A standard curve using 10-fold serial dilutions of methylated or unmethylated template was generated to determine the efficiencies of the CDO1 and ��-actin primer/probe efficiencies, respectively. MEK162 ARRY-438162 Mixtures of methylated and unmethylated DNA (100% methylated, 50:50 methylated:unmethylated, and 100% unmethylated) were used to control PCR bias between methylated and unmethylated templates. A Ct of less than 35 was considered positive methylation. Statistical analysis Box plot figures were produced using an online tool (www.physics.csbsju.edu). The expression differences between two categories were assessed for significance using the two-sided Mann-Whitney U test, available at www.elegans.som.vcu.edu. Differences were considered statistically significant at P < 0.05.

Results Patients and tumor characteristics A total of 64 de-identified liposarcoma specimens were used to determine expression levels of CDO1 transcript (Table 1). Of these, 32 liposarcomas were WDLS, with an equal frequency of males and females. WDLS were resected from the retroperitoneal space and from the extremities (thigh, buttock, axilla, and upper limbs) with equal frequency. In all, 21 cases of WDLS presented as primary tumors whereas 11 cases were recurrent WDLS. Our cohort of patients contained 20 cases of DDLS, of which 11 cases were female and 9 cases were male. All of the DDLS were resected from the retroperitoneal space, 8 were primary whereas the remaining 12 cases were recurrent. The study included 12 cases of PLS, of which 9 cases were male and 3 cases were female.

Resections from the extremities accounted for 9 cases of the 12 PLS tumors, while the remaining 3 tumors were resected from the retroperitoneal space. There were 10 primary PLS and 2 recurrent PLS. The median age at diagnosis was 66, 71, and 69 years for WDLS, DDLS, and PLS, respectively. Thus, this cohort is representative of liposarcomas in general. The WDLS and DDLPS represented on the TMA were primarily from deep retroperitoneal locations. Table 1 Characteristics of liposarcoma specimens. CDO1 gene expression is higher in WDLS than in DDLS CDO1 gene expression was examined in 30 liposarcoma specimens (12 cases of WDLS, 10 cases of DDLS, and 8 cases of PLS) and 3 cell lines from the adipogenic lineage using pre-existing gene expression microarray data.26 In WDLS, high CDO1 mRNA levels were observed (median = 1,757, range = 239�C2847, Fig.

1A). The distribution of CDO1 gene expression in WDLS was asymmetrical and appeared biphasic (Supplementary Fig. 1A). CDO1 mRNA levels were lower in the DDLS specimens (median = 186, AV-951 range = 21�C897). The majority of PLS (7/8 tumors analyzed) were in the same range as DDLS, with one tumor exhibiting very high CDO1 gene expression (median = 495, range = 15�C2677).