Periostin and Epithelial–Mesenchymal Transition Score As

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 2020 American Association for Cancer Research. selected. Three primary STS cell cultures were isolated and analyzed

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at 80C 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 migration 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 confirmed leiomyosarcoma, myxofibro- previously (12). sarcoma, or undifferentiated pleomorphic sarcoma; availability of The expression of genes linked to an epithelial state (CDH1/E- fresh or frozen tumor tissue from untreated patients at the time of cadherin and ZO-1/ZO-1) and to a mesenchymal state (SNAI1/SNAIL, diagnosis; histologic confirmation of representativeness of the sample; SNAI2/SLUG, ZEB1/ZEB1, SIP1/SIP1, ACTA2/alpha-smooth muscle and follow-up of at least 3 years for the screening set (follow-up period actin or ASMA, VIM/vimentin, CDH2/N-cadherin and POSTN/peri- end: November 2018). Exclusion criteria were neoadjuvant treatment, ostin) was assessed by quantitative real-time PCR (qRT-PCR). First- ambiguous histopathologic diagnosis, and history of other cancers. strand cDNA synthesis was performed with 500 ng of total RNA using Clinical data of all retrospective cases were reviewed. RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific) Results obtained in the discovery stage were further evaluated using in a total volume of 20 mL containing the reaction buffer, 20 U of two different approaches. First, the selected marker was evaluatedbyIHC RiboLock RNase inhibitor, 1 mmol/L dNTPs, 200 U of reverse in an independent set of formalin-fixed and paraffin-embedded (FFPE) transcriptase, and 0.2 mg of random primers (Invitrogen, Thermo tissue samples retrospectively selected from the archives of the Anatomy Fisher Scientific); reactions were carried out according to the manu- and Pathological Histology Unit (IOV). This set included 31 leiomyo- facturer's protocol. qRT-PCR was carried out using Platinum SYBR sarcomas, 6 leiomyomas, and 15 normal muscular tissues (NMT). Green qPCR SuperMix-UDG (Invitrogen) in a 7900HT Real-Time Second, the circulating levels of the marker were quantified in serum/ PCR System (Applied Biosystems, Thermo Fisher Scientific). Melting plasma samples of 67 patients with STS (47 of the screening set and 20 curves were analyzed for each reaction to ensure the specificity of the prospectively enrolled with the same inclusion/exclusion criteria). The amplicons. The same positive control for each analyzed gene was run time period was April 1998–November 2015 for all cases included in the in each plate to monitor interplate variability and measurement initial discovery set, and September 1995–November 2017 for those reproducibility. Primer sets specific for intron-spanning amplicons included in the subsequent IHC evaluation and those additional patients for CDH1, SNAI1, SNAI2, ZEB1, SIP1 and ACTA2 used in qRT-PCR included in the measurement of the circulating levels of the marker. were published previously (13). Primer pairs were designed for Histologic diagnosis of all retrospectively selected cases was transcripts specific for ZO-1 (forward: 50-AGGTCTGCCGGGA- reviewed and confirmed by two expert pathologists (M.C. Montesco CAACA-30; reverse: 50-AGGATCACAGTGTGGTAAGCG-30), VIM and R. Cappellesso), according to the current WHO classification (2). (forward: 50-CGAAAACACCCTGCAATCTT-30; reverse: 50-CTGGA Biobank samples were stored at 80C until RNA extraction. Fresh TTTCCTCTTCGTGGA-30), CDH2 (forward: 50-GGACAGTTCCT- surgical tissue samples were snap frozen in liquid nitrogen and stored GAGGGATCA-30; reverse: 50-CTTGGAGCCTGAGACACGAT-30),

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and POSTN (forward: 50-TGTGGACAGAAAACGACTGTG-30; IHC reverse: 50-ATGCCCAGAGTGCCATAAACA-30). Expression levels IHC was performed automatically using the Bond Polymer Refine of the analyzed genes are reported as relative quantification (RQ) Detection kit (Leica Biosystems) in the BOND-MAX system (Leica calculated using human porphobilinogen deaminase gene as the Biosystems) on a 4-mm–thick section from each tissue sample with the DDC endogenous control with the 2 t method (14). Normalized RQ primary antibody anti-periostin (rabbit polyclonal, Abcam; dilution (nRQ) were calculated on the global mean (15). 1:800). Sections were then counterstained with hematoxylin. Appro- priate positive and negative controls were run concomitantly. IHC for miRNA quantification periostin was jointly analyzed and scored for positivity in the cells and To better evaluate the profile of the epithelial component, rarely in the stroma by two pathologists (R. Cappellesso and M.C. Montesco) foundinSTS,weincreasedthenumberofpossibleepithelialmarkers blinded to clinical data. A four-tier system was applied to score the through the selection of miRNAs known to affect EMT and to be maximum intensity of the staining in both the cells and the stroma of aberrantly expressed in STS (16, 17). miRNAs linked to EMT (miR- each tissue sample: 0 ¼ total absence, 1 ¼ weak, 2 ¼ moderate, and 3 ¼ 100–5p; ref. 18), to MET (miR-200b-3p, miR-30b-5p, and miR-30c- strong. 5p; refs. 19, 20), and myo-miRs (miR-1, miR-133a-3p, and miR- Cell pellets of primary STS cultures were formalin-fixed and 133b) were quantified using miRNA-specificlockednucleicacids paraffin-embedded using the Shandon Cytoblock Cell Block Prepa- (LNATM) PCR primer sets (Exiqon A/S). The three myogenic ration System (Thermo Fisher Scientific), and IHC was performed on miRNAs were included as further independent control of diagnosis 4-mm–thick sections from each cell block as described above using the and tumor representativeness, as they were previously shown to be primary mouse mAbs anti-ASMA (clone 1A4, Merck KGaA), anti- highly expressed in leiomyosarcoma samples (16, 17). Ten nano- desmin (D33, Abcam), anti-CD34 (QBEND-10, Abcam), anti-MDM2 grams of RNA was reverse transcribed to cDNA using the miRCURY (IF2, Thermo Fisher Scientific), and the rabbit polyclonal anti-S100 LNA Universal Reverse Transcription miRNA PCR cDNA Synthesis antibody (Dako). Kit II (Exiqon) according to the manufacturer's protocol. The assays were performed in duplicate and each miRNA was quantified in two Quantification of circulating levels of periostin independent runs using the ExiLENT SYBR Green mastermix (Exi- Circulating amounts of periostin were measured in serum or plasma qon) in a 7900HT Real-Time PCR System. To monitor the quality samples from 67 patients diagnosed with leiomyosarcoma, myxofi- and efficiency of RNA extraction, cDNA synthesis, and PCR ampli- brosarcoma, or undifferentiated pleomorphic sarcoma. Matched tis- fication, synthetic small RNA molecules (spike-ins, Exiqon) were sue-blood samples were available for 43 patients. Samples were stored added into each sample prior to RNA isolation (UniSp2, UniSp4, at 80C before testing. Quantification was carried out using a UniSp5) and prior to cDNA synthesis (UniSp6) according to the sandwich ELISA kit (DuoSet ELISA Development System Human manufacturer's protocol. Spike-in amplification was carried out for Periostin/OSF-2, R&D Systems) according to the manufacturer's each sample, and samples not ensuring the highest sensitivity were instructions. All samples were analyzed using an initial 1:25 dilution. not included in the analysis. miRNA expression levels were calcu- DDC lated using U6 as the endogenous control with the 2 t meth- EMT score analysis od (14). nRQ were normalized on the global mean (15). Criteria used The EMT score was calculated as reported previously (21), and for identifying outliers during quantification of gene-specific tailored on the markers selected as follows. A descriptive network mRNAs and miRNAs and for excluding unreliable discoveries analysis based on Pearson correlation between the logarithm levels of due to technical variability have been applied and previously all analyzed markers was performed to identify the strength and reported (12). direction of existing structures and patterns. The expressed epithelial

Table 1. Clinical and pathologic characteristics of patients with STS of the initial discovery and subsequent evaluation stages.

Patients Histotype LMSa Variable N (%) LMS MFS UPS N (%)

Age (years) ≤70 33 (60.0%) 20 (71.4%) 8 (61.5%) 5 (35.7%) 21 (67.7%) >70 22 (40.0%) 8 (28.6%) 5 (38.5%) 9 (64.3%) 10 (32.3%) Gender M 28 (50.9%) 13 (46.4%) 8 (61.5%) 7 (50.0%) 12 (38.7%) F 27 (49.1%) 15 (53.6%) 5 (38.5%) 7 (50.0%) 19 (61.3%) Grade G1 3 (5.5%) 1 (3.6%) 2 (15.4%) 0 7 (22.6%) G2 14 (25.4%) 10 (35.6%) 4 (30.8%) 0 7 (22.6%) G3 38 (69.1%) 17 (60.7%) 7 (53.8%) 14 (100%) 17 (54.8%) Tumor size ≤5 cm 13 (23.6%) 10 (35.7%) 1 (7.7%) 2 (14.3%) 16 (51.6%) >5 cm 42 (76.4%) 18 (64.3%) 12 (92.3%) 12 (85.7%) 15 (48.4%) Metastasis at diagnosis Yes 8 (14.6%) 8 (28.6%) 0 0 0 No 47 (85.4%) 20 (71.4%) 13 14 31 Survival status Alive 21 (38.2%) 8 (28.6%) 9 (69.2%) 4 (28.6%) 19 (61.3%) Dead 34 (61.8%) 20 (71.4%) 4 (30.8%) 10 (71.4%) 12 (38.7%) Histotype LMS 28 (50.9%) MFS 13 (23.6%) UPS 14 (25.5%)

Abbreviations: LMS, leiomyosarcoma; MFS, myxoﬁbrosarcoma; UPS, undifferentiated pleomorphic sarcoma. aSamples for subsequent IHC evaluation.

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variable (ZO-1) was scaled and centered and the identified mesen- had measurable values for the markers selected; specifically, 4 leio- chymal variables were linearly combined in the first principal com- myosarcomas, 3 myxofibrosarcomas, and 1 undifferentiated pleomor- ponent using the larger eigenvector extracted from the correlation phic sarcoma were excluded from this analysis. matrix as weights (21). To compute the EMT score, the difference between the mesenchymal principal component and the scaled epi- Chemosensitivity and cell migration of primary STS cell lines thelial variable was calculated and its prognostic role was assessed both Chemoresistance to epirubicin was tested by MTT assay (22). Cells in univariate and multivariable models. The EMT score was calculated were seeded in 96-well plates (3 104/well) and treated with epirubicin using the expression profile of 47 tumor tissues of the screening set that (2-fold dilutions from 40 mmol/L to 0.62 mmol/L for S78 and S79 cells,

Figure 1. Detection and prognostic significance of periostin in leiomyosarcoma, myxofibrosarcoma, and undifferentiated pleomorphic sarcoma. A, Kaplan–Meier curves for OS according to the level of expression of periostin (dichotomized according to the median nRQ value of expression) measured in tumor tissue samples obtained from 55 patients with STS of the screening set. Each sample was run in duplicate in three independent plates. The log-rank test was used to calculate the P value. HR, 95% CI, and the associated P value are also indicated. B, Represen- tative photomicrographs of the immunoreactions for periostin performed on FFPE samples of the normal muscular layer (a), leiomyoma (b), and leiomyosarcoma (c and d). Scale bars, 200 mm. Original magnification 100. C and D, Kaplan–Meier OS curves according to periostin detection in the stromal compartment or tumor cells, respectively. P values, HR, and 95% CI were calculated as reported above.

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m m and from 40 mol/L to 0.31 mol/L for S57) for 48 hours. IC50 was Table 2. Multivariable analysis for association of periostin determined by a nonlinear regression analysis using the GraphPad expression in tumor tissues and clinical variables with OS. Prism software (6.07 for Windows). Cell migration ability was analyzed by cell scratch assay (23). Cells Multivariable OS a P were seeded in 6-well plates (3 105/well) and allowed to reach Variable HR (95% CI) fl con uence. Scratching was performed with a sterile pipette tip, and the Age (years) ≤70 1 cells were incubated with complete medium with or without recom- >70 2.8 (1.4–5.8) 0.0047 binant TGFb1 (TGFb1, 1 ng/mL; R&D Systems). Phase-contrast Grade G1þG2 1 photographs of each scratch were taken at 24 and 48 hours after G3 3.3 (1.2–9.0) 0.0189 scratching. The percentage of wound closure was calculated as Periostin (nRQ) ≤1.6 1 described previously (23). At 48 hours, cells were detached by trypsin >1.6 2.9 (1.3–6.9) 0.0134 treatment (Thermo Fisher Scientific), washed with cold PBS (Oxoid) fi and processed for total RNA extraction. Abbreviation: nRQ, normalized relative quanti cation (median value). aOnly statistically significant variables were reported. Statistical analyses Quantitative variables were described as median and interquartile Fig. S2; Supplementary Table S2), advanced age, high grade, large range; categorical variables were summarized as counts and percen- tumor, and leiomyosarcoma histotype remained independently asso- tages. The median follow-up time was based on the reverse Kaplan– ciated with an unfavorable outcome in a multivariable analysis (Sup- Meier estimator. Clinical outcome was analyzed in terms of overall plementary Table S2). Evaluation of the effect of periostin adjusted for survival (OS), which was the time from the date of diagnosis to death these clinical variables confirmed that high periostin expression in from any cause. Patients who did not develop a survival event during tumor tissue was an independent, unfavorable prognostic factor, in the study period were censored at the date of last observation. The addition to advanced age and high grade (Table 2). survival probabilities were estimated using the Kaplan–Meier method and compared among strata using the log-rank test. The HRs were IHC for periostin in leiomyosarcoma obtained from univariate Cox proportional hazards regression models, To further investigate the association between periostin and out- after checking any deviation from the proportional hazards assump- come, IHC for this protein was performed on FFPE tissue samples of 31 tion. Quantitative markers entered the models as dichotomous vari- leiomyosarcomas, 6 leiomyomas, and 15 NMT. Characteristics of this ables, categorized according to their median value. Statistically sig- independent set of patients with leiomyosarcoma are shown nificant variables were entered into a multiple Cox proportional in Table 1. The estimated median follow-up time was 3.4 years hazards regression model to backward select predictors independently (1.9–9.5), with a 5-year OS of 46.2% (21.3–68.1) and a 10-year OS associated with the outcome. of 37.0% (13.6–60.9). At the time of this analysis, 38.7% (12/31) of the The association of IHC score with tissue samples was verified using patients had died. the Likelihood Ratio x2 test. The distributions of circulating levels of Tissuesampleswereclassified as either low (scores 0–1) or high periostin were compared across clinical characteristics using the (scores 2–3) for periostin detection in stroma and cells. Fig. 1B Kruskal–Wallis test. Differences in migratory capacity were assessed shows representative IHC staining for periostin in FFPE sections of using the Student t test. P values were adjusted for multiple compar- NMT (a), leiomyoma (b), and leiomyosarcoma (c and d). Stromal isons using the Benjamini–Hochberg method. periostin detection was significantly different among the three tissue All statistical tests used a two-sided 5% significance level and samples (P ¼ 0.0484; Supplementary Table S3), and significantly association measures were provided with their 95% confidence interval higher in the stroma of leiomyomas (P ¼ 0.0490) and leiomyo- (CI). Statistical analyses were performed using the SAS statistical sarcomas (P ¼ 0.0447) compared to NMT. The difference between package (SAS, rel. 9.4; SAS Institute Inc.) and RStudio (RStudio: leiomyomas and leiomyosarcomas was not significant. Neverthe- Integrated Development for R. RStudio Inc.). less, patients with leiomyosarcoma with high stromal periostin detection showed significantly reduced OS and increased risk (Fig. 1C) compared to patients with low stromal staining. Periostin Results detection in cells was lower (Supplementary Table S3) and the Prognostic value of periostin expression in leiomyosarcoma, association with OS and HR in leiomyosarcomas did not reach myxofibrosarcoma, and undifferentiated pleomorphic sarcoma significance (Fig. 1D). Characteristics of the patients included in the screening are shown in Table 1. The expression level of EMT/MET-related factors was Circulating levels of periostin analyzed in tumor tissue from 55 untreated patients. The estimated Periostin was quantified in serum/plasma samples from 67 median follow-up time was 8.2 years, (95% CI, 4.4–14.1), with a 5-year patients with STS, whose characteristics are reported in Supple- OS of 51.2% (36.9–63.8) and a 10-year OS of 33.7% (19.0–49.1). At the mentary Table S4. The estimated median follow-up time was time of this analysis, 61.8% (34/55) of the patients had died. 7.3 years (4.4–14.1), with a 5-year OS of 55.7% (42.2–67.3) and E-cadherin was not found to be expressed in any of the analyzed a 10-year OS of 34.8% (20.6–49.4). At the time of this analysis, samples. Among the expressed markers, high periostin mRNA levels 55.2% (37/67) of the patients had died. were found to be significantly associated with decreased OS and As for some patients only serum samples were available, we increased HR (Fig. 1A), whereas none of the other biomarkers was preliminarily measured protein concentration in matched serum and significantly associated with OS in univariate analysis (Supplementary plasma samples available for 12 patients. Mean levels were analyzed Table S1). and found to be comparable (meanserum ¼ 22.70, meanplasma ¼ 21.67; Among the clinicopathologic variables significantly associated with paired t test, P ¼ 0.7), suggesting that, in our hands, the type of sample decreased OS and increased risk in univariate analysis (Supplementary does not influence the assessment of periostin concentration in

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Figure 2. Circulating levels of periostin in 67 STS patients. A, Dot plot graphs showing the distribution of the levels of periostin, expressed as ng/mL, measured in serum/plasma samples according to patient characteristics. Statistical signiﬁcance of the different distribution is also reported and was calculated by the Kruskal–Wallis test. P values were adjusted for multiple comparisons using the Benjamini–Hochberg method. Median and interquartile range are also indicated. Each serum/plasma sample was tested in duplicate in two independent ELISA assays. LMS, leiomyosarcoma; MFS, myxoﬁbrosarcoma; UPS, undifferentiated pleomorphic sarcoma. B, Kaplan– Meier curves for OS according to the circulating levels of periostin. The P value was calculated by the log-rank test. HR, 95% CI, and the associated P value are also reported.

r ¼ P ¼ peripheral blood. Figure 2A shows the distribution of periostin levels tissues (Spearman correlation, s 0.435, 0.0036), suggesting that according to patient characteristics. High amounts, that is, above the tumor tissue may be the main source of circulating periostin. median concentration, of circulating periostin were significantly associated with G3 tumors and leiomyosarcoma and undifferentiated EMT score pleomorphic sarcoma histotypes. Elevated levels of circulating peri- We combined periostin expression with that of other EMT/MET ostin were significantly associated with a shorter OS (Fig. 2B), and factors in a global EMT score to study the prognostic value of the were confirmed to be an independent risk factor in a multivariable overall contribution of epithelial and mesenchymal factors in STS. model, in addition to high grade (Table 3A). Network analysis was performed to study the strength and direc- A positive and significant correlation was found between circulating tion of correlations among the EMT/MET-related factors measured levels of the soluble protein and transcripts in 43 matched tumor in our samples (Fig. 3A). This analysis showed that the most

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Table 3A. Univariate and multivariable analyses for association of all clinical variables and circulating periostin with OS.

Univariate Multivariable Variable Events/N HR (95% CI) P HR (95% CI) P

Age (years) ≤70 17/39 1 >70 20/28 1.9 (1.0–3.6) 0.0566 Gender M 19/36 1 F 18/31 1.8 (0.9–3.4) 0.0853 Grade G1þG2 9/23 1 1 G3 28/44 3.2 (1.4–6.9) 0.0040 2.4 (1.1–5.6) 0.0352 Tumor size ≤5 cm 5/12 1 >5 cm 32/55 2.2 (0.8–5.7) 0.1068 Metastasis at diagnosis No 31/60 1 1 Yes 6/7 3.2 (1.3–.9) 0.0106 2.0 (0.8–5.1) 0.1280 Histotype LMS 20/30 1 MFS 7/23 0.3 (0.1–0.7) 0.0073 UPS 10/14 1.2 (0.6–2.7) 0.5773 Circulating periostin ≤20 ng/mL 13/35 1 1 >20 ng/mL 24/32 3.1 (1.6–6.1) 0.0012 2.6 (1.3–5.1) 0.0086

Abbreviations: LMS, leiomyosarcoma; MFS, myxoﬁbrosarcoma; UPS, undifferentiated pleomorphic sarcoma.

Table 3B. Multivariable model for association of statistically signiﬁcant clinical variables and EMT score with OS.

Multivariable OS Variablea HR (95% CI) P

Age (years) ≤70 1 >70 6.0 (2.5–14.6) <0.0001 Grade G1þG2 1 G3 4.6 (1.7–12.7) 0.0034 EMT Score Tran 1 Mes 5.2 (2.1–13.0) 0.0005

Abbreviations: Mes, mesenchymal; Tran, transitioning. aOnly statistically significant variables were shown. positively inter-correlated variables were ZO-1, ASMA, ZEB1, sarcoma subtypes under study, as evidenced by the network analysis r – SIP1, vimentin, N-cadherin, and periostin ( p range: 0.40 0.87, that showed that ZO-1 expression was strongly positively correlated P ≤ 0.009), in descending order. ZO-1 expression was found to to that of mesenchymal factors. Therefore a more appropriate, mainly increase concurrently with the expression of ASMA, ZEB1, sarcoma-tailored classification should only include a transitioning and SIP1. Similarly, miR-100-5p was positively correlated to 3 and a mesenchymal state. Consequently, the first two tertiles were epithelial miRNAs, whereas the three myo-miRNAs were highly joined into a single transitioning state and tumor samples were intercorrelated and their expression was mainly linked to that of subdivided into transitioning and mesenchymal. In univariate ZEB1 and SIP1 (Fig. 3A). Conversely, the expression of two analysis, patients with a mesenchymal score showed an increased mesenchymal markers, specifically SNAIL and SLUG, was nega- risk and reduced OS (Fig. 3B). In a multivariable model, a mes- tively correlated to that of all other mesenchymal factors (Fig. 3A, enchymal EMT score was confirmed to be an independent, unfa- r P ≤ p range: 0.35 to 0.49, 0.0241). Therefore, the mesenchymal vorable prognostic factor, in addition to advanced age and G3 grade component was defined by clustering the gene expression data by (Table 3B). The distribution of samples according to the calculated principal component analysis of all mesenchymal markers except EMT score is shown in Fig. 3C. Interestingly, a cluster of leiomyo- SNAIL and SLUG. The epithelial component was defined using sarcoma samples was frankly mesenchymal. scaled ZO-1 expression levels. Moreover, miRNAs were excluded from this analysis as their relevance in calculating the global EMT In vitro chemoresistance and migration of primary STS cells status of tumors needs to be further evaluated, as reported Three primary sarcoma cell lines were isolated: S57, derived from a previously (21). myxofibrosarcoma, and S78 and S79, derived from two leiomyosar- Each tumor sample was then quantitatively scored by subtracting comas. S57 and S79 formed a uniform monolayer at confluence, the epithelial component from the mesenchymal component. The whereas the S78 culture was composed of long spindle-shaped cells obtained EMT scores, ranging from 2.68 to 5.36, were initially with a pattern of multilayered growth at confluence (Supplementary subdivided into tertiles, to adjust for the three “canonical” EMT Fig. S3A). S57 became a stable, self-immortalized cell line after the first states, that is, epithelial (from 2.68 to -0.55), transitioning/hybrid 30 passages (p), and it maintained a reliable proliferative rate. S79, after (from 0.55 to 0.15), and mesenchymal (higher than 0.15). a period of slow growth, reached p37, whereas S78 showed late However, a canonical, “pure” epithelial profile does not apply to the senescence at p21. STR profiles of these cell lines (Supplementary

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Figure 3. EMT score in STS tissues, and chemoresistance and migration of primary STS cell lines. A, Pearson correlation network analysis of the analyzed biomarkers. Each node in the network represents an epithelial (E), a mesenchymal (M), or a myo-miR (My) biomarker. The thickness of the edge between two nodes represents the strength of correlation. Positive correlations are green, negative correlations are red. Correlations ranged from 0.5 to 1. Correlations with absolute value less than 0.4 are not drawn. B, Kaplan–Meier curves for overall survival according to the EMT score. P values were calculated as reported above. C, Vertical bar charts of EMT scores of 47 samples of STS aligned from the most transitioning to the most mesenchymal. Leiomyosarcoma (L) are reported in red, myxoﬁbrosar- coma (M) in green, and undifferentiated pleomorphic sarcoma (U) in blue, and the histotype is also indicated under each bar. D, Dose–response curves of epirubicin sensitivity of primary STS cell lines. The results, obtained in S57 cells, derived from a myxoﬁbrosarcoma (MFS), in S78 and S79 cells, derived from two leiomyosarcomas (LMS), are the mean SD of two independent experiments performed in quadruplicate. Dotted line indicates 50% cell viability relative to untreated cells. The experiments were performed at passage (p) 90 and p92 of S57, p14–15 of S78, and p10–12 of S79. EMT score values of cell lines are reported in the legend. E, Represen- tative images of cell scratch assays performed in S57 (p87–90) and S79 (p35–37) cells after 24 and 48 hours of

mock- or TGFb1 treatment. The bar charts on the right side of the images show the mean SD of two independent experiments performed in duplicate at 24 and 48 hours. Differences in migratory capacity were assessed using the Student t test and P values were adjusted for multiple comparisons (, P < 0.05).

Table S5) and IHC analyses (Supplementary Fig. S3B) confirmed their EMT profile, whereas S78 and S79 exhibited a mesenchymal score, unique identity and their derivation. as reported in Fig. 3D. As sarcoma aggressiveness may be linked to increased therapeu- To examine the migration ability of S57 and S79 cells, cell scratch tic resistance, we investigated the in vitro sensitivity of these cell assays were performed in complete medium (mock) and after EMT fi b lines to epirubicin, an anthracycline used as rst-line chemotherapy induction by TGF 1 treatment (Fig. 3E). Mock-treated S79 cells in aggressive STS subtypes. As shown in Fig. 3D, S57 cells were showed a significantly faster migration into the scratch compared foundtobemoresensitivetothecytotoxic activity of epirubicin with S57 cells at 24 hours. EMT induction significantly enhanced ¼ m (IC50 2.5 0.5 mol/L) compared to the two leiomyosarcoma cell migration in both cell lines at 24 hours, and promoted a marked m lines, as S79 showed a 5.6-fold higher IC50 (13.9 0.61 mol/L) and morphologic change in S57 cells, characterized by a criss-cross pattern S78 was resistant to all analyzed doses. qRT-PCR analyses and EMT of growth (Fig. 3E). qRT-PCR analyses confirmed a transcriptional score computation showed that S57 was characterized by a hybrid reprogramming in EMT-induced cells, resulting in a shift to a more

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mesenchymal profile, as indicated by the EMT score, even though S57 ECM components, but not periostin, responsible for tissue stiffness, maintained a hybrid state (Fig. 3E). which could represent new therapeutic targets for treatment of this common pathology (33). Therefore, periostin too might be a relevant player in this pathologic condition, and further studies are needed to Discussion prove this incidental, interesting finding. Periostin is a matricellular protein implicated in EMT and tumor The lack of expression of E-cadherin in all our tissue samples may be progression (24). Under physiologic conditions, it localizes at linked to the number of analyzed samples as well as to the high collagen-rich regions to contribute to the mechanical strength of expression levels of mesenchymal transcriptional repressors of this connective tissues (24), that is, the site of sarcoma origin. Diverse structural epithelial protein. In parallel, we measured the expression of normal fetal and adult tissues express periostin at variable levels, another epithelial marker, ZO-1, which was found to be positively mainly depending on stromal abundance (25), but its marked intercorrelated with mesenchymal markers in the three histotypes, as upregulation and secretion have been evidenced in sites of injury previously shown for E-cadherin (6). This finding suggests that some and inflammation as well as in several tumor types, highlighting STS might express hybrid, metastable profiles between the epithelial and its relevance in the neoplastic process (26). Its protumorigenic mesenchymal states (4, 5) not necessarily linked to the presence of activity is linked to integrin binding with subsequent activation of E-cadherin. Hybrid states of EMT were initially demonstrated in Akt/PKB- and FAK-mediated signaling pathways as well as to its circulating carcinoma cells and were shown to characterize more ability to promote remodeling of extracellular matrix (ECM), invasive carcinomas (34, 35). We applied an EMT scoring method (21) processes implicated in cell survival, invasiveness, metastasis, and based on the expression of a small set of genes including periostin, and EMT (24, 27–29). Indeed, periostin overexpression and increased subdivided tumor samples into transitioning and mesenchymal. Our detection, mainly in tumor stroma, were associated with poor data showed that a transitioning EMT score identified STS patients with prognosis in many neoplasms, including colorectal carcinoma, a better survival. These findings, although limited to a small STS cohort, epithelial ovarian cancer, esophageal adenocarcinoma, non–small are inagreementwith in silico RNA-seq data analyses of 250 STSsamples cell lung cancer, breast cancer, hepatocellular carcinoma, and that correlated a hybrid EMT signature with a favorable outcome (36). prostate cancer (refs. 24, 26, and references therein). Recent studies conducted in carcinomas highlighted that Concerning sarcoma, to date periostin expression was shown to EMT is frequently not complete in tumor cells, which were shown be associated with poor survival in patients with osteosarcoma (30). to exploit multiple transitioning states (9, 10). It is conceivable that Interestingly, periostin was found to be mainly localized in tumor different hybrid forms might be linked to opposite outcomes cells in osteosarcoma tissues, and correlated with vessel densi- according to the epithelial or mesenchymal state ab initio.Infact, ty (31). Scanty data are instead available on its detection in in epithelial tumors, hybrid forms with a higher content of epithelial STS (32). Our results clearly show for the first time that this proteins and limited mesenchymal factors might exhibit an aggres- multifunctional protein may be overexpressed in tissue samples of sive, metastatic behavior (37). The term “epithelial–mesenchymal the three studied subtypes. Moreover, it may accumulate at plasticity” resumes the multiple bidirectional EMT/MET pathways high levels in the stromal compartment of leiomyosarcomas. High controlling the flexible attitude of a tumor cell in terms of metastatic expression in ex vivo STS tumor samples and high accumulation in potential, stemness, and therapeutic resistance (9, 10). In light of leiomyosarcoma stroma were significantly associated with poor these recent findings, plasticity programs in sarcoma cells need to be survival, indicating that periostin may be a reliable indicator of an fully investigated. Partial EMT states in sarcoma might derive from active tumor also in patients affected by leiomyosarcoma, myxofi- the acquisition of few epithelial features in a massive mesenchymal brosarcoma, or undifferentiated pleomorphic sarcoma. As the landscape, thus possibly attenuating the aggressive nature of these sample size of this pilot study is small, further studies are needed tumors. To explore this hypothesis, we isolated three primary to validate our findings in an independent and larger STS cohort, sarcoma cell lines that showed different, intrinsic EMT states, in possibly also including patients affected by other STS histotypes, line with our data on tumor tissues. A self-immortalized cell line and to assess whether this matricellular component might repre- derived from a myxofibrosarcoma exhibited a transitioning EMT sent a new therapeutic target in aggressive STS. profile and showed a sensitivity to epirubicin higher than that As other matricellular proteins, periostin is measurable in body measured in two leiomyosarcoma cell lines, characterized by a fluids, and its increased circulating amounts were associated more mesenchymal state. Moreover, an intrinsic or induced mes- with aggressiveness in different tumor types (24). Here, data obtained enchymal state was found to be associated with increased migration. in 67 STS patients supported the prognostic role of this protein, Although preliminary, these findings suggest that the attenuated likely secreted by the tumor tissues, as circulating marker. Given the aggressiveness of sarcomas residing in a hybrid state might be large amount of data indicating that periostin, measured in solid and/ linked to reduced chemoresistance and migration. or liquid biopsies, might be a prognostic factor in several tumor types, In conclusion, results of this exploratory study suggest that expres- its usefulness as a biomarker for risk assessment on a routine basis sion and circulation of periostin might support clinic-pathologic should be considered. It has to be pointed out that analyses of parameters in risk stratification of patients with the three analyzed association with OS in our exploratory study are data driven, and STS histotypes. An EMT score including periostin might also add further analyses are needed to establish whether universal cut-off valuable information. Interestingly, our data highlight that, while a values for periostin expression and plasmatic levels can be determined transitioning EMT state in carcinomas is associated with tumor or, alternatively, adjusted according to the clinical setting. aggressiveness, a hybrid profile in STS might predict a more favorable No relevant differences were found between detection of periostin in outcome, suggesting that the clinical relevance of the hybrid EMT leiomyomas and leiomyosarcomas by IHC. Despite the low number of phenotypes might depend on tumor type. In vitro studies suggested cases analyzed, this is not a surprising result since leiomyoma is a that a hybrid EMT state might attenuate aggressiveness of sarcoma fibrotic, benign tumor mainly characterized by massive ECM accu- cells by reducing therapeutic resistance and migration. Further clinical mulation and remodeling (33). Attention has been focused on several and biological studies are needed.

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Disclosure of Potential Conflicts of Interest Acknowledgments No potential conflicts of interest were disclosed. The authors thank Giuseppe Opocher for constant support, Maura Digito and Clara Benna of the biobank of tumor tissues of the Department of Surgery, Oncology and Gastroenterology of the University of Padua (Padua, Italy), all the persons who Authors’ Contributions contributed to the establishment and governance of the biobank, Sandro Pasquali and Conception and design: A. Brunello, M.L. Calabro Mark Boyd for helpful comments, Claudia Sedda for contributing to the assessment of Acquisition of data (provided animals, acquired and managed patients, provided circulating periostin, Christina Drace for help in preparing the manuscript and facilities, etc.): A. Brunello, R. Cappellesso, P. Del Fiore, M. Rastrelli, A. Sommariva, Pierantonio Gallo for artwork. This work was supported by intramural grants from 5 M.C. Montesco, C.R. Rossi, V. Zagonel 1000-IOV2010 (grant CUP no. J94G13000140001, to M.L. Calabro and A. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Brunello) and from 5 1000-IOV2012 (grant CUP no. J96D16000040005, to M. computational analysis): M.A. Piano, A. Brunello, R. Cappellesso, P. Del Bianco, L. Calabro, A. Brunello, M.C. Montesco, and C.R. Rossi). M.A. Piano, A. Mattiolo, C. A. Mattiolo, C. Fritegotto, B. Montini, C. Zamuner, G.L. De Salvo, M.C. Montesco, Fritegotto, B. Montini, and C. Zamuner were recipients of a Ricerca Corrente M.L. Calabro fellowship, Italian Ministry of Health (IMH). IMH funding was also used for payment Writing, review, and/or revision of the manuscript: M.A. Piano, A. Brunello, of the publication fee. R. Cappellesso, P. Del Bianco, A. Mattiolo, B. Montini, A. Sommariva, M.C. Montesco, M.L. Calabro The costs of publication of this article were defrayed in part by the payment of page Administrative, technical, or material support (i.e., reporting or organizing data, charges. This article must therefore be hereby marked advertisement in accordance constructing databases): A. Brunello, C. Zamuner, P. Del Fiore with 18 U.S.C. Section 1734 solely to indicate this fact. Study supervision: G.L. De Salvo, C.R. Rossi, V. Zagonel Other (biological assays, molecular and biological data analyses): M.A. Piano Received July 15, 2019; revised December 17, 2019; accepted February 28, 2020; Other (molecular and biological analyses): A. Mattiolo, C. Fritegotto, B. Montini published first March 3, 2020.

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Periostin and Epithelial−Mesenchymal Transition Score as Novel Prognostic Markers for Leiomyosarcoma, Myxofibrosarcoma, and Undifferentiated Pleomorphic Sarcoma

Maria Assunta Piano, Antonella Brunello, Rocco Cappellesso, et al.

Clin Cancer Res Published OnlineFirst March 3, 2020.

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