodies were carried out according to GTVision™ Kit instructions (GK600711, Gene Tech, Shanghai, China). Antigen extraction was performed for each antibody according to the manufacturer's instructions. Nonspecific stain blocker was used to block endogenous peroxidase activity. Sections were incubated with each antibody and HRP enzyme-conjugated goat anti-mouse/rabbit IgG polymer. 3.3′-diaminobenzidine (DAB) was used to develop the color, then hematoxylin was used to counterstain.
Histochemical stain
Alcian blue and periodate Schiff's reaction (AB-PAS) staining fluid (BA4121, BaSO Companh, Zhuhai, China) was used for histochemical stain. Sections were routinely dewaxed to water and washed with distilled water, dyed with alcian blue staining fluid (PH2.5) for 10–20 min, washed with water and removed excess water, oxidized by periodic acid solution for 10 min, rinsed with distilled water and removed excess water,dyed with schiff reagent for 10–15 min, rinsed with water and removed excess water, dyed the nucleus with hematoxylin solution for 2–3 min, rinsed with water and dried excess water,finally sealed with neutral gum after conventional dehydration transparency.
FISH detection
CDKN2A/p16 (9p21) two-color fluorescence deletion probe kit (F.01265-01, Anbiping Company, Guangzhou, China) was applied for FISH. Tissue sections were pretreated with pure water at 88–92 °C for 30 min and digested with pepsin at 37 °C for 15–20 min after baking and dewaxing, then further rinsed, fixed, and dehydrated. 10 μL of the probe working solution was placed onto the tissue specimen and the edges sealed with rubber cement after the tissue sections naturally drie. Tissue sections were put in a hybridization apparatus, denatured at 78 °C for 5 min, and hybridized at 42 °C for 12–18 h. Tissue sections were washed, dried and added 10 μL 4′,6-diamidino-2-phenylindole after hybridization, then put at room temperature for 15 min. The BioView automatic scanning image analysis system (Duettm) was used to store and analyze the fluorescence FISH images. Finally, the FISH slides were stored at − 20 °C in the dark for future experiments. For each slide, 100 nuclei of tumor cells were analyzed with two observers. 0 red and 2 green represented homozygous deletion, which were considered positive. When the cut-off was greater than or equal to 10%, the specimen was diagnosed as CDKN2A/p16 (9p21) gene deletion.
Case presentation
Case 1
A 58-year-old woman presented with a one-month history of intense abdominal pain. Abdominal ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) all suggested the presence of a 3-cm long mass in the right adnexal area (Fig. 1a) and multiple uterine nodules (Fig. 1b). The mass in the right adnexal area was cystic and solid, and had enhancement due to tumor vascularity from MRI, which suggested malignant potential. The patient had elevated levels of c-reactive protein (CRP, 48.75 mg/L) and thyroid-stimulating hormone (TSH, 6.22 uIU/mL). The plasma levels of tumor markers, including CA125, HE4, CA199, CA724, CEA, CA153, SCC, AFP, AFU, and NSE, were normal. Her medical and surgical history revealed that she had been diagnosed with Hashimoto's thyroiditis > 30 years ago, undergone a minor surgery to remove a laryngeal polyp 15 year ago, and undergone laparoscopic left salpingo-oophorectomy 8 years ago. She had a 30-year history of smoking 10 cigarettes daily, but no history of alcohol consumption. The patient had had 11 pregnancies in her lifetime, 10 of which resulted in miscarriages (G11P1).
Macroscopically, the dimensions of the right ovariectomized specimen were 3 cm × 3 cm × 2 cm. The specimen had a partial defect, which resulted from the intraoperative frozen examination of the specimen. The central area of the specimen, which comprised normal ovarian tissue measuring about 2 cm × 1.5 cm × 1 cm, was tough with a porcelain white color. The tumor was present on the circumferential surface. The mass had a greyish-yellow color. A cut surface of the tumor revealed a mucus-containing honeycomb-like structure with a soft texture.
Microscopically, the histological morphology of intraoperative frozen pathological and postoperative paraffin sections was basically the same. At low magnification, the tumor tissue had an interspersed distribution of macrocystic, microcystic, and solid growth patterns, which were well-defined from normal ovarian parenchyma (Fig. 2a). At high magnification, the tumor tissue comprised multiple mutually anastomosing adenoid spaces, which were lined with flat, cubic, or short columnar epithelioid cells with mild atypia. The cell cytoplasm was vacuolated, and occasionally contained basophilic substances. Signet-ring cells were seen locally (Fig. 2b). Thread-like bridging strands were present within the luminal spaces (Fig. 2c). Hyaline connective tissues were present in trace quantities. No eosinophilic cytoplasm and inflammatory lymphocytic infiltration were present in this case.
Because the patient had a history of laparoscopic left salpingo oophorectomy, and morphology lacked of evidence of malignant tumor, such as obvious cell atypia and invasive growth pattern, it was considered as a benign lesion. Subsequently, she underwent laparoscopic hysterectomy and right salpingo-oophorectomy based on intraoperative pathological diagnosis.
The IHC analysis revealed that CK, CK7, Calretinin (Fig. 2d), WT-1, D2-40, L1CAM (Fig. 2e), and BAP1 (Fig. 2f) were fully expressed. CK5/6 and Cyclin D1 were focally expressed; β-catenin was fully expressed in the membrane. CD56 was focally expressed, and P16 was patchy positive. CD34 had a positive expression in vessels, and Ki67 was about 3% positive. However, EMA, CEA, MOC-31, Ber-EP4, Vimentin, SALL4, inhibin-α, SF-1, CD10, CD99, S-100, SMA, MelanA, PAX-2, GATA-3, ER, PR, AR, and CK20 were negative. AB-PAS staining revealed an AB-positive stain. Upon FISH analysis, CDKN2A/p16 gene deletion test was shown to be negative (Fig. 2g).
Based on the histomorphological, IHC, histochemical, and FISH detection findings, a final diagnosis of AT of the right ovary and multiple leiomyoma of the uterus was made. From her medical history, she had already been diagnosed with AT of the left ovary after multiple hospital consultations. The microscopic appearance of the left ovary is shown in Fig. 2h. The histomorphological features of both ovaries were similar. The present postoperative follow-up time was 13 months, and no recurrence or metastasis was found.
Case 2
A 41-year-old woman was found to have a mass in her right adnexal area during a physical examination, which had been gradually increasing in size for two years. Ultrasound and CT of the abdomen suggested the presence of a 3-cm long mass in the right adnexal region, which was also cystic and solid, and color dopper flow image (CDFI) from ultrasound showed increased flow signals. Plasma levels of tumor markers were normal. The patient had no prior medical or surgical history, and no history of smoking or alcohol consumption.
Macroscopically, the diameter of the removal right ovarian mass specimen was approximately 2.5 cm. The specimen was partially defective due to intraoperative frozen sampling. The cut surface was alternately cystic and solid. The solid area was 1.5 cm in diameter, yellowish white in color and soft in texture.
Microscopically, the tumor tissue showed multiple growth patterns such as large cysts, small cysts, adenoid and solids under low magnification (Fig. 3a). The tumor cells were flat, cuboidal or short columnar epithelioid cells with mild to moderate cellular atypia under high magnification. The cell cytoplasm was eosinophilic and occasionally vacuolated. Signet-ring cells and thread-like bridging strands were seen locally (Fig. 3b). There was a certain amount of hyaline stroma and no inflammatory lymphocytic infiltration. The intraoperative frozen pathological diagnosis was benign lesion, and she subsequently underwent laparoscopic right salpingo-oophorectomy.
The IHC analysis revealed that CK, CK7, Calretinin (Fig. 3c), WT-1, CK5/6, D2-40, L1CAM (Fig. 3d), and BAP1 (Fig. 3e) were fully expressed. Cyclin D1 and CD56 were focally positive. β-catenin was fully expressed in the membrane. P16 was patchy positive. CD34 had a positive expression in vessels, and Ki67 was approximately 5% positive. However, EMA, CEA, MOC-31, Ber-EP4, Vimentin, SALL4, inhibin-α, SF-1, CD10, CD99, S-100, SMA, MelanA, PAX-2, GATA-3, ER, PR, AR, and CK20 were negative. AB-PAS staining revealed an AB-positive stain. CDKN2A/p16 gene deletion was negative by FISH analysis (Fig. 3f). The postoperative follow-up time was 26 months, and no recurrence or metastasis was found.
