Published OnlineFirst March 3, 2020; DOI: 10.1158/1078-0432.CCR-19-2297 CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING Periostin and Epithelial–Mesenchymal Transition Score as Novel Prognostic Markers for Leiomyosarcoma, Myxofibrosarcoma, and Undifferentiated Pleomorphic Sarcoma Maria Assunta Piano1, Antonella Brunello2, Rocco Cappellesso3, Paola Del Bianco4, Adriana Mattiolo1, Chiara Fritegotto1, Barbara Montini1, Carolina Zamuner5, Paolo Del Fiore6, Marco Rastrelli6, Antonio Sommariva6, Gian Luca De Salvo4, Maria Cristina Montesco5, Carlo Riccardo Rossi6,7, Vittorina Zagonel2, and Maria Luisa Calabro1 ABSTRACT ◥ Purpose: Interpatient clinical variability in soft-tissue sarcomas Results: High expression of periostin, a mesenchymal matri- (STS) highlights the need for novel prognostic markers supporting cellular protein, in sarcoma tissues (P ¼ 0.0024), its high patient risk stratification. As sarcomas might exhibit a more mes- stromal accumulation in leiomyosarcomas (P ¼ 0.0075), and enchymal or a more epithelial state, we focused on epithelial– increased circulation (>20 ng/mL, P ¼ 0.0008) were associated mesenchymal and mesenchymal–epithelial transitions (EMT/ with reduced OS. High periostin expression [HR 2.9; 95% MET) for prognostic clues, and selected three histotypes with confidence interval (CI), 1.3–6.9; P ¼ 0.0134] and circulation variable aggressiveness. (HR 2.6; 95% CI, 1.3–5.1; P ¼ 0.0086), and a mesenchymal Experimental Design: The expression of EMT/MET-related EMT score (mesenchymal vs. transitioning; HR, 5.2; 95% CI, factors was measured by qRT-PCR in 55 tumor samples from 2.1–13.0, P ¼ 0.0005) were associated with increased risk in patients with leiomyosarcoma, myxofibrosarcoma, or undifferen- multivariable models. An intrinsic or induced mesenchymal tiated pleomorphic sarcoma. The identified marker was further state enhanced chemoresistance and migration in sarcoma evaluated by IHC in 31 leiomyosarcomas and by measuring its cell lines. circulating levels in 67 patients. The prognostic value of a sarcoma- Conclusions: Although limited to a pilot study, these findings tailored EMT score was analyzed. Epirubicin chemosensitivity and suggestthatperiostinmightcontributeprognosticinformationin migration were studied in primary STS cultures. Associations with the three studied STS histotypes. Moreover, a transitioning EMT overall survival (OS) were assessed using Kaplan–Meier and Cox score measured in the tumor might predict a less active and a more regression methods. chemosensitive disease. Introduction solitary fibrous tumors, a multifaceted genetic, transcriptomic, and regulomic landscape characterizes most sarcomas (3), including leio- Soft-tissue sarcomas (STS) are rare tumors characterized by variable myosarcoma, myxofibrosarcoma, and undifferentiated pleomorphic aggressiveness and high heterogeneity, as histopathology differentiates sarcoma, which share an intrinsic molecular complexity that governs a more than 50 subtypes (1, 2). While well-defined molecular alterations relevant interpatient clinical variability. Biomarkers with accurate drive oncogenesis in few histotypes, such as synovial sarcoma and prognostic value supporting clinicopathologic parameters in patient risk stratification are urgently needed. Sarcomas are mesenchymal tumors that may express epithelial 1Immunology and Molecular Oncology, Veneto Institute of Oncology IOV–IRCCS, markers, such as E-cadherin or ZO-1, indicating the occurrence of Padua, Italy. 2Medical Oncology 1, Veneto Institute of Oncology IOV–IRCCS, mesenchymal–epithelial transition (MET) during sarcomagenesis 3 Padua, Italy. Surgical Pathology and Cytopathology, Department of Medicine, (4–6). The clinical value of epithelial and mesenchymal markers in 4 University of Padua, Padua, Italy. Clinical Trials and Biostatistics, Veneto STS needs to be fully understood. In fact, detection of E-cadherin or Institute of Oncology IOV–IRCCS, Padua, Italy. 5Anatomy and Pathological Histology, Veneto Institute of Oncology IOV–IRCCS, Padua, Italy. 6Surgical SNAIL, a mesenchymal transcriptional repressor, in tumor cells was Oncology, Veneto Institute of Oncology IOV–IRCCS, Padua, Italy. 7Department associated with favorable or poor clinical outcome in patients with of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy. leiomyosarcoma and other sarcomas, respectively (7, 8). Similarly, Note: Supplementary data for this article are available at Clinical Cancer intense staining for vimentin, a critical organizer of shape and motility Research Online (http://clincancerres.aacrjournals.org/). in mesenchymal cells, showed negative prognostic value in patients with leiomyosarcoma (7). Therefore, some histotypes exhibit a more M.A. Piano and A. Brunello contributed equally to this article. mesenchymal or a more epithelial state, which in turn governs clinical Corresponding Author: Maria Luisa Calabro, Immunology and Molecular Oncol- course, highlighting the possible clinical implication of epithelial and ogy, Veneto Institute of Oncology IOV–IRCCS, via Gattamelata 64, Padua I- 35128, Italy. Phone: 39-049-8215883; Fax: 39-049-8072854; E-mail: mesenchymal factors in sarcoma evolution. [email protected] In this monocentric, pilot study, we investigated whether the expression of epithelial–mesenchymal transition (EMT)/MET- Clin Cancer Res 2020;XX:XX–XX related factors, as single or combined parameters, are correlated with doi: 10.1158/1078-0432.CCR-19-2297 patient outcome. Three subtypes with variable aggressiveness were Ó2020 American Association for Cancer Research. selected. Three primary STS cell cultures were isolated and analyzed AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 3, 2020; DOI: 10.1158/1078-0432.CCR-19-2297 Piano et al. at À80C until RNA extraction. Histopathologic diagnosis of all Translational Relevance prospectively collected tumor samples was performed by the same Soft-tissue sarcomas (STS) are rare mesenchymal tumors of pathologists (M.C. Montesco and R. Cappellesso). relevant molecular and clinical heterogeneity. Sarcomas may Clinical, pathologic, and follow-up data of all patients were collected exhibit a more mesenchymal or a more epithelial state, which by a data manager (P. Del Fiore). This study was examined and governs clinical behavior. Thus, we focused on epithelial– approved by the IOV Ethics Committee (protocol no. 2014/91) and mesenchymal transition (EMT) for prognostic clues and per- was conducted in compliance with the guidelines of the Declaration of formed a pilot study to search for biomarkers supporting patient Helsinki. Written informed consent for the collection and analysis of risk stratification in three potentially aggressive histotypes. Mul- biological samples was obtained from all biobank participants and tivariable models showed that periostin expression and circulation prospectively enrolled patients. levels were significantly associated with increased risk in patients with STS. Moreover, stromal accumulation of periostin was asso- Isolation and characterization of primary STS cell cultures ciated with shorter survival in patients with leiomyosarcoma. After surgical resection, tumor samples were immediately processed Periostin was combined with other EMT markers in the compu- under sterile conditions by mechanical dissociation. The obtained cell tation of a sarcoma-tailored EMT score. A transitioning EMT suspension was cultured in DMEM-F12 medium (Sigma-Aldrich) score, previously shown to denote aggressive carcinomas, predicted supplemented with 20% FCS (Gibco, Thermo Fisher Scientific), a better outcome in STS, suggesting that the clinical behavior of 2 mmol/L L-glutamine (Gibco), 50 mg/mL gentamicin (Sigma- hybrid states might be related to tumor type. Moreover, a transi- Aldrich), 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 tioning state might attenuate chemoresistance and migration in mg/mL amphotericin (Gibco). This complete medium was replaced primary sarcoma cell lines. twice a week, and when cells reached confluence, they were maintained through serial passages. Short tandem repeat (STR) profiling of the primary STS cultures was performed by BMR Genomics (https://www. bmr-genomics.it/). The isolated primary cell lines were Mycoplasma- for chemoresistance and migration, properties linked to epithelial– free, as confirmed by periodical PCR check (every 10 passages for the mesenchymal plasticity (9, 10). first 30 passages, then every 30 passages), and were characterized by IHC, EMT/MET transcriptional profile, chemoresistance, and migra- tion ability. Materials and Methods Patients Quantitative mRNA analyses A screening phase was conducted to molecularly characterize 55 Total RNA, enriched with low molecular weight molecules, was tumor tissue samples retrospectively selected from a biobank of the extracted using the NucleoSpin miRNA isolation Kit (Macherey- Department of Surgery, Oncology and Gastroenterology of Padua Nagel GmbH & Co. KG) according to manufacturer's protocol University (DISCOG), and prospectively obtained during the initial from 15–30 mg of tumor tissue that was ground to a fine powder part of the study. A REMARK flow diagram of samples and analyses is under liquid nitrogen and from cell pellets of primary STS shown in Supplementary Fig. S1 (11). Criteria for patient inclusion cultures. RNA concentration and purity were assessed as reported were: primary histologically