Paraffin-embedded OS tissue specimens were taken from October 2009 to July 2019. The paraffin specimens of 51 patients with OS who were pathologically diagnosed after surgical resection in the Department of Orthopedics at Second Affiliated Hospital of Shanxi Medical University were used as the experimental group, of which 10 bone tissues adjacent to the tumor (3 cm from the edge of the tumor shown by MRI, no tumor cells confirmed by pathological examination) served as control group. The diagnosis of the 51 patients with OS were certified by pathologists.
The inclusion criteria for this study are as follows: (1) limb osteosarcoma, (2) American Joint Cancer Committee (AJCC) stage II and III patients, (3) preoperative neoadjuvant chemotherapy and radical treatment arranged in our hospital Surgery, then adjuvant chemotherapy, (4) Incision biopsy specimens for immunohistochemistry and PAS staining. The exclusion criteria are as follows: (1) secondary osteosarcoma, (2) extraosseous osteosarcoma, (3) periosteum or paraosseous osteosarcoma, (4) skipping or metastatic disease at diagnosis, (5) no postoperative patient Re-examination, no follow-up information.
Among the 51 patients, 28 were males and 23 were females, their ages range from 10 to 67 (mean 27.04). There were 44 cases of ordinary osteosarcoma, 2 cases of chondroblastic osteosarcoma, 2 cases of telangiectatic osteosarcoma, 2 cases of giant cell osteosarcoma, and 1 case of low-grade central osteosarcoma. Ennecking staging: 34 cases in stage ⅡA, 13 cases in stage ⅡB, and 4 cases in stage Ⅲ. The patient's limb X-ray and chest CT were reviewed every 3 months after the operation, and the whole-body bone scan was reviewed every 6 months. The follow-up time of 51 patients range 4 to 78 months. During the follow-up period, 36 patients died, 37 patients suffered from metastasis or recurrence in situ (lung metastasis, pelvic metastasis, and multiple metastasis), 5 patients survived with tumor, and 10 patients survived without tumor. Record the clinical pathological variables including age, gender, the maximum diameter of the tumor on the initial magnetic resonance image, Ennecking stage, local recurrence, and lung metastasis. Follow up the clinical results were obtained from the day of surgery to the day of death or until March 2021. Survival time is defined as the time span from diagnosis to death caused by the primary tumor or the related reason.
Bmi1 antibody (dilute at 1:300, ab38295) and KLF4 antibody (dilute at 1:400, ab106629) were purchased from American Abcam. Hematoxylin and eosin staining kit and neutral glial fixation sheet were purchased from Shanghai Weiao Biotechnology Co., Ltd., streptavidin-biotin complex (SABC) immunohistochemical staining kit, Diaminobenzidine (DAB) color development kit and phosphate buffered saline (PBS) were purchased from Wuhan Bosite Bioengineering Co., Ltd. The biomarker goat anti-rabbit IgG was purchased from Beijing Zhongshan Bridge. 0.25% Tripsin-EDTA was purchased from Gibco, USA.
After the sections were made, HE staining was routinely performed firstly, then the cell morphology was observed using a microscope. Then immunohistochemical detection was performed, briefly, xylene dewaxing, ethanol solution rehydration, and EDTA antigen retrieval solution high pressure repair. Then the sections were blocked with PBS, and incubated with Bmi1, KLF4 antibody at 4 °C overnight. At the end of incubation, the sections were washed 3 times with PBS, and then incubated with biotin-labeled goat anti-rabbit IgG at 37 °C for 1 hour. Then, SABC was added, and incubated at 37 °C for 30 minutes. DAB solution was used to develop, hematoxylin solution was used to stain, hydrochloric acid alcohol was applied to turn blue, and the slide was mounted with neutral gum. The results of anti-Bmi1 and KLF4 staining were evaluated by Pannorama MIDI digital slide scanner and scanner control software (3DHISTECH, Ltd., Budapest, Hungary).
Determination of positive results: Bmi1 and KLF4 are positive cells with brown particles in the nucleus, respectively. Image analysis with Image J. Five fields of view were randomly selected under high magnification (x 400), and the average gray value (staining intensity) of positive cells and the percentage of positive area (stained area) were used as IHC measurement indicators. Finally, four scores were given: High positive (3+), Positive (2+), Low Positive (1+) and Negative (0). The final score of 0 ~ 1.5 and > 1.5 is defined as low expression and high expression, respectively.
SPSS 22.0 and Graphpad prism 8.0 statistical software were used to analyze the data and draw, respectively. The categorical data was analyzed by χ2 test for comparison between groups. Survival analysis was calculated by Kaplan-Meier. Statistical differences in survival rate between each factor group were analyzed by Log-rank test. Multivariate survival analysis on survival data were performed by Cox proportional hazard regression model. P<0.05 was considered as statistically significant.