Role of CDK4 as prognostic biomarker in Soft Tissue Sarcoma and synergistic effect of its inhibition in dedifferentiated liposarcoma sequential treatment

Abstract

Soft tissue sarcomas represent an heterogeneous group of rare mesenchymal tumors comprising 1% of all solid malignancies. Among them, liposarcoma is one of the most common histotypes with atypical lipomatous tumor/well differentiated liposarcoma and dedifferentiated liposarcoma (ALT/WDLPS and DDLPS) as the major sub-entities. The unavailability of predictive, prognostic and druggable biomarkers makes the management of these lesions challenging. In recent years CDK4 and its inhibitors have emerged as potential agents for these lesions especially for ALT/WDLPS and DDLPS but the results are not conclusive and need to be elucidated. This study involved 21 ALT/WDLPS and DDLPS patients. Histological analyses of MDM2 and CDK4 were carried out. Moreover, a DDLPS patient-derived cancer model was established in vitro and in vivo assessing the efficacy of palbociclib in combination and sequential treatment. Finally, in silico analyses on CDK4 expression were carried out. The results showed a higher expression of CDK4 and MDM2 in DDLPS compared to ALT/WDLPS. Moreover, no correlation between MDM2 expression and CDK4 was observed. Next, in vitro analysis of CDK4 inhibitor palbociclib showed an antagonistic effect when combined to other chemotherapeutics, while it exhibited a significant synergy when administered in sequential schedule with lenvatinib. Next, in vivo analysis on DDLPS xenotransplanted embryos assessing the efficacy and safety profile of the in vitro tested schedules confirmed the observed data. This proof-of-concept study sheds light on the natural history of ALT/WDLPS and DDLPS and provides the rationale for the clinical applicability of sequential treatment with palbociclib in the management of DDLPS.

Fig. 1

CDK4 expression in STS. (A) CDK4 alteration frequency among solid tumors. Red: amplification; Green: Mutation; Grey: Multiple alterations; Blue: Deep deletion. Tumor type groups were included with n > 30 cases; (B) Overall survival correlation with CDK4 alteration in 206 Soft Tissue Sarcoma; (C) Representative images of hematoxylin and eosin (HE) and IHC staining. Upper row: HE of three LPS patients’ surgically resected tumor specimens (10× and 20X magnification). Middle and bottom rows: IHC of MDM2 and CDK4 in LPS patients’ surgically resected tumor specimens (10× and 20X magnification); (D) Scatter Plot showing the percentage of expression of MDM2 and CDK4 in ALT/WDLPS and DDLPS, with standard deviation. Significant difference was accepted for p < 0.001 (**)

Fig. 2

Pharmacological analysis of palbociclib activity in DDLPS patient-derived cells. (A) Representative images of DDLPS patient’s frontal CT-scan and resected DDLPS tumor mass and related processed specimen. (B) HE and CDK4 IHC staining of patient tumor tissue and patient-derived primary tumor cells. The morphology of the primary culture analyzed through HE staining (upper right) recapitulates the one observed in the patient tumor (upper left) with markedly atypical cells. Moreover, CDK4 expression resulted positive in both the patient tumor and primary culture (lower right and left) further corroborating the establishment of DDLPS patient-derived primary culture model. (C) Pharmacological analysis of palbociclib in vitro activity in combination and (D) in sequential treatment with chemotherapy in DDLPS patient-derived cells. (n = 8 for each condition) (E) Representative fluorescence microscopy images of zebrafish embryos xenotransplanted with DDLPS patient-derived primary cells. Representative images of 2 hpi xenotransplanted embryos and images of embryos untreated (CTR) and exposed to tested drugs at 72 hpi, scale bar 1000 μm. (F) Mean fluorescence signal of DDLPS xenotransplanted embryos, arbitrary units (n average = 15 for each condition). (G) Tumor-growth inhibition rate between tested drugs. Significant differences between treatments were accepted for p < 0.05 (*), (n average = 15 for each condition)