Detection of SS18-SSX1/2 fusion transcripts in circulating tumor cells of patients with synovial sarcoma
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COMMENTARY
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Detection of SS18-SSX1/2 fusion transcripts in circulating tumor cells of patients with synovial sarcoma Joanna Przybyl1*, Matt van de Rijn1 and Piotr Rutkowski2
Abstract A recent study on 15 patients with synovial sarcoma demonstrated very low prevalence of tumor-specific fusion transcripts in peripheral blood specimens. Our results in an independent cohort of 38 patients with synovial sarcoma support these findings. Synovial sarcoma patients could greatly benefit from a non-invasive monitoring of tumor burden by liquid biopsies. However, given the low detection rate of SS18-SSX1/2 in circulation, we conclude that alternative markers other than the tumor-type specific fusion transcripts should be considered. Keywords: Liquid biopsy, Circulating tumor cells (CTCs), Nested RT-PCR, SS18-SSX fusion transcript, Synovial sarcoma
Introduction We have read with great interest the study on release of circulating tumor cells and cell-free nucleic acids in synovial sarcoma by Mihály et al. published in Diagnostic Pathology [1]. The authors reported the presence of SS18-SSX2 fusion transcript in circulation of 1 of 15 patients (6.7%), which suggests that the presence of tumor-derived cell-free RNA or circulating tumors cells (CTCs) is a rare event in patients with synovial sarcoma. We performed a study in an independent cohort of 38 patients with synovial sarcoma and our results support the findings of Mihály et al. [1]. Patients and methods We analyzed 38 blood specimens collected between 2008 and 2011 from 28 females and 10 males with the median age of 35 years at diagnosis (range: 23–62 years). The size of primary tumor ranged from 1.5 to 15 cm. Approximately 9 ml (range: 5–14.5 ml) of peripheral blood was collected into EDTA tubes before the diagnostic biopsy and before the initiation of treatment. Blood cells were separated from plasma by centrifugation (3000 rpm, 10 min, at room temperature) within 2 h of the blood draw and the bottom fraction containing * Correspondence: [email protected] 1 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA Full list of author information is available at the end of the article
erythrocytes, leukocytes, platelets, and CTCs was used to extract total RNA with TRI Reagent BD (Sigma). We employed nested RT-PCR assay that allows for detection of one CTC in 1 ml of whole blood (estimated based on the serial dilutions of HS-SY-II cells carrying SS18-SSX1 fusion in the whole blood of a healthy donor). One microgram of total RNA was reverse-transcribed with oligo(dT)12–18 primers and random hexamers using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). cDNA quality was assessed by PCR using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) primers as described previously [2]. SS18-SSX1/2 fusion junctions were detected using AmpliTaq Gold DNA Polymerase (Thermo Fisher Scientific) using primers described previously by Panagopoulos et al. [3]. The first round of amplification was performed with 2 μl of cDNA and the seco
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