PHOSPHORILATION OF RB FAMILY GENES IN OSTEOSARCOMA PATIENTS: PATTERN OF EXPRESSION AND CORRELATION WITH OUTCOME

Sangiorgi L.*, Gobbi G.A.*, Magagnoli G.*, Benassi M.S.*, Lucarelli E.*, Baldi A.^o, Giordano A.^o, Picci P.*

*Laboratory of Oncology Research, Rizzoli Orthopedic Institute, Bologna, Italy, 40136 and ^oPathology, Anatomy and Cell Biology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA., 19107 USA

Introduction: The retinoblastoma tumor suppressor gene (Rb1) and its relatives, p107 and p130 (Rb2), encode a family of proteins that share several properties, including the capacity to regulate E2F-dependent transcription and inhibit cell-cycle progression. Although Rb1 inactivation is implicated in osteosarcoma, the growth regulatory function of p107 and p130 (Rb2) and the functional relationship within the gene family have not been explored in this tumor. The aim of this study is to investigate the interaction between the Retinoblastoma family genes and to verify if their phosporilation status is a marker able to discriminate among different prognosis for osteosarcoma patients.

Methods: We have analyzed the biopsy specimen of 60 homogeneously treated osteosarcoma patients (all pts have been treated with the same chemotherapy regimen and had surgery in the same institution) with a minimum follow-up of 6 years. Formalin-fixed and paraffin-embedded samples were prepared for histological diagnosis and for immunohistochemical studies (IHC) and analyzed with antibodies that recognize the unphosphorilated form (the form that acts as tumor suppressor) of the Rb1, p107 and p130/Rb2 genes. The protein distribution was rated as follows: negative, no or less then 10% stained tumor cells positive, positive more than 11%. Intensity of immunoreaction was scored as follow: negative (-) weak (+) moderate (++) strong (+++). Positive and negative controls were performed for each stained series. Of the 60 primary biopsies, 4 were not evaluable.

Results: In 56 primary biopsies from osteosarcoma patients we observed an appreciable distinction between the 3 genes status. In fact, except for 4 samples that were negative for all the 3 genes, we observed that those samples that were positive for Rb1 or p107, were negative for p130 and the samples that were positive for p130 were negative for either Rb1 or p107. Moreover, we observed that the phosphorilation status of p130 is related to survival. In fact, of the 23 positive cases, 19 pts were alive and 4 dead while of the 33 negative cases 8 pts were alive and 25 dead (p=0.0001). We observed the same pattern for 3 local recurrences (LR) and 12 pulmonary metastases (M) that occurred lately in the same group of patients.

Conclusion: These data could be consistent with a coordinated interaction between the 3 Rb family genes and Rb2 seems to play a new role in cell cycle progression in the osteosarcoma patients. Moreover, these data clearly show (considering those samples that were negative for all the 3 Rb-family member) that other genes are involved in the osteosarcoma progression.

We are currently evaluating the relation of Rb family genes with the E2F transcription factors and other players in the G1 checkpoint (like cyclin D1 and CDK4).