Evaluation of Salivary Exosomal Chimeric GOLM1-NAA35 RNA As a Potential Biomarker in Esophageal Carcinoma

Published OnlineFirst February 11, 2019; DOI: 10.1158/1078-0432.CCR-18-3169

Precision Medicine and Imaging Clinical Cancer Research Evaluation of Salivary Exosomal Chimeric GOLM1- NAA35 RNA as a Potential Biomarker in Esophageal Carcinoma Yusheng Lin1,2,3, Hongmei Dong1,3,4, Weilun Deng3, Wan Lin3, Kai Li3, Xiao Xiong3, Yi Guo5, Fuyou Zhou6, Changchun Ma7, Yuping Chen8, Hongzheng Ren3, Haijun Yang9, Ningtao Dai9, Lang Ma10, Stephen J. Meltzer11, Sai-Ching J.Yeung12,13, and Hao Zhang1,2,3,4

Abstract

Purpose: Transcriptionally induced chimeric RNAs are an qRT-PCR and analyzed for diagnostic accuracy, longitudinal important emerging area of research into molecular signatures monitoring of treatment response, and prediction of pro- for biomarker and therapeutic target development. Salivary gression-free survival (PFS). exosomes represent a relatively unexplored, but convenient, Results: Exosomal G-NchiRNA was readily detectable in and noninvasive area of cancer biomarker discovery. However, ESCC cells and nude mouse ESCC xenografts. SeG-NchiRNA the potential of cancer-derived exosomal chimeric RNAs in levels reﬂected tumor burden in vivo and correlated with tumor saliva as biomarkers is unknown. Here, we explore the poten- G-NchiRNA levels. In prospective studies of a training cohort tial clinical utility of salivary exosomal GOLM1-NAA35 (n ¼ 220) and a validation cohort (n ¼ 102), seG-NchiRNA chimeric RNA (seG-NchiRNA) in esophageal squamous cell levels were substantially reduced after ESCC resection. More- carcinoma (ESCC). over, seG-NchiRNA was successfully used to evaluate chemo- Experimental Design: In a retrospective study, the prog- radiation responsiveness, as well as to detect disease progres- nostic signiﬁcance of G-NchiRNA was determined in ESCC sion earlier than imaging studies. Changes in seG-NchiRNA tissues. The correlation between seG-NchiRNA and circulat- levels also predicted PFS of patients after chemoradiation. ing exosomal or tumoral G-NchiRNA was ascertained in Conclusions: SeG-NchiRNA constitutes an effective candi- cultured cells and mice. In multiple prospective cohorts date noninvasive biomarker for the convenient, reliable assess- of patients with ESCC, seG-NchiRNA was measured by ment of therapeutic response, recurrence, and early detection.

Introduction 1Institute of Precision Cancer and Pathology, Jinan University Medical College, Cancer-specific macromolecules, such as mutated or chimeric 2 Guangzhou, Guangdong, China. Department of Immunotherapy and Gastro- nucleic acids and proteins, often find their way into the blood and intestinal Oncology, Affiliated Cancer Hospital of Shantou University Medical fl 3 various bodily uids, fostering the development of a "liquid College, Shantou, Guangdong, China. Cancer Research Center, Shantou Uni- – versity Medical College, Shantou, Guangdong, China. 4Department of Pathology, biopsy" for cancer (1). Exosomes are small (30 100 nm) mem- Jinan University Medical College, Guangzhou, Guangdong, China. 5Endoscopy brane-bound vesicles containing RNA and protein cargo, and Center, Affiliated Cancer Hospital of Shantou University Medical College, Shan- secreted by eukaryotic cells into circulation (2). The contents of tou, Guangdong, China. 6Department of Thoracic Surgery, Anyang Tumor cancer cell–derived exosomes may potentially serve as a source of 7 Hospital, Anyang, Henan, China. Department of Radiation Oncology, Affiliated tumor biomarkers. The saliva is a readily accessible bodily fluid, Cancer Hospital of Shantou University Medical College, Shantou, Guangdong, and salivary exosomal contents have recently been investigated China. 8Department of Thoracic Surgery, Affiliated Cancer Hospital of Shantou University Medical College, Shantou, Guangdong, China. 9Department of Pathol- for diagnosis and prognosis (3, 4). ogy, Anyang Tumor Hospital, Anyang, Henan, China. 10Department of Gastro- Golgi membrane protein 1 (GOLM1, also known as Golgi enterology, University of Texas MD Anderson Cancer Center, Houston, Texas. phosphoprotein 2 and Golgi membrane protein GP73) is a type 11Division of Gastroenterology, Department of Medicine, Johns Hopkins Univer- II Golgi membrane protein of unclear function, but its involve- sity School of Medicine and Sidney Kimmel Comprehensive Cancer Center, ment in hepatocellular carcinoma and ability to promote cancer 12 Baltimore, Maryland. Department of Emergency Medicine, University of Texas metastasis have been amply documented (5). The GOLM1 gene is MD Anderson Cancer Center, Houston, Texas. 13Department of Endocrine Neoplasia and Hormonal Disorders, University of Texas MD Anderson Cancer located on human chromosome 9q21.33, along with its closest Center, Houston, Texas. neighbor, the Na-acetyltransferase 35 gene (NAA35, also known as MAK10). NAA35 is an auxiliary subunit of the N-terminal Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). acetyltransferase C complex. We previously screened a set of 32 chimeric RNAs known to be differentially expressed in cancer (6). Y. Lin, H. Dong, and W. Deng are the co-first authors of this article. In esophageal squamous cell carcinoma (ESCC), GOLM1-NAA35 Corresponding Author: Hao Zhang, Jinan University Medical College, 601 chimeric RNA (G-NchiRNA) is highly expressed relative to Huangpu Avenue West, Guangzhou, Guangdong 510632, China. Phone/ matched adjacent nonmalignant esophagus as well as to normal Fax: 8620-8522-3480; E-mail: [email protected] esophagus from subjects without ESCC (7). Mechanistic studies doi: 10.1158/1078-0432.CCR-18-3169 have shown that G-NchiRNA encodes a secreted chimeric protein, 2019 American Association for Cancer Research. and is generated by transcription read-through/splicing or trans-

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included a discovery cohort, a training cohort, and a validation Translational Relevance cohort. In the discovery phase, 10 patients with ESCC who were to Because most solid tumors are currently diagnosed by receive surgery were recruited at CHSUMC, and 8 healthy volun- invasive and/or imaging procedures, noninvasive assays are teers were recruited in Shantou from May 1, 2015 to June 30, urgently needed. Here, we describe the discovery and evalu- 2015. From July 1, 2015 to September 30, 2016, a training cohort ation of a transcription-induced chimeric RNA as an exosomal of 275 patients with ESCC and 65 healthy volunteers were biomarker of cancer in cells, mice, and prospective patient similarly recruited in Shantou. Concurrently with the recruitment cohorts. Salivary exosomal levels of G-NchiRNA (designated for the training cohort, a validation cohort of 124 patients with seG-NchiRNA) accurately detected early- and advanced-stage ESCC were recruited at ATH and 42 healthy volunteers were esophageal squamous cell carcinomas (ESCC) in training and recruited in Anyang. From July 1, 2018 to November 10, 2018, validation cohorts, reflected therapeutic response, and pre- another cohort including 10 patients with ESCC who were to dicted progression-free survival in patients undergoing che- receive surgery were recruited at CHSUMC, and 10 healthy moradiation. These discoveries highlight a seG-NchiRNA assay volunteers were recruited in Shantou to evaluate the distribution that does not require a blood draw and enables serial testing pattern of seG-NchiRNA in salivary fractions (cell pellet, exo- during the clinical course of patients with ESCC. To our somes, and exosome-depleted supernatant). All healthy subjects knowledge, this is the first report of an exosomal chimeric were approached for participation in the study at public places RNA as a disease biomarker. This biomarker assay has the (e.g., parks, senior activity centers, and shopping areas); they were potential to be developed for cancer surveillance, early diag- matched to at least one ESCC case for gender, age, and tobacco nosis, and treatment response, with minimal patient discom- usage and were not eligible if they had any history of malignancy, fort and cost. severe oral disease, diabetes, lung disease, renal/hepatic dysfunc- tion, severe immune alterations, and cardiovascular event in the past 6 months. Inclusion and exclusion criteria of training and validation cohorts are shown in Supplementary Fig. S8. The cases splicing, but not by mutational events that produce a fusion were selected on the basis of new pathologic diagnosis of ESCC, gene (7). without anticancer treatment. The median follow-up time was 15 ESCC is the third most common cancer of the gastrointestinal months (range: 8–22). tract and the sixth leading cause of cancer-related death world- The pathologic stage was assessed according to the Union wide (8–11). Currently, there are no widely accepted biomarkers for International Cancer Control (UICC) Tumor-Node-Metastasis for ESCC screening, treatment response, or recurrence. Endoscop- (TNM) staging system (7th edition)(12). Patients with stage I/IIa ic examination/biopsy and imaging studies are widely used were classified as early-stage patients and patients with stage IIb/ diagnostic and monitoring approaches; these approaches are III/IV were classified as late-stage ones. At CHSUMC and ATH, 87 either invasive (endoscopy) or nonsensitive (imaging) as screen- and 59 patients, respectively, underwent surgical resection of ing modalities. Recently, minimally invasive technologies such as ESCC, and were further analyzed as the surgical subcohorts. Forty cytosponge or transnasal endoscopy have become available. patients underwent chemoradiation at CHSUMC, and were fur- However, these tests have not been popularized because of ther analyzed as the chemoradiation subcohort. There were only cost and discomfort. Hence, specific and convenient biomarkers 10 chemoradiation patients at ATH, which were too few to be are badly needed to optimize diagnosis and prognosis of ESCC. analyzed as a subcohort (Supplementary Fig. S8B). The chemor- Here, we evaluated the diagnostic potential of exosomal adiation regimen was standard-of-care treatment that consisted G-NchiRNA in tissues and saliva from mice and patients with of paclitaxel (45 mg/m2) and cisplatin (25 mg/m2) on days 1–3 ESCC. Because saliva collection is noninvasive and painless, and intensity-modulated conformal radiotherapy (60 Gy in we further evaluated the clinical utility of salivary exosomal 30 fractions). Responses to chemoradiation were assessed accord- G-NchiRNA (seG-NchiRNA) as a candidate convenient, robust ing to Response Evaluation Criteria in Solid Tumors (RECIST). biomarker assay for therapeutic response, recurrence, and early Written informed consents were obtained from all participants in detection in two prospective ESCC patient cohorts. accordance with the principles established by the Helsinki Dec- laration. The clinical study was approved by the Institutional Ethics Committees and conducted under Institutional Review Materials and Methods Board–approved protocols of CHSUMC (IRB serial number: Study population #04-070) and ATH (AZLL022016008161201), which included Shantou, Guangdong, China is located in a coastal region with patients from the respective hospitals and healthy subjects from a high incidence of ESCC. Anyang, Henan, is located in another public places. high-incidence region in central China. This study involves a retrospective tissue archive, and a prospective observational clin- Statistical analysis ical study including three cohorts from two institutions: (i) All statistical analyses were performed using the SPSS 19.0 Cancer Hospital of Shantou University Medical College statistical software package (SPSS Inc.) and R V.3.33 (The R (CHSUMC, Shantou, Guangdong, China) and (ii) Anyang Tumor Project for Statistical Computing, http://www.r-project.org). Hospital (ATH, Anyang, Henan, China). Summary statistics reporting means, SE, and 95% confidence The retrospective tissue archive consisted of 120 pairs of frozen intervals were stated as appropriate. Statistical methods used human ESCC specimens and adjacent tissues that were banked included t test, one-way ANOVA, Shapiro–Wilk test, Brown– from June 1, 2009 to August 31, 2011 at CHSUMC. The demo- Forsythe test, Wilcoxon matched-pair signed-rank test, Pearson graphics and clinicopathologic characteristics of this cohort are correlation, logistic regression, ROC analysis, Kaplan–Meier summarized in Supplementary Table S1. The prospective study survival analysis, and Cox proportional hazard modeling.

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Details about statistical analysis are provided in online bases in Fig. 1A), was enriched in human ESCC versus nonneo- Supplementary Materials and Methods. plastic esophageal tissues, suggesting this chimeric RNA as a Details for cells, animals, exosomes, and biochemical assays are promising biomarker for ESCC (7). Therefore, we performed included in online Supplementary Materials and Methods. a retrospective study to investigate the association between tissue G-NchiRNA levels and ESCC prognosis in 120 patients. An optimal discriminative cut-off value according to Youden index Results (i.e., relative tissue G-NchiRNA expression ¼ 0.019) was chosen to Aberrant G-NchiRNA levels in ESCC tissue predict prognosis classify patients into either high expression (n ¼ 57) or low We previously showed that G-NchiRNA, which contains a expression (n ¼ 63) group (Supplementary Table S1). High discernable 50 splice donor site from GOLM1 fused to a 30 splice G-NchiRNA expression was associated with poor differentiation acceptor site from NAA35 at its RNA–RNA junction (capitalized and lymph node metastasis (P ¼ 0.001 and P ¼ 0.017,

Figure 1. G-NchiRNA in human ESCC and its detection in exosomes. A, Schematic diagram of the G-NchiRNA. Coding exons are represented by tall blocks, introns by horizontal lines, and 50-and30- UTR by short blocks. Arrows indicate the direction of transcription of parental genes. Transcription read-through and splicing produce a chimeric mRNA with substitution of the last 25 amino acids of GOLM1 with 26 new amino acids resulting from the antisense DNA sequence in the 30 UTR of NAA35. Splice junctions are shown in capital letters. Sequences in black are in introns; red, from NAA35; blue, from antisense GOLM1. B, The relative level of G-NchiRNA in ESCC tissues was measured by qRT-PCR. Kaplan–Meier analysis shows that the OS was significantly better in patients with low expression of G-NchiRNA than those with high expression (P < 0.001, log rank test). C, Immunoblotting of exosomal membrane markers in exosomes purified from media conditioned by NE2, HK2-shCtrl, HK2-shG-N cells, TE1-shCtrl and TE1-shG-N cells. D, Transmission electron micrograph of exosomes purified from media conditioned by ESCC cells. Scale bar, 200 nm. E, The expression of G-NchiRNA (relative to NE2 cells) in cell lysate (left) and exosomes in conditioned media (middle) from NE2, HK2-shCtrl, and HK2-shG-N cells. Ratio of secreted exosomal G-NchiRNA to cell lysate G-NchiRNA from above cells (right). F, The expression of G-NchiRNA (relative to NE2 cells) in cell lysate (left) and exosomes (middle) from NE2, TE1-shCtrl, and TE1-shG-N cells. Ratio of secreted exosomal G-NchiRNA to cell lysate G-NchiRNA from above cells (right). Samples shown are representative of three independent experiments. Error bars, SEM (, P < 0.001 by one-way ANOVA with post hoc Dunnett test).

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2 respectively; x test; Supplementary Table S1). Moreover, Kaplan– and saliva was collected at 4-day intervals thereafter (Fig. 2A). Meier analysis demonstrated that high G-NchiRNA expression Knockdown of G-NchiRNA decreased tumor growth [P < 0.01 for was significantly associated with shorter overall survival (OS) all timepoints beyond 4 days (one-way ANOVA with post hoc than low G-NchiRNA expression (P < 0.001, log-rank test; Fig. 1B). Tukey test; Fig. 2B, left)] as well as tumor weight (P < 0.001, Multivariable Cox regression analysis showed that G-NchiRNA t test; Fig. 2B, right), suggesting that G-NchiRNA promoted ESCC expression in ESCC tissues was a significant independent predic- progression (Fig. 1B; Supplementary Tables S1 and S2). Isolation tor of OS (HR ¼ 2.49, 95% CI: 1.51–4.11; P < 0.001; Supple- of exosomes from mouse saliva was confirmed by immunoblot- mentary Table S2). ting and TEM (Fig. 2C and D). G-NchiRNA from saliva, blood, and tumor lysate of each mouse was measured by qRT-PCR. Exosomal G-NchiRNA levels correlate with intracellular SeG-NchiRNA increased with tumor growth only in HK2-shCtrl G-NchiRNA levels in ESCC cells mice, but not in HK2-shG-N mice (P < 0.05 for all timepoints; We hypothesized that G-NchiRNA is exported from ESCC cells one-way ANOVA with post hoc Tukey test; Fig. 2E). These results by exosomes. To validate this hypothesis, G-NchiRNA was first demonstrated that seG-NchiRNA increased in parallel with silenced in two ESCC cell lines (HKESC-2 and TE1) (7) by two tumor growth, and that xenografts were a specific source of specific shRNAs (shG-N #1 and #2; Supplementary Fig. S1A seG-NchiRNA. At the end of each experiment, we collected saliva, and S1B), while parental genes were not significantly affected blood, and tumor tissues from each mouse; the amount of (Supplementary Fig. S1A, S1C, and S1D). As shown by qRT-PCR, G-NchiRNA in tumor lysate significantly correlated with its both shRNAs efficiently knocked down G-NchiRNA levels in two amount in salivary exosomes and in serum exosomes (r ¼ cell lines (P < 0.001 for both, one-way ANOVA with post hoc 0.691 and P ¼ 0.003; r ¼ 0.610 and P ¼ 0.008, respectively, Dunnett test; Supplementary Fig. S1B); shG-N #2 (more efficient, Pearson correlation test; Fig. 2F, left and middle). In cells treated hereafter designated shG-N) was chosen for subsequent experi- with shG-N, the ratio of G-NchiRNA in salivary exosomes relative ments. Next, we isolated exosomes from conditioned media of to tumor tissue [0.805 0.147 (SD)] was comparable with the either ESCC cell line or immortalized human esophageal epithe- ratio of serum exosomes relative to tumor tissue (0.649 0.261) lial cell line (NE2). Isolation of exosomes was confirmed by and not statistically different from each other; similar results were immunoblotting of exosomal markers (ALIX, CD9, TSG101, observed in cells treated with shCtrl (0.547 0.227 vs. 0.429 and CD63) (13) (Fig. 1C) and transmission electron microscopy 0.232, respectively; Fig. 2F, right). Taken together, these in vivo (TEM; i.e., spherical membrane-bound particles with diameters data suggest that seG-NchiRNA correlates with tumor extent and between 30 and 100 nm) (14, 15) (Fig. 1D). G-NchiRNA was can be used to monitor tumor burden. significantly more abundant in both ESCC cells versus nonneo- plastic NE2 cells, and shG-N decreased G-NchiRNA levels versus a Diagnostic potential of seG-NchiRNA in early- and advanced- nonspecific control shRNA (shCtrl) in both cell lines (P < 0.001 stage pretreated patients with ESCC for indicated comparisons; Fig. 1E and F, left). G-NchiRNA was To translate our findings from cells and mice to patients, an also significantly more abundant in exosomes from conditioned investigation was carried out in a discovery cohort of 10 patients media from both ESCC cell lines than in exosomes from NE2 with ESCC and 8 healthy subjects (Fig. 3A–F). Isolated salivary cell–conditioned medium, and its levels were significantly low- exosomes were first characterized by immunoblotting and TEM ered by shG-N versus shCtrl (P < 0.001 for the indicated (Fig. 3A and B). Nanoparticle tracking analysis further confirmed comparisons; Fig. 1E and 1F, middle). Of note, ratio of exosomal that human exosomes had an average diameter of 99 nm to cellular G-NchiRNA in NE2 cells [0.290 0.011 (SD)] was (Fig. 3C) (18, 19). SeG-NchiRNA levels were 2.1-fold higher comparable with that in HK2-shCtrl cells (0.274 0.019) or that in patients with ESCC than in healthy subjects (P ¼ 0.005, in HK2-shG-N cells (0.322 0.034) and none of intergroup t test; Fig. 3D). SeG-NchiRNA levels also significantly correlated comparisons were statistically significantly different (Fig. 1E, with tumoral G-NchiRNA levels (r ¼ 0.881 and P < 0.001, Pearson right). Similar results were obtained by similar experiments correlation test; Fig. 3E). We next evaluated the pattern of replacing HK2 cells with TE1 cells (NE2: 0.317 0.018, G-NchiRNA distribution in fractions of saliva. qRT-PCR using RNA TE1-shCtrl: 0.290 0.011, TE1-shG-N: 0.272 0.013; Fig. 1F, from different fractions of saliva showed that the levels of exosomal right). These results confirmed that intracellular G-NchiRNA G-NchiRNA were significantly higher in patients ESCC than healthy closely correlated with exosomal G-NchiRNA. Therefore, we rea- controls (P < 0.001, t test; Supplementary Fig. S2A). There was no soned that exosomal G-NchiRNA should reflect intracellular significant difference in G-NchiRNA in the salivary cell pellet content of G-NchiRNA in ESCC. (presumably mostly cells from the buccal mucosa), and no significant difference in exosomes-depleted salivary supernatant. Fur- SeG-NchiRNA levels correlate with circulating exosomal and thermore, we found that the seG-NchiRNA was resistant to RNase tumoral G-NchiRNA levels in an ESCC mouse model digestion but susceptible to RNase when exosomal membranes Exosomes can be secreted into the circulation by tumors (16), were broken by Triton X-100 (i.e., RNase and Triton X-100 simul- but saliva has also been put forth as a convenient, noninvasive taneously; P < 0.001, one-way ANOVA with post hoc Dunnett test; source of biomarkers of systemic diseases and cancer (4, 17). We Supplementary Fig. S2B), supporting that exosomal G-NchiRNA therefore ascertained whether exosomes containing G-NchiRNA was stable and suitable for measurement by qRT-PCR. To deter- could also be detected in exosomes from saliva in ESCC-bearing mine the minimum amount of exosomal RNA required for reliable animals. HK2-shG-N cells (i.e., HKESC-2 cells in which qPCR, different exo-RNA inputs were used to detect seG-NchiRNA. G-NchiRNA had been stably knocked down by transfection with SeG-NchiRNA was reliably detected using 50 ng or more, but not shG-N) and HK2-shCtrl cells (i.e., HKESC-2 cells stably trans- detectable using only 20 ng of exosomal RNA (Fig. 3F; Supple- fected with a control vector) were xenografted into nude mice. The mentary Figs. S3–S5). Four of 105 reactions showed nonspecific day on which tumors became palpable was designated as day 0, amplification in reactions using 50 ng exo-RNA, while only 1 of 105

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Figure 2. SeG-NchiRNA in mice bearing ESCC tumors. A, HK2-shCtrl and HK2-shG-N cells were subcutaneously injected into nude mice (n ¼ 5 per group). The scheme indicates the timing of xenografting and longitudinal sample collection. B, Tumor growth curves show the measured tumor volumes over time (left; error bars, SEM; , P < 0.01; , P < 0.001 by one-way ANOVA with post hoc Tukey test). Representative tumors and the box plots of the weights of all tumors harvested on day 24 (right) were shown. , P < 0.001 by Student t test. C, Immunoblotting showed the exosomal membrane markers in exosomes isolated from mouse saliva. D, Transmission electron microscopy of exosomes isolated from mouse saliva. Scale bar, 100 nm. E, SeG-NchiRNA expression (relative to shCtrl group on day 4) in ESCC-bearing mice (n ¼ 5 per group) at indicated times after xenografting. Error bars, SEM. , P < 0.05; , P < 0.01; , P < 0.001 by one-way ANOVA with post hoc Tukey test. F, SeG-NchiRNA (left) and serum exosomal G-NchiRNA (middle) were correlated (Pearson correlation test) with tumor tissue G-NchiRNA DC expressioninpatientswithESCC; chimeric RNAexpressioncalculated as2 t relative to GAPDH. Ratio of seG-NchiRNA relative to G-NchiRNA in tumor tissue and ratio of circulating exosomal G-NchiRNA relative to G-NchiRNA in tumor tissue were plotted for cells treated with shCtrl or shG-N (right).

reactions showed speciﬁcampliﬁcation in reactions using 20 ng value for healthy controls was about 33 (Supplementary Fig. S6). exo-RNA (Supplementary Figs. S3–S5). On the basis of the distri- Therefore, the Ct values of all subjects were in a reliable range. bution of the Ct value of seG-NchiRNA among all subjects, the Circadian variability among different times of day was low (Sup- median Ct value for cancer cases was about 30, and the median Ct plementary Fig. S7A). The ICV was 7.03%–10.85% among 5

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Figure 3. Detection of seG-NchiRNA in patients with ESCC. A, Immunoblotting showed the exosomal membrane markers in exosomes isolated from the saliva of two patients with ESCC (P-01 and P-02) and two healthy subjects (H-01 and H-02). B, Transmission electron microscopy of exosomes isolated from human saliva. Scale bar, 100 nm. C, Exosome concentration and size distribution by NanoSight analysis of human saliva. D, SeG-NchiRNA expression (relative to healthy controls) was measured by qRT-PCR in a discovery cohort of patients with ESCC (n ¼ 10) and healthy controls (n ¼ 8). Error bars, SEM. , P < 0.01 by Student t test. E, SeG-NchiRNA correlated (Pearson correlation test) with tumor tissue G-NchiRNA expression in patients with ESCC; chimeric RNA expression calculated DC as2 t relative to GAPDH. F, SeG-NchiRNA was measured by qRT-PCR using 50 or 100 ng of puriﬁed exosomal RNA (exo-RNA) from healthy controls (n ¼ 8) or patients (n ¼ 8). Error bars, SEM. , P < 0.001 by Student t test. G, Box and scatter plots of seG-NchiRNA in the training cohort consisting of 220 patients with ESCC (61 early-stage patients and 159 advanced stage patients) and 55 healthy subjects (left). Receiver operator characteristic (ROC) analysis of seG-NchiRNA in the training cohort (middle). The red circle indicates the optimal cutoff point for dichotomous categorization (presence or absence of ESCC), resulting in an AUC of 0.912. The red reference line indicates a reference with an AUC of 0.5. Evaluation of seG-NchiRNA in a validation cohort of 102 patients with ESCC (38 early-stage patients and 64 advanced stage patients) and 35 healthy subjects (right). Error bars, SEM (, P < 0.001 by one-way ANOVA with post hoc Dunnett test).

patients. SeG-NchiRNA levels at different times of day significantly We then evaluated the diagnostic performance of seG-NchiRNA correlated with tumoral G-NchiRNA levels, with excellent correla- in two cohorts from two independent institutions: (i) CHSUMC tion coefficients (r ¼ 0.827 and P ¼ 0.032 for 9 A.M., r ¼ 0.958 and (Shantou, Guangdong, China) and (ii) ATH (Anyang, Henan, P ¼ 0.003 for 3 P.M., r ¼ 0.983 and P ¼ 0.001 for 9 P.M., Pearson China; Supplementary Fig. S8). A total of 399 patients were correlation test; Supplementary Fig. S7B). These results suggested enrolled and 581 saliva samples were collected from 322 eligible that seG-NchiRNA represents a potential cancer biomarker for patients, including 220 of 275 (80%) eligible patients from human ESCC that lacks significant circadian variability, adding to CHSUMC as a training cohort and 102 of 124 (82.3%) eligible its suitability. patients from ATH as a validation cohort (Supplementary Fig. S8).

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The qRT-PCR data from the patient cohorts with the amplification validation cohorts were 16.52 (95% CI: 5.42–36.26) and 26.57 profile data (Ct values and amplification plots) were provided in (95% CI: 6.83–60.83), respectively. These analyses revealed the the separate Supplementary "zip" archive files available for down- abilities of seG-NchiRNA to effectively distinguish early-stage load. The cut-off value for seG-NchiRNA as a diagnostic test was patients from healthy subjects. Collectively, our data demonstrate determined in the training cohort (n ¼ 220), and then its diag- that seG-NchiRNA accurately detects early- and advanced-stage nostic performance was confirmed in the validation cohort ESCC, supporting the strong clinical potential of seG-NchiRNA as (n ¼ 102). Among demographic and clinicopathologic character- a convenient, noninvasive diagnostic test. istics, age, gender, tobacco use, location of tumors, lymph node metastasis, distant metastasis, and stage did not show any signi- SeG-NchiRNA accurately reflects tumor burden before versus ficant differences between the two cohorts (Supplementary after ESCC resection 2 Table S3). However, tumor depth (T criterion; P ¼ 0.010, x test) Given that seG-NchiRNA level correlated with tumor burden in was significantly different (Supplementary Table S3). our mouse model, we examined whether its level reflected dynam- In the training cohort from CHSUMC, seG-NchiRNA in early- ic changes in ESCC before versus after surgery. In two subcohorts stage patients (0.037 0.016, n ¼ 61) was higher in healthy of patients who underwent surgical resection at CHSUMC (n ¼ 87, subjects (0.019 0.010, n ¼ 55), but lower in advanced-stage Supplementary Table S5) or ATH (n ¼ 59, Supplementary Table patients (0.071 0.024, n ¼ 159; P < 0.001 for both, one-way S6), salivary specimens were collected 7 days before surgery and ANOVA with post hoc Dunnett test; Fig. 3G, left). On the basis of an on the seventh day after surgery (Supplementary Fig. S10). First, in ROC curve, which had an AUC of 0.912, an optimal cut-off value the CHSUMC surgical subcohort, preoperative seG-NchiRNA (i.e., 0.035) as a binary classifier was chosen by Youden index to levels in tumors with length 5 cm (20) were significantly higher 2 discriminate patients (i.e., early- and advanced-stage) from than those in tumors with length < 5cm(P ¼ 0.004, x test; healthy subjects; seG-NchiRNA expression was significantly Supplementary Table S5); a similar result was observed in the ATH 2 different between these two groups (P < 0.001, Mann–Whitney surgical subcohort (P ¼ 0.036, x test; Supplementary Table S6). U test; Fig. 3G, middle). Using the cut-off value of 0.035, sensi- Second, a paired comparison of pre- versus postoperative levels tivity for identifying ESCC was 89.1%, while specificity was 89.1% showed that seG-NchiRNA levels decreased significantly after (Table 1). In the validation cohort, seG-NchiRNA levels in early- surgery in both subcohorts (CHSUMC surgical subcohort: medi- stage patients (0.048 0.021, n ¼ 38) were significantly different an preoperative level ¼ 0.064, median postoperative level ¼ from healthy subjects (0.018 0.012, n ¼ 35) and advanced-stage 0.023, P < 0.001; ATH surgical subcohort: median preoperative patients (0.070 0.028, n ¼ 64; P < 0.001 for both, one-way levels ¼ 0.044, median postoperative level ¼ 0.017, P < 0.001; ANOVA with post hoc Dunnett test; Fig. 3G, right). The perfor- Wilcoxon matched-pair signed-rank test for both). Intriguingly, mance of the 0.035 cut-off value was then tested in the validation seG-NchiRNA levels increased in 8 of 87 cases and 5 of 59 cases in cohort from ATH, where sensitivity for identifying ESCC was the CHSUMC and ATH subcohorts, respectively (Supplementary 85.3% and specificity was 91.4% (Table 1). Diagnostic ORs Fig. S10). Among these 13 outlier cases, 3 of the 8 CHSUMC cases (DOR) for the training and validation cohorts, respectively, were with a postoperative increase in seG-NchiRNA were later diag- 66.69 (95% CI: 24.68–85.98) and 61.87 (95% CI: 16.76–99.36). nosed with ESCC recurrences at their 15-month follow-up Thus, seG-NchiRNA could effectively identify patients with ESCC appointment after surgery. Similarly, 2 of the 5 ATH cases with from healthy subjects. a postoperative increase in seG-NchiRNA were diagnosed To further examine the potentials of seG-NchiRNA for early with ESCC recurrence at their 11-month follow-up. Furthermore, detection of ESCC, ROC analysis was performed to differentiate Fisher exact test showed that the patients with increased early-stage patients (stage I/IIa) and healthy subjects. The ROC seG-NchiRNA expression after surgery were associated with post- had an AUC of 0.790; an optimal cut-off value (i.e., 0.030) was surgical recurrence in both the training (P ¼ 0.001; Supplementary chosen by Youden index to discriminate early-stage patients from Table S7) and validation cohorts (P ¼ 0.006; Supplementary healthy subjects; seG-NchiRNA expression was significantly dif- Table S8). Therefore, we concluded that seG-NchiRNA levels ferent between early-stage patients and healthy subjects (P < reflect ESCC tumor burden in the majority of cases, with substan- 0.001, Mann–Whitney U test; Supplementary Fig. S9). In the tial potential implications for postsurgical recurrence monitoring. training cohort, according to this screening cut-off value, the sensitivity for identifying early-stage ESCC was 73.8%, while the SeG-NchiRNA levels reflect recurrence and predict specificity was 85.5% (Supplementary Table S4). In the validation chemoradiation response cohort, the performance of this screening cut-off value was val- In a subcohort of 40 patients who underwent chemoradiation idated, and the sensitivity was 81.6% and the specificity was at CHSUMC (Supplementary Table S9), seG-NchiRNA levels were 85.7% (Supplementary Table S4). DORs for the training and measured longitudinally and correlated with clinical response to

Table 1. Performance of seG-NchiRNA test to identify patients with ESCC in training and validation cohorts Cohorts Cancer Test positive (n) Test negative (n) Total (n) Sensitivity Speciﬁcity Training Absent 6 49 55 89.1% 89.1% Present 196 24 220 Total 202 73 275 Validation Absent 3 32 35 85.3% 91.4% Present 87 15 102 Total 90 47 137 NOTE: The cutoff value calculated in training cohort (i.e., 0.035) was applied in the validation cohort. Test Positive in this analysis is based on a seG-NchiRNA level >0.035; the remaining individuals were classiﬁed as Test Negative.

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Figure 4. Prediction of clinical response to chemoradiation by dynamic changes of seG-NchiRNA in patients with ESCC. A, Saliva was collected 7 days before initiation of chemoradiation and half month after its completion (Note: Scheme not drawn to a time scale). The seG-NchiRNA was signiﬁcantly reduced after chemoradiation (n ¼ 40, P ¼ 0.015 by Wilcoxon matched-pair signed-rank test). Pre- and posttherapy seG-NchiRNA levels are plotted in two groups: patients with a decrease in seG-NchiRNA after therapy (n ¼ 31) and those without (n ¼ 9). The red lines connect the pre- and posttherapy values in patients with SD or PD; blue, CR or PR. B, Kaplan–Meier analysis of PFS. After receiving chemoradiation, patients with a negative DseG-NchiRNA (i.e., a fall in seG-NchiRNA after therapy) had signiﬁcantly longer PFS than those with a positive DseG-NchiRNA (i.e., a rise in seG-NchiRNA after therapy; P < 0.001, log-rank test). C, Time schedule of saliva collection relative to chemoradiation. Baseline samples were collected 7 days before chemoradiation. D, Case 01 (stage III, T4N0M0): decrease in seG-NchiRNA preceded radiological evidence of response to therapy. The cut-off value for discriminating patients and healthy subjects is 0.035. The red arrows indicate tumor locations on contrast esophagram and CT images. The pink-shaded area indicates the treatment duration. E, Case 07 (stage III, T3N1M0) is presented in the same format above. A rise in seG-NchiRNA preceded radiologic evidence of resumption of tumor growth.

chemoradiation according to RECIST criteria. Comparing saliva change in seG-NchiRNA levels pre- and postchemoradiation samples collected pretherapy and at one half-month posttherapy [i.e., DseG-NchiRNA ¼ (seG-NchiRNA after chemoradiation) – (Fig. 4A, top), 7 of 9 (77.8%) patients without a fall in (seG-NchiRNA before chemoradiation)] was a predictor of clin- seG-NchiRNA after therapy were chemoradiation nonresponders ical response (OR ¼ 17.67, 95% CI: 2.39–130.74; P ¼ 0.005) [stable disease (SD) or progressive disease (PD)], whereas 80.6% while adjusting for demographic and clinicopathologic character- (25/31) of those with a fall in seG-NchiRNA after therapy istics (Supplementary Table S11). Thus, we conclude that seG- seG-NchiRNA were responders [complete response (CR) or partial NchiRNA levels are potentially promising for monitoring the 2 response (PR); P < 0.001, x test; Fig. 4A; Supplementary Table therapeutic response of patients with ESCC. S10]. Paired comparison of pre- and postchemoradiation levels Kaplan–Meier analysis demonstrated that a positive showed that seG-NchiRNA signiﬁcantly decreased after chemor- DseG-NchiRNA (i.e., a rise in seG-NchiRNA after therapy) was adiation (P ¼ 0.015, Wilcoxon matched-pair signed-rank associated with a signiﬁcantly shorter (P < 0.001, log-rank test; Fig. 4A). Logistic regression analysis revealed that test; Fig. 4B) PFS than a negative DseG-NchiRNA (i.e., a fall in

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seG-NchiRNA after therapy). Multivariable Cox regression anal- and alternative RNA splicing (25–28). Here, we showed that the ysis revealed that DseG-NchiRNA was an independent predictor aberrant splicing–induced G-NchiRNA found in ESCC tissues was of PFS of patients with ESCC undergoing chemoradiation associated with malignant progression (Supplementary Table S1) (HR ¼ 3.97; 95% CI: 1.29–12.20; P ¼ 0.016; Supplementary and short overall survival (Fig. 1B). Furthermore, we demonstrat- Table S12). Therefore, DseG-NchiRNA is predictive of prognosis in ed the direct relevance of G-NchiRNA expression to ESCC pro- patients with ESCC undergoing chemoradiation. gression by experimentally inhibiting its expression, which sig- Longitudinal measurement of seG-NchiRNA levels was carried nificantly slowed tumor growth in vivo (Fig. 2). Finally, salivary out according to the scheme outlined (Fig. 4C). Fourteen patients chimeric RNA (seG-NchiRNA) levels accurately reflected the with ESCC who had both evaluable longitudinal clinical data and dynamic change in tumor burden or growth in mice and human seG-NchiRNA data were analyzed. Seven patients were nonre- patients with ESCC. sponders (PD), and 7 others were responders (CR or PR). Dynam- In this study, >90% of patients with ESCC showed a consid- ics of seG-NchiRNA levels paralleled, or in some cases preceded, erable drop in seG-NchiRNA after surgery in cohorts from two the trends of the longest tumor dimension as measured on high ESCC incidence regions in China. These findings imply that contrast esophagram (Fig. 4D and E; Supplementary Figs. S11 seG-NchiRNA can serve as a biomarker of tumor burden, with and S12). Case 01 (Fig. 4D) was a patient who had a remarkable substantial potential in postsurgical recurrence monitoring. In drop in seG-NchiRNA levels two months ahead of noticeable addition, 13 patients from these cohorts exhibited increased levels tumor shrinkage, as judged by radiography. Case 07 (Fig. 4E) was after surgery (Supplementary Fig. S10): 5 of these 13 developed a patient who initially did not show disease progression, as recurrence soon after follow-up. Whether the remaining 8 will examined by X-ray and CT scan until 4 months after initiation develop recurrence remains to be observed. Thus, seG-NchiRNA of therapy. However, this patient had a significant rise of levels may even presage clinical detection of postoperative tumor seG-NchiRNA levels 2 months after initiation of therapy, and recurrences. continued to increase, suggesting that seG-NchiRNA levels could Like most solid tumors, ESCC detection currently involves detect disease progression earlier than radiological studies. Addi- invasive (e.g., endoscopy/biopsy), minimally invasive (e.g., trans- tional examples, including case 16 with CR (Supplementary nasal endoscopy/biopsy or cytosponge), or expensive clinical Fig. S11A), case 04 with PR (Supplementary Fig. S12A), case 17 procedures (e.g., CT scan with contrast and endoscopy/biopsy) with PD (Supplementary Fig. S11B), and case 14 with PD (29, 30). Compared with the existing options, our salivary test is (Supplementary Fig. S12B) demonstrated that the dynamics of preferred because of economy, comfort, convenience, and patient seG-NchiRNA levels paralleled the clinical response to chemor- acceptance. Our study shows that seG-NchiRNA levels permit adiation over time. Wilcoxon matched-pair signed-rank test repeated testing for primary and secondary detection over the revealed that the seG-NchiRNA assay might predict PD earlier entire clinical course of ESCC. Moreover, our results suggest that than radiologic imaging by a median of 35 days in nonresponders seG-NchiRNA may predict ESCC recurrence or disease progression for chemoradiation (P ¼ 0.018; Supplementary Table S13). Thus, earlier than standard radiological methods. We demonstrate that seG-NchiRNA level offers substantial potential as a biomarker to DseG-NchiRNA acts as an independent predictor of PFS after longitudinally evaluate the clinical effectiveness of chemoradia- chemoradiation. Although our data showed that increased seG- tion in ESCC. NchiRNA after surgery was associated with postsurgical recurrence and that increased seG-NchiRNA after chemoradiation was associated with disease progression, larger cohorts are needed for Discussion confirmation. Thus, seG-NchiRNA levels show promise as a Tumor-secreted exosomes may contain transcriptional, trans- molecular assay for close monitoring of ESCC, with minimal lational, or epigenetic information about the cancer and have patient discomfort and avoidance of radiation exposure or other emerged as a liquid biopsy substrate for cancer diagnosis or complications (e.g., anesthesia complications during endoscopy, prognosis (1). While circulating tumor cells and nucleic acids are intravenous contrast allergy, and intravenous contrast nephrop- mainly analyzed using blood samples, exosomes are present in a athy). SeG-NchiRNA levels may be useful for evaluating efficacy of variety of biological fluids that may be more accessible than cancer therapies in conjunction with standard methods, while blood. Unlike miRNAs, which are inherently stable in biological simultaneously decreasing the frequency of invasive monitoring fluids, mRNAs are easily degraded unless they are inside exo- procedures or costly tests. somes (21, 22). In support of this point, circulating exosomal Even after surgery with curative intent, the 5-year survival of mRNAs have been reported as a biomarker for the diagnosis and ESCC is only 26.2%–49.4% (31). Studies have suggested that prognosis of pancreatic cancer (16). In this study, we demon- survival will be dramatically improved if ESCC is diagnosed at strated that a chimeric mRNA was present in salivary exosomes of an early stage or chemoradiation response is monitored closely patients with ESCC and in animal saliva, and that levels of this (32, 33). Unlike endoscopy or imaging methods, seG-NchiRNA chimeric RNA in salivary exosomes served as a noninvasive measurement is based on exosome purification and qRT-PCR, biomarker for early and advanced stage ESCC detection, as well which constitute a noninvasive, low-cost, convenient platform for as for postoperative surveillance, therapeutic response, and tumor ESCC screening. Although several serum biomarkers, including recurrence. To our knowledge, this is the first report of a salivary squamous cell cancer antigen (SCC-Ag), carcinoembryonic anti- exosomal chimeric RNA as a disease biomarker. gen (CEA), and cytokeratin 21–1 fragment (CYFRA21-1) have Fusion transcripts (e.g., BCR-ABL) and their products have been been proposed for ESCC diagnosis (34, 35), their implementation employed as cancer biomarkers for many years (23, 24). With in clinical settings have been challenged by insufficient sensitivity recent advances in next-generation RNA sequencing and bioin- and limited specificity (34, 35). Recently, serum exosomal formatics, mechanisms producing chimeric RNAs besides gene miRNA-21 has been investigated in a study involving 51 ESCC fusion have been discovered, such as transcriptional read-through cases and 41 benign controls, revealing an association of

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circulatory exosomal miRNA-21 with ESCC progression (36). On assays can serve as a noninvasive approach for cancer molecular the basis of the training and validation cohorts in our prospective detection even at early stages, for monitoring of tumor burden, study (322 cases and 90 controls in total), sensitivity of and for surveillance of treatment response to tailor therapeutic seG-NchiRNA is 85.3%–89.1% and specificity is 89.1%–91.4% decisions. Further analysis with regard to clinical outcomes of for ESCC diagnosis, while DOR is 61.87–66.69. Notably, ESCC will be possible as outcome data for the two clinical seG-NchiRNA distinguished early-stage patients from healthy cohorts become available in the next few years. Further large subjects in both training and validation cohorts. As a screening independent prospective studies from different regions of test to distinguish early-stage (stage I/IIa) patients from healthy China and other cohorts in other countries are needed to validate volunteers, a cut-off value of 0.030 can achieve a sensitivity of these results. 73.8%–81.6% and specificity of 85.5%–85.7% (DOR: 16.52– 26.57). These results supported seG-NchiRNA as a potential Disclosure of Potential Conflicts of Interest biomarker for early disease detection. Given a high pretest prob- S.J. Meltzer is a consultant/advisory board member for TwoXAR, Inc. ability for ESCC in high-incidence regions, multiple-cohort clin- S.-C.J. Yeung is a consultant/advisory board member for Celgene, and ical trials are now warranted to determine whether seG-NchiRNA reports receiving commercial research grants from DepMed and Bristol- Myers Squibb ARISTA. No potential conflicts of interest were disclosed by can serve as a cost-effective screening biomarker for ESCC. the other authors. One limitation of our study was the relatively small sizes of our patient cohorts, which limited the number of covariates that could Authors' Contributions be included in multivariate statistical models. In addition, there Conception and design: H. Zhang was no nonsurgical comparison group for the surgical subcohorts Development of methodology: H. Zhang to show whether changes in seG-NchiRNA levels were due to Acquisition of data (provided animals, acquired and managed patients, debulking the tumor: in this context, it would have been unethical provided facilities, etc.): Y. Lin, H. Dong, W. Deng, K. Li, X. Xiong, Y. Guo, to randomize patients with ESCC to a no-treatment group. F. Zhou, C. Ma, Y. Chen, H. Ren, H. Yang, N. Dai, H. Zhang Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Moreover, patients who choose supportive care only are computational analysis): Y. Lin, H. Dong, W. Deng, W. Lin, L. Ma, S.J. Meltzer, usually end-stage patients with poor performance status, who do S.-C.J. Yeung, H. Zhang not follow up in the oncology or surgery clinics. A large, multi- Writing, review, and/or revision of the manuscript: Y. Lin, H. Dong, W. Deng, center, double-blinded (i.e., clinical evaluators blinded to the S.J. Meltzer, S.-C.J. Yeung, H. Zhang seG-NchiRNA results, and performers of the seG-NchiRNA assay Administrative, technical, or material support (i.e., reporting or organizing blinded to the clinical status) prospective study should now be data, constructing databases): Y. Lin, H. Dong, W. Deng, W. Lin, K. Li, X. Xiong, Y. Guo, F. Zhou, C. Ma, Y. Chen, H. Yang, H. Zhang performed to provide a definitive validation of this biomarker in Study supervision: H. Zhang several clinical contexts: for primary cancer detection, for prognosis, and early secondary detection marker of recurrence/pro- Acknowledgments gression. Because exosomes are present in bodily fluids other than The authors thank the surgeons, radiotherapists, physicians, and patients blood and saliva, that is, most importantly urine, the performance who participated in these studies. This work was supported in part by National of exosomal G-NchiRNA in spot urine or 24-hour urine samples Natural Science Foundation of China (81572876, 81773087, 81071736 and for ESCC diagnosis and prognosis should be compared with 30973508 to H. Zhang). seG-NchiRNA. To further expand the translation of use of this biomarker to other types of cancer, it should be evaluated in other The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked cancer types. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate G-N In summary, se chiRNA levels, which do not require a blood this fact. draw or other invasive testing, constitute a convenient and reliable biomarker of human ESCC. Our prospective investigations in Received October 3, 2018; revised December 3, 2018; accepted February 1, training and validation cohorts demonstrated that seG-NchiRNA 2019; published first February 11, 2019.

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Evaluation of Salivary Exosomal Chimeric GOLM1-NAA35 RNA as a Potential Biomarker in Esophageal Carcinoma

Yusheng Lin, Hongmei Dong, Weilun Deng, et al.

Clin Cancer Res Published OnlineFirst February 11, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3169

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