Seventy-one patients were selected who were diagnosed with uterine leiomyosarcoma, and were treated, and had regular follow-ups at the Asan Medical Center, Seoul, Korea, between January 2000 and December 2014. Overall, 42 paraffin blocks of the patients who were diagnosed with uterine leiomyosarcoma were preserved. Clinicopathological data were reviewed and the manufacture of tissue microarray (TMA) was performed under the approval of the Institutional Review Board of Asan Medical Center (approval number: 2009-0450). It thus meets the standards of the Declaration of Helsinki in its revised version of 1975 and its amendments of 1983, 1989, and 1996 [22]. FIGO 2009 uterine sarcoma classification was adopted to stage uterine leiomyosarcoma [23].

Tumor tissues obtained during surgery for routine diagnostic pathologic examinations were used for immunohistochemistry (IHC) studies. The most apparent and dense part of the tumors in paraffin blocks were obtained using a 2-mm diameter puncture needle, and were sent to Bio-Resource Center (BRC) in the hospital to make TMA. Three punctures at different parts of tumor were performed in each case to minimize selection bias and control quality of results by confirming tumor heterogeneity. Formalin-fixed, paraffin-embedded tissue sections were stained by immunohistochemical method for antibodies using a BenchMark ULTRA automatic immunostaining device (Ventana Medical Systems, Tucson, AZ, USA) with an OptiView 3,3′-diaminobenzidine (DAB) IHC Detection Kit (Ventana Medical Systems) according to the manufacturer's manual in each case. Four-micrometer-thick sections which were obtained with a microtome, were placed onto silanized charged slides and allowed to dry for 10 minutes at room temperature, followed by 20 minutes in an incubator at 65°C. Sections were performed by the heat-induced epitope retrieval (HIER) method using a Cell Conditioning 1 (CC1) buffer for 32 minutes and incubated for 16 minutes with rabbit anti-AR (1:100; AC-0071; Epitomics, Inc., Burlingame, CA, USA), rabbit anti-cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) (1:50; PAB2971; Abnova Corporation, Taipei, Taiwan), mouse anti-ERα (1:100; NCL-L-ER-6F11, Novocastra™; Leica Biosystems, Newcastle, UK), rabbit anti-ERβ (1:100; SC-8974; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-gonadotropin releasing hormone (GnRH) receptor (1:25; NLS209; Novus Biologicals, San Diego, CA, USA), mouse anti-PR (1:200; NCL-L-PGR-312; Zymed, Carlsbad, CA, USA). Antigen-antibody reactions were visualized using Ventana OptiView DAB IHC Detection Kit (Optiview HQ Linker 8 minutes, Optiview horseradish peroxidase [HRP] Multimer 8 minutes, Optiview hydrogen peroxidase [H₂O₂]/DAB 8 minutes, Optiview Copper 4 minutes). Counterstaining was performed by using Ventana Hematoxylin II and bluing reagent for 32 and 4 minutes, respectively. All slides were removed from the stainer, dehydrated, and coverslipped for interpretation. For positive controls for each antibody, adequate immune-reactive prostate tissue samples were used. By omission of the primary antibodies, negative controls were produced. Immune-reaction of AR, ERα, ERβ, and PR were evaluated by observing expression pattern of nucleus. Immune-reaction of GnRH and CYP19A1 were assessed by analyzing both nucleus/cytoplasm and membrane, respectively (Fig. 1).

Slides were reviewed by a pathologist subspecializing in gynecologic oncology and a gynecologic oncologist without access to the clinical data of the patients. An assessment of the immunohistochemical results was performed using a semi-quantitative scoring system. This was calculated by multiplying the intensity of stain and the percentage of the total width (0 [0–20 points], 1+ [21–80 points], 2+ [81–180 points], and 3+ [181–300 points]). Patient group was categorized according to the expression of biomarkers by negative (0) versus positive (1+, 2+, and 3+) cases in ERα, PR, and AR. In ERβ and CYP19A1, patients group was categorized by moderate (2+) versus strong (3+) positive cases because all of them showed moderate to strong expression in this study. Patient categorization was not possible in GnRH because all of the patients showed strong (3+) positivity, and this was not included in the analysis.

For univariate analyses, Mann-Whitney U test and Student's t-test were used to analyze median and mean value of each variable, respectively (Table 1). Chi-square test and Fisher's exact test were used to test for statistical differences among frequencies of each variable (Tables 1 and 2). The Kaplan-Meier method was used to analyze survival outcome, and the difference between each group was analyzed by log-rank test (Fig. 2) and Cox's proportional regression analysis (Table 3). Disease-free survival (DFS) was defined as from the date of operation to the date of recurrence or the last follow-up, and OS was defined as from the date of operation to the date of death or the last follow-up. Statistical significance was defined as p<0.05. SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis.

Twenty-six patients (61.9%) were FIGO stage I. Recurrence and death occurred in 23 (54.8%) and 14 (33.3%) of 42 patients with uterine leiomyosarcoma, respectively. Distant metastasis with/without loco-regional recurrence was observed in 14 of 23 recurrent patients (61.9%). The lung (n=11) was one of the most common site of distant metastasis (lung=8, abdominal peritoneum=1, lung/bone=1, lung/liver=1, and lung/liver/spleen/pancreas/neck/thigh/bone=1). Two patients showed metastasis at both loco-region and distant site (pelvis/abdominal wall/omentum=1 and pelvis/abdominal peritoneum=1). A hysterectomy was performed in all of the patients. Lymphadenectomy was performed in 19 (45.2%) of all patients. Lymph node metastasis (LNM) was found in 1 case (5.3%). Twenty-seven patients (64.3%) received adjuvant treatment. Adjuvant treatment was given when patients had extra-uterine tumor extension (FIGO II–IV). Omission of adjuvant treatment was decided by consulting the patients when they were diagnosed as FIGO stage I and had a good compliance to make a regular follow-up. Chemotherapy was performed in 25 patients (59.5%) and was the most common type of treatment (Table 1). Among them, the majority of the patients received ifosfamide based regimen (ifosfamide+cisplatin=11, etoposide+ifosfamide+cisplatin=3, and ifosfamide=1). The other regimens were doxorubicin based (paclitaxel+doxorubicin+cisplatin=2, doxorubicin+cisplatin=4, and doxorubicin=3) except one patient who received docetaxel+gemcitabine. AR expression group showed significantly less recurrences with no death events. Expression of AR showed no significant relevance with other clinic-pathological characteristics.

Of the 42 patients with uterine leiomyosarcoma, overall immune-positivity of ERα, ERβ, PR, GnRH, and CYP19A1 were 18 (42.9%), 42 (100%), 20 (47.6%), 42 (100%), and 42 (100%), respectively. Eleven patients (26.2%) showed immunoreactivity in AR. Among them, 7 patients (16.7%) showed moderate to strong immune-reaction (2+ to 3+). GnRH showed strong immune-reaction (3+) in all cases. ERβ and CYP19A1 showed a high proportion of strong immunoreactivity (3+), 35 cases (83.3%) and 35 cases (83.3%), respectively. ERα and PR showed low rates of strong immuno-reaction (3+) with 9 cases (21.4%) and 12 cases (28.6%), respectively (Fig. 1, Table 2).

The mean follow-up period was 42.6±41.2 months (range, 2.6–172.6 months). In terms of tumor size and mitotic count, previous studies showed that tumor size or mitotic count greater than 10 cm or 10 mitoses/10 high-power fields were strongly associated with worse survival outcome [2425], and we adopted these as cut-off values for more accurate analysis. In univariate analysis, tumor size, mitotic index, FIGO stage, lymphadenectomy, PR, AR, and CYP19A1 were significantly associated with DFS. The multivariate analysis was performed with variables that were statistically significant in univariate analyses, and FIGO stage and AR were an independent prognostic factor for DFS. Menopause, tumor size, FIGO stage, PR, and AR were all statistically significant in the univariate analyses for OS. There were no events in 5-year OS in uterine leiomyosarcoma patients showing AR immune-reaction compared to the no expression group (100% vs. 54.8%; p=0.014), and patients having a co-expression of AR with ER and/or PR showed statistically significant better 5-year DFS and OS compared with ER and/or PR positive patients with negative AR expression. However, FIGO stage was the only independent prognostic factor for OS in the multivariate analyses (Fig. 2, Table 3).