Abstract 447: Circulating TP53 tumor DNA as a potential biomarker for pre-operative identification of uterine leiomyosarcoma
Authors: Lopategui Jean R. , Balzer Bonnie , Wang Yizhou , Santiskulvong Chintda , Hyunh Carissa A. , Ong Chun Yat , Arana Manuel , Gayhart Matthew , Amersi Farin , Silberman Allan W. 
Published: July 2021

Abstract
Uterine leiomyosarcoma (LMS) is a highly aggressive but rare malignancy with a dismal prognosis. In nearly all cases, LMS is unsuspected prior to resection for a presumed leiomyoma. The risk of occult uterine LMS is reported to be 0.2% (1 in 500) in the largest cohort study of women undergoing surgery for presumed fibroid disease. The major problem is that there is no reliable preoperative method to distinguish LMS from a leiomyoma (LM) or STUMP (smooth muscle tumor of uncertain malignant potential), and the patient's prognosis for LMS is even poorer if the tumor is transected, which occurs in procedures performed for LM. Effective therapy for LMS is lacking and most patients eventually succumb to the disease. Thus, there is a considerable need for a reliable preoperative test to triage patients into the proper surgical procedure for LMS. TP53 alterations are reportedly associated with uterine LMS in about 40% of cases. We screened plasma for TP53 variants in patients with pathologically confirmed LMS. We evaluated TP53 circulating tumor DNA (ctDNA) by deep next-generation sequencing in a cohort of 7 patients with LMS, 1 patient with STUMP, and 3 patients with LM. Clinical data were reviewed to assess disease burden. LMS patients' tumor burden ranged from no evidence to extensive metastatic disease. DNA extraction was performed using the QIAamp DNA Micro Kit, QIAamp DNA FFPE Tissue Kit, or QIAamp MinElute ccfDNA Mini Kit. DNA quality was assessed using the TapeStation Genomic DNA kit. NGS libraries targeting the entire exonic region of the p53 gene were prepared using the QIASeq Targeted DNA Custom Panel. Sequencing was performed on the MiSeq system using 150bp paired end sequencing with a minimum read depth of 2x0.5M reads for plasma and 2x1M reads for formalin-fixed paraffin embedded and fresh-frozen tissue samples. Raw sequencing data were demultiplexed by bcl2fastq to generate the FASTQ files. Then raw sequencing reads were aligned to the human reference genome GRCh38 by BWA-MEM 0.7.9a-r786. SmCounter2 was used for p53 variants calling and variants quality control. We discovered TP53 ctDNA variants in 5 of 7 (71.4%) LMS cases including p.E258V, p.R248Q, P.R337C, p.E221*, p.R174_R175delinsWC, but not in the control cases of LM or STUMP. In the 5 plasma samples with TP53 mutated LMS, the patients had extensive metastatic disease and in the 2 TP53 wild-type LMS and STUMP cases, the patients had no evidence of disease. To our knowledge, this is the first pilot study to demonstrate the comparative use of TP53 ctDNA in patients with LMS, LM, and STUMP. Further study of these rare LMS is needed to determine the preoperative utility of NGS TP53 ctDNA mutational status as a useful molecular biomarker to help guide surgery and avoid unwarranted manipulation and pelvic contamination of undetected LMS. Supported by the Gottlieb, Buss and Snyder Endowments in Surgical Oncology
Citation Format: Jean R. Lopategui, Bonnie Balzer, Yizhou Wang, Chintda Santiskulvong, Carissa A. Hyunh, Chun Yat Ong, Manuel Arana, Matthew Gayhart, Farin Amersi, Allan W. Silberman. Circulating TP53 tumor DNA as a potential biomarker for pre-operative identification of uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 447.