YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma

We enrolled 41 osteosarcoma patients (stage II-IVa UICC/AJCC classification) from the Department of Pathology of the Istituto Ortopedico Rizzoli (Bologna, Italy) and from the Division of Surgical Pathology, Istituto Nationale Tumori, Fondazione G. Pascale (Naples, Italy), under their Local Ethical Committee approval. We used the bioptic samples of primary tumor before any treatment (see below). Of the 41 patients, 14 had metastasis at the first visit (synchronous), 15 developed metastasis during follow-up (metachronous) and 12 were metastasis-free. Metastases were localized in lung and the primary sites were in extremity bones. Extraskeletal, periosteal, and paraosteal osteosarcomas were excluded from this study. All slides of the cases were reviewed by two pathologists to confirm diagnosis. Patients received preoperative, postoperative or no chemotherapy according to degree of tumor stage. Necrosis area was defined by using the Huvos grading system, as described in detail [21, 22]. Accordingly, we subdivided patients into two groups (<90%) and (≥90%) based on chemonecrosis area as indicated by the European Cooperative Osteosarcoma Study Group coordinated by the Istituto Ortopedico Rizzoli (COSS) [21, 22], a partner of the present study.

Chemotherapy protocols included methotrexate (12 g/m²) with leucovorin rescue, cisplatin (90-150 mg/m²), doxorubicin (60-90 mg/m²), and ifosfamide (6-10 g/m²). The scheduled duration of chemotherapy ranged from 24 to 38 weeks. For chemotherapy patients, surgery was scheduled to take place between weeks 9 and 11 and radiotherapy was not used. We collected clinical data from all patients including age, sex, tumor site, necrosis area after chemotherapy and surgical stage.

Biopsies before chemotherapy were fixed and paraffin embedded. Conventional immunohistochemical studies were performed on 5-6 μm section, as previously described in detail [16, 22]. Briefly, slides were immersed in a water bath (W-cap Bioptica) and incubated for 30 minutes at 95°C, then cooled for 20 minutes at room temperature. Sections were then incubated for 10 minutes in 3% hydrogen peroxide in distilled water, and washed in PBS for 5 minutes. Slides were incubated with the primary antibody (mouse-monoclonal YY1 diluted 1:100 sc7341 Santa Cruz) for 30 minutes in a Dako Autostainer device. The signal was then visualized with streptavidina-biotina Dako REAL Detection System Peroxidase/DAB, Rabbit/Mouse Dako Autostainer. Slides were counterstained with hematoxylin. Washing steps were included after each incubation. All samples were stained with vimentin as tissue quality controls while positive and negative conventional controls were used to test antibody as already reported [16]. All controls gave satisfactory and reproducible results. Digital photos were obtained with Olympus Vanox AHBS3. The staining of tumor cells for evaluation of YY1 was scored from 0 to 4 (0=no staining; 1=weak 1-25% positive cells; 2= moderate 26%-50% positive cells; 3= strong 51%-75% positive cells; 4= very strong 76%-100% positive cells. This score was checked by two independent observers blinded for clinical outcome or other biological tumor features. Discrepant cases were decided on consensus.

Data were analyzed with SPSS 13.0 software. Results are reported as mean ± SD. YY1 was considered as a discrete variable and the patients were stratified into two classes: high score as 3-4, (>51-100% positive cells) and low score as 0-2, (0-<50% positive cells) of YY1. ANOVA was used to assess difference for continuous variables (age and mean follow-up) while Pearson chi square test was used to examine differences between YY1 expression and each clinical variable: gender, tumor site, histotype, chemonecrosis, presence of metastasis at diagnosis and occurrence of metastasis at follow-up (n = 41). Survival periods were counted in months from the date of first visit to date of death or last follow-up before study closure. Follow-up data started at time of diagnosis allowing a minimum of 3.8 months (mean 47.0 ± 25.3 month range, 3.8-95 months). Mean age of the sample was 19.4 ± 15.5 with a range from 4 to 76 years. Metastasis-free period was counted in months from the date of surgery to date of detection of first metastasis, since no local relapses occurred. We used life-table method to estimate the metastasis-free survival and overall survival for low and high levels of YY1 expression. Multivariate Cox regression analysis was used to evaluate the effect of YY1 expression on metastasis occurrence and survival independently of age, gender, histotype and chemonecrosis. A p value <0.05 was considered as statistically significant.

Immunohistochemistry revealed that all normal bone tissues analyzed (n = 10) were almost negative to YY1 antibody or in some cases normal bone showed faint and diffuse cytoplasm staining, as previously reported [16]. Figure 1 shows two representative examples of osteosarcoma with the low score (2) and high score (4) of YY1 antibody localized predominantly in nuclei (95%). Table 1 shows YY1 scoring with respect to clinical variables. Table 2 summarizes the characteristics of the study population stratified by high and low YY1 expression (see methods). A total of 61% of tumor samples were positive to YY1 staining (nuclear staining) with strong intensity (score 3-4, >51-100% positive cells) while 39% showed a lower degree of YY1 staining (score 1-2, <50% of positive cells). Distal femur and tibia were the most common sites (53.7% and 19.5% respectively). The majority of tumors arose on extremities. There was no difference in degree of YY1 expression in histological subgroups (i.e. chondroblastic, fibroblastic and osteoblastic types, p = 0.557) and age. Twenty-seven patients were free of metastasis at baseline while 15 (55.6%) developed metastasis during follow-up. Of these, 80% were positive to YY1 (51-100% positive cells) (p = 0.004). In univariate analysis, death occurred in 64% of YY1 strong positive patients (p = 0.005). Importantly, multivariate Cox regression analysis revealed that a high level of YY1 expression (score 3-4) was predictive of poor metastasis-free survival (HR = 4.690, 95%CI = 1.079-20.396; p = 0.039) (Table 3 and Figure 2). The estimated overall survival (after 60 months of follow-up) calculated by life-table method (Table 4) was 34% for patients with a high YY1 score and 79% for those with a low YY1 score. Multivariate Cox regression analysis indicated that YY1 expression is predictive of mortality (HR = 8.353, 95%CI = 1.863-37.451, p = 0.006) as an independent variable with respect to age, gender, histotype and chemonecrosis (Figure 3 and Table 5).