Cycle threshold values were averaged across triplicate samples. homogenous immunoreactivity for PRAME. There was a variable, focal expression of MAGEA1 (11%) and SSX2 (16%) and no expression of ACRBP. Quantitative real-time PCR demonstrated and transcripts in all eight samples: six tumors with high mRNA levels; two tumors with low mRNA levels. The gene expression of was not detected in the majority of cases. In conclusion, myxoid and round-cell liposarcomas consistently express PRAME by immunohistochemistry as well as and AEZS-108 by qualitative real-time PCR. This supports the use of cancer-testis antigen-targeted immunotherapy in the treatment of this malignancy. (encodes LAGE-1), (encodes NY-ESO-1), and various transcripts.18C20 Furthermore, increased and mRNAs have been reported specifically in the myxoid and round-cell subtype.20,21 More recently, overexpression of the highly immunogenic cancer-testis antigen NY-ESO-1 was reported in myxoid and round cell liposarcomas by both immunohistochemistry and quantitative real-time PCR.22,23 Expression was seen in 90C100% of samples tested and immunoreactivity was strong and homogenous in the majority of positive cases. Of note, occasional expression was also reported in the pleomorphic and dedifferentiated liposarcoma subtypes. Cancer-testis antigen-expressing tumors frequently demonstrate a coordinated expression of cancer-testis antigens, meaning more than one cancer-testis antigen is expressed.6 Given the consistent over-expression of NY-ESO-1 in myxoid and round cell liposarcoma, we evaluated for the expression of the cancer-testis antigens MAGEA1, ACRBP, PRAME, and SSX2 by immunohistochemistry and and by quantitative real-time PCR. Materials and methods Rationale Frequent and homogenous expression of NY-ESO-1, a highly immunogenic cancer-testis antigen, in myxoid and round cell liposarcoma has been recently documented.20,22,23 Herein, we sought to explore the expression of other cancer-testis antigens as a rationale for a potential polyvalent immunotherapeutic target in the treatment of this neoplasm. The MAGE antigens including MAGE1 and MAGE3 are attractive targets previously explored in immune-based clinical trials in solid organ malignancies. SSX2 and ACRBP are highly immunogenic antigenic targets and so are PRAME and CTAG2. There are immunotherapy-based clinical trials targeting PRAME, CTAG2 (in combination with CTAG1B) and SSX2 expression in various hematologic and solid organ malignancies. Case Material Myxoid and round cell liposarcomas (gene rearrangement determined by fluorescence hybridization and/or karyotype analysis demonstrating a t(12;16) (q13;p11) translocation. In addition, frozen tissue of myxoid and round cell liposarcomas ((encodes LAGE-1), (encodes PRAME), and (encodes MAGE-A3) was measured by qualitative real-time PCR. Of note, the mRNA expression of Rabbit polyclonal to IGF1R.InsR a receptor tyrosine kinase that binds insulin and key mediator of the metabolic effects of insulin.Binding to insulin stimulates association of the receptor with downstream mediators including IRS1 and phosphatidylinositol 3′-kinase (PI3K). (encodes NY-ESO-1) in these samples was reported previously.22 RNA was extracted from frozen sarcoma samples using Ribozol (Amresco, Solon, OH, USA) and a modified manufacturers protocol for RNA extraction using Trizol reagent (Ambion Life Technologies, Grand Island, NY, USA). RNA was quantitated using a NanoDrop-ND 1000 (Thermo Fisher Scientific, Wilmington, DE, USA). One microgram of RNA per sample was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Qualitative real-time PCR was performed in 10(Hs00266705_1), (Hs00535628_m1), (Hs01022301_m1), and (Hs.PT.39a.22214836) were used. Input cDNA was doubled for the due to low expression. Each sample was measured in triplicate. No template controls and no reverse transcriptase controls for each sample were included. Cycle threshold values were averaged across triplicate samples. was used to calculate percentage relative expression of each sample. Samples were further normalized to the expression of the testis-positive control. Standard deviations were calculated by comparing delta cycle thresholds for each well in triplicate. Immunohistochemistry A representative formalin-fixed, paraffin-embedded block was obtained for each tumor (total gene expression by quantitative real-time PCR in eight myxoid and round cell liposarcoma samples and one testis-positive control sample (Figures 1 and ?and2).2). Quantitative real-time PCR demonstrated and mRNA in all eight myxoid and round cell liposarcomas: six samples with high levels and two samples with low levels. As previously reported, seven (88%) samples showed gene expression of and The gene expression was detected in only two samples, both of which contained much lower levels compared with the testis control even when input cDNA was doubled. As several of the myxoid and round cell liposarcoma samples showed high levels of expression, we also assessed the level of in three well-differentiated liposarcomas and three dedifferentiated liposarcomas. One of the well-differentiated liposarcomas showed PRAME expression similar to the testis control. None of the dedifferentiated liposarcomas showed high levels of expression (Supplementary Figure AEZS-108 1). AEZS-108 Open in a separate window Figure 1 Quantitative real-time PCR.
