Circulating PIWI-Interacting Rnas Pir-5937 and Pir-28876 Are Promising Diagnostic Biomarkers of Colon Cancer

Published OnlineFirst July 5, 2018; DOI: 10.1158/1055-9965.EPI-18-0318

Research Article Cancer Epidemiology, Biomarkers Circulating PIWI-Interacting RNAs piR-5937 and & Prevention piR-28876 Are Promising Diagnostic Biomarkers of Colon Cancer Petra Vychytilova-Faltejskova1,2, Karolina Stitkovcova1, Lenka Radova1, Milana Sachlova2, Zdenka Kosarova1, Katerina Slaba1, Zdenek Kala1,3, Marek Svoboda1,2, Igor Kiss2, Rostislav Vyzula2, William C. Cho4, and Ondrej Slaby1,2

Abstract

Background: The early detection of colon cancer is one of 28876, it was possible to differentiate between cancer patients the main prerequisites for successful treatment and mortality and healthy donors with high sensitivity and specificity. More- reduction. Circulating PIWI-interacting RNAs (piRNA) were over, both piRNAs exhibited satisfactory diagnostic perfor- recently identified as novel promising biomarkers. The pur- mance also in patients with stage I disease and enabled pose of the study was to assess the profiles of piRNAs in blood detection of colon cancer with higher sensitivity than currently serum of colon cancer patients with the aim to identify those used biomarkers CEA and CA19-9. Finally, the expression of with high diagnostic potential. analyzed piRNAs in blood restored significantly 1 month after Methods: Blood serum samples from 403 colon cancer the surgical resection. patients and 276 healthy donors were included in this 3-phase Conclusions: Based on our findings, piRNAs are abundant biomarker study. Large-scale piRNA expression profiling was in human blood serum. Furthermore, their levels in colon performed using Illumina small RNA sequencing. The diagnos- cancer have been observed to be significantly deregulated. tic potential of selected piRNAs was further validated on inde- However, their involvement in carcinogenesis must be further pendent training and validation sets of samples using RT-qPCR. established. Results: In total, 31 piRNAs were found to be significantly Impact: piRNAs could serve as promising noninvasive bio- deregulated in serum of cancer patients compared with markers for early detection of colon cancer. Cancer Epidemiol healthy donors. Based on the levels of piR-5937 and piR- Biomarkers Prev; 27(9); 1019–28. 2018 AACR.

Introduction Currently, fecal occult blood testing and endoscopic app- roaches are the predominant screening methods used for early Colon cancer accounts for approximately for 7% of all cancers, detection of colon cancer. Although it has been shown that these and it is one of the most common causes of cancer-related deaths. methods significantly contribute to reduced risk of colon cancer– The prognosis of patients depends mostly on tumor–node– associated mortality (2), screening effectiveness is limited by test metastasis (TNM) stage at the time of diagnosis as well as on the performance, high costs, and invasiveness as well as a suboptimal possibility of curative surgical resection, which can be accom- screening compliance. Thus, the development of a simple blood- plished only in patients with localized disease (1). Thus, the early based test, with a specimen drawn during the routine medical detection of colon cancer or, even better, precancerous lesions is check-up, could improve the screening rates. Lately, various one of the main prerequisites for mortality reduction and more molecules including DNA (3), proteins (4), mRNA (5), or miR- successful treatment. In addition, novel biomarkers suitable for NAs (6, 7) have shown a great potential to serve as new molecular monitoring of disease progression as well as treatment response markers for the development of noninvasive and accurate tests for are in great demand. colon cancer screening. Recently, several studies have indicated the abundance of novel 1Centre for Molecular Medicine, Central European Institute of Technology, class of small noncoding RNAs called PIWI-interacting RNAs fl Masaryk University, Brno, Czech Republic. 2Department of Comprehensive (piRNA) in various types of body uids (8, 9). These 26-32 Cancer Care, Masaryk Memorial Cancer Institute, Faculty of Medicine, Masaryk nucleotide-long molecules have been shown to participate in the University, Brno, Czech Republic. 3Department of Surgery, Institutions Shared epigenetic regulation of cancer and other diseases and are key with the Faculty Hospital Brno, Faculty of Medicine, Masaryk University, Brno, elements of cellular homeostasis (10). Furthermore, they play an 4 Czech Republic. Cancer Research Unit, Department of Clinical Oncology, Queen important role in tumor formation, proliferation, and migration Elizabeth Hospital, Kowloon, Hong Kong, PR China. of the cells (11, 12). Similar to miRNAs, piRNAs posttranscrip- Note: Supplementary data for this article are available at Cancer Epidemiology, tional regulation occurs in the cytoplasm. The piRISC protects the Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). integrity of the genome by binding the transposable elements as Corresponding Author: Ondrej Slaby, Masaryk University, Kamenice well as mRNAs or lncRNAs (13). Moreover, these molecules may 753/5, Brno 625 00, Czech Republic. Phone: 00420549496876; E-mail: regulate gene expression through histone modifications and DNA [email protected] methylation (14). Unlike miRNAs, piRNAs are extremely diverse, doi: 10.1158/1055-9965.EPI-18-0318 with at least hundreds of thousands of mature species transcribed 2018 American Association for Cancer Research. from thousands of genomic loci (15). However, similarly to

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miRNAs, they are short enough to not be easily degraded by validation phase of the study. Clinical and pathologic character- ribonucleases, and they can pass through the cell membranes istics are summarized in Table 1. The concentrations of CEA and (16). Studies on the biological functions and possible clinical CA19-9 were measured at Department of Laboratory Medicine at relevance of piRNAs in colorectal cancer (CRC) are still in the MMCI using electrochemiluminescence immunoassay and beginning. In 2015, Chu and colleagues (17) identified 7 com- ELECSYS 2010 system (Roche), and during the study, cutoffs mon single-nucleotide polymorphisms in 9 known piRNAs. used in clinical routine were used (CEA: 5 ngmL 1; CA19-9: Further, they revealed that piR-015551 (DQ591252) may be 27 UmL-1). All blood serum samples were collected prior to generated from long noncoding RNA LNC00964-3, which is surgery. Serum samples from 276 healthy donors involved in this significantly downregulated in CRC tissues and may be involved study were collected at the Department of Preventive Oncology in disease development. In addition, rs11776042 in piR-015551 (Masaryk Memorial Cancer Institute, Brno, Czech Republic); was associated with a decreased risk of CRC. This year, piR-25447 these donors had no prior diagnosis of any malignancy. The (DQ558335), piR-23992 (DQ556880), and piR-1043 samples of cases and controls in all phases of the study were (DQ540931) were found to be overexpressed in tumor tissue balanced regarding the age and sex. In addition, 20 paired samples compared with adjacent mucosa, whereas piR-28876 from colon cancer patients before and 1 month after the surgery (DQ598676) was significantly underexpressed. Furthermore, (7 men, 13 women; mean age 72 years) were included (Faculty 27 piRNAs were differentially expressed between adjacent tissue Hospital Brno, Czech Republic). All subjects enrolled in the study and CRC metastases (18). Recently, piR-823 (DQ571031) was were of the same ethnicity (European descent), and colon cancer found to be upregulated in CRC tissues, and its inhibition patients did not receive any neoadjuvant treatment. Ten hemo- suppressed cell proliferation, arrested the cell cycle in G1 phase, lytic serum samples were excluded from the study before the and induced apoptosis in DLD-1 and HCT-116 cells. Importantly, groups were selected. Written informed consent was obtained the authors further revealed that this piRNA plays a tumor- from all participants, and the study was conducted in accordance promoting role by upregulating phosphorylation and transcrip- with Declaration of Helsinki and approved by the local Ethics tional activity of HSF1, the common transcription factor of heat Board at Masaryk Memorial Cancer Institute. shock proteins (19). Interestingly, piR-823 was also observed to be deregulated not only in tissue, but also in blood serum of RNA extraction patients with renal cell carcinoma (8) and gastric cancer (20). Before RNA extraction, all samples were checked for hemo- Currently, next-generation sequencing (NGS) is widely used to lysis using the Harboe's spectrophotometric method (25). identify known as well as novel piRNAs with deregulated expres- Only the samples with hemoglobin concentration lower than sion in cancer. In 2013, Huang and colleagues (21) demonstrated 5mgdL 1 were further used. Total RNA enriched for small that a wide variety of RNA species including piRNAs are embed- RNAs was isolated from blood serum using Qiagen miRNeasy ded in the circulating vesicles. This observation was confirmed 3 Serum/Plasma Kit (Qiagen, GmbH; catalog number 217184) years later when Freedman and colleagues (22) found 144 dif- according to the modified manufacturers' protocol. Briefly, ferent piRNAs to be stably present in human plasma. Up today, 250 mL of serum was thawed on ice and centrifuged at only one study analyzed the expression of circulating piRNAs in 14,000 g at 4C for 5 minutes to remove cellular debris. colorectal cancer (23). They found significant deregulation of piR- Subsequently, 200 mL of supernatant was lysed with 1 mL of 019825 (DQ597218) in plasma samples of patients compared QIAzol Lysis Reagent. For each sample, 1.25 mLof0.8mg∙mL 1 with healthy donors. MS2 RNA carrier (Roche; catalog number 10165948001) was In this three-phase study, large sets of serum specimens from added to 1 mL of QIAzol solution. The elution of RNA was patients with colon cancer as well as from healthy controls were performed twice with volumes of 20 mL (total volume of eluted analyzed using NGS and subsequent RT-qPCR validation with the RNA was 40 mL) using preheated Elution Solution. For the aim to identify piRNAs with deregulated expression that could purposes of library preparation, RNA pools were used. Each potentially serve as novel noninvasive biomarkers for early detec- RNA pool was gained using 12 serum samples of colon cancer tion of the disease. During the study, piRNAs' labeling according patients or healthy donors (12 250 mL; in case of colon to piRBase (24) is used together with unique GenBank accession cancer patients, all 12 patients were of the same stage). The number (DQ identifier) when mentioned first. Similarly, all lysis with QIAzol was performed separately for each sample as names of cited piRNAs are accompanied by this identifier. described previously. After phase separation, upper aqueous phase from all 12 samples was combined, mixed with 1.5 volume of 100% ethanol and pipetted into one RNeasy MinE- Materials and Methods lute spin column. Elution was performed using 14 mLof Patients' samples and study design preheated Elution Solution. The concentration and purity of The study design consisted of three phases. All thresholds were RNA were determined spectrophotometrically by measuring its determined to enable potential analytical applicability of the optical density (A260/280 > 2.0; A260/230 > 1.8) using biomarker (Fig. 1). In total, a consecutive set of 403 patients with NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Sci- histopathologically verified colon cancer, who underwent resec- entific). Further, the concentrations and quality of RNA of tion from 2010 through 2014 at Masaryk Memorial Cancer pooled samples for NGS were also measured using Qubit 2.0 Institute (MMCI, Brno, Czech Republic), was included in the Fluorometer (Thermo Fisher Scientific) and Agilent 2100 study. These patients were further proportionally divided into Bioanalyzer (Agilent Technologies). screening, training, and validation sets based on TNM stage. In total, 144 cases and 96 controls were included in the screening Small RNA library construction and sequencing phase, 80 cases and 80 controls were included in the training In total, 144 cases and 96 controls were sequenced by preparing phase, and 179 cases and 100 controls were analyzed in the 20 libraries, each containing pooled RNA of 12 different cases (for

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Figure 1. Flow diagram of the study design illustrating how the patients and controls were divided into screening, training, and validation phase of the study.

a total of 12 libraries) or 12 controls (for a total of 8 libraries). All package (26) and further between-sample normalized by the libraries were prepared using the Illumina TruSeq Small RNA voom function in LIMMA package. After the normalized expres- Library Prep Kit (Illumina; catalog number RS-200-0012) follow- sion levels were determined, differentially expressed piRNAs were ing the manufacturer's instructions. The concentration of pre- screened applying linear model fitting and a Bayes approach. The pared libraries was measured using High Sensitivity DNA chip and obtained P values were adjusted for multiple testing using the Agilent 2100 Bioanalyzer. Equimolar amounts of each library Benjamini–Hochberg method. were pooled at a final concentration of 2 nmolL 1 cDNA, and samples were sequenced on a flowcell with 50-bp single-end reads Reverse transcription and quantitative real-time PCR using MiSeq sequencer (Illumina). Complementary DNA was synthesized from total RNA using customized piRNA-specific primers (Supplementary Table S1) Sequencing data processing and differential piRNA analyses and10ngofRNAsampleaccordingtotheTaqManMicroRNA Count-based piRNA expression data were generated by the CLC Assay protocol (TaqMan MicroRNA Reverse Transcription kit, Genomic Workbench from fastq files. All sequences were adapter Applied Biosystems; catalog number 4366597). Reaction mix- trimmed and mapped against piRBase (www.regulatoryrna.org/ tures were incubated for 30 minutes at 16C, 30 minutes at database/piRNA; ref. 24) allowing up to two mismatches per 42C, 5 minutes at 85C,andthenheldat4C (T100 Thermal sequence. Further analyses were performed using R/Bioconductor Cycler; Bio-Rad). Real-time PCR was performed using the packages. PiRNAs having less than 1 read per million in more than TaqMan (NoUmpErase UNG) Universal PCR Master Mix (cat- 17 pooled samples were dropped out. The read counts were alog number 4440040) and QuantStudio 12K Flex Real-Time prenormalized by adding normalization factors within edgeR PCR system (all from Applied Biosystems). Reactions were

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Table 1. Clinicopathologic characteristics of study subjects Characteristics Screening phase Training phase Validation phase Colon cancer cases Number 144 80 179 Age (mean SD), years 65 12 65 12 66 12 Sex, number (%) Male 82 (57) 44 (55) 94 (53) Female 62 (43) 36 (45) 85 (47) TNM stage, number (%) Stage I 36 (25) 19 (24) 29 (16) Stage II 36 (25) 21 (26) 62 (35) Stage III 36 (25) 21 (26) 48 (27) Stage IV 36 (25) 19 (24) 40 (22) Grade, number (%) Grade 1 43 (30) 22 (28) 49 (27) Grade 2 74 (51) 42 (53) 91 (51) Grade 3 24 (17) 13 (16) 38 (21) Unknown 3 (2) 3 (3) 1 (1) Location, number (%) Distal 85 (59) 47 (59) 100 (56) Proximal 58 (40) 33 (41) 79 (44) Unknown 1 (1) 0 (0) 0 (0) Tumor size, number (%) <50 mm 84 (58) 46 (57) 93 (52) 50 mm 45 (31) 30 (38) 75 (42) Unknown 15 (11) 4 (5) 11 (6) Pre-CEA levelsa, number (%) < 5ng mL1 44 (31) 21 (26) 53 (30) 5ng mL1 40 (28) 19 (24) 50 (28) Unknown 60 (41) 40 (50) 76 (42) Pre-CA19-9 levelsb, number (%) < 27 U mL1 70 (49) 31 (39) 75 (42) 27 U mL1 15 (11) 13 (16) 26 (15) Unknown 59 (40) 36 (45) 78 (43) Healthy donors Number 96 80 100 Age (mean SD), years 62 11 60 759 7 Sex, number (%) Male 48 (50) 47 (59) 48 (48) Female 48 (50) 33 (41) 52 (52) aPre-CEA—preoperative levels of carcinoembryonic antigen. bPre-CA19-9—preoperative levels of CA19-9.

incubated in a 96-well optical plate at 95C for 10 minutes, (R Development Core Team). P values of less than 0.05 were followed by 40 cycles at 95C for 15 seconds and 60Cfor1 considered statistically signiﬁcant. minute.

Data normalization and statistical analyses Results The threshold cycle data were calculated by QuantStudio 12K Identiﬁcation of deregulated piRNAs using small RNA Flex software. All real-time PCR reactions were run in triplicates. sequencing The average expression levels of all measured piRNAs were nor- In the screening phase of the study, small RNA sequencing of malized using piR-28131 (DQ597916) and subsequently ana- RNA isolated from blood serum samples from 144 colon cancer D lyzed by the 2- Ct method. Statistical differences between the patients and 96 healthy controls was carried out using MiSeq levels of analyzed piRNAs in serum samples of colon cancer sequencer (Illumina). In total, 20 small RNA libraries were patients and healthy donors were evaluated by two-tailed non- prepared and sequenced (12 libraries per colon cancer patients, parametric Mann–Whitney test. Paired samples before and after 8 libraries per healthy controls). On average, more than 96% of the surgery were analyzed using two-tailed nonparametric Wil- the reads had Q-score higher than 30, thus the obtained data coxon test for paired samples. Further, receiver operating curve were considered to be of high quality. The sequenced (ROC) analyses were performed to ascertain the diagnostic per- samples contained on average 8.925.000 2.139.597 reads, and formance of analyzed markers, and the maximum Youden index 8.219.664 1.954.177 reads passed the ﬁlter. Mapping data to was used to obtain optimal cutoff values. The diagnostic score the piRBase, 163.355 91.338 reads were annotated, being a (DXscore) was established using logistic regression. The Spear- proportion of 2.11 1.31% of the total sequenced reads (Sup- man correlation was performed to assess the relation between plementary Table S2). Of the 854 different piRNAs that were analyzed piRNAs. All calculations were performed using Graph- found to be present in serum samples, 482 piRNAs had more than Pad Prism version 5.00 (GraphPad Software) and R environment 1 read per million in more than 3 samples and were involved in

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Table 2A. The list of piRNAs signiﬁcantly deregulated in pooled serum samples of colon cancer patients compared with healthy donors (P < 0.01; ordered by fold change) Downregulated Fold change P value Upregulated Fold change P value piR-hsa-24672 0.114 4.03 1011 piR-hsa-30937 3.775 1.42 104 piR-hsa-5937 0.125 8.46 1012 piR-hsa-8226 3.758 7.44 104 piR-hsa-28876 0.194 8.51 109 piR-hsa-6746 3.233 2.43 103 piR-hsa-28846 0.241 2.14 104 piR-hsa-19076 2.584 6.97 103 piR-hsa-32158 0.246 2.12 103 piR-hsa-8079 2.561 7.30 103 piR-hsa-28019 0.260 3.26 103 piR-hsa-30715 2.496 3.30 103 piR-hsa-32159 0.303 3.02 105 piR-hsa-26872 2.218 2.62 103 piR-hsa-23209 0.321 3.79 105 piR-hsa-2107 1.978 5.07 103 piR-hsa-32162 0.337 3.98 106 piR-hsa-23210 0.374 3.86 105 piR-hsa-29716 0.400 3.39 103 piR-hsa-28190 0.413 7.75 105 piR-hsa-32161 0.433 1.33 103 piR-hsa-1242 0.434 4.31 104 piR-hsa-32187 0.443 2.29 103 piR-hsa-32238 0.462 3.00 103 piR-hsa-32167 0.464 1.27 103 piR-hsa-29218 0.469 1.12 103 piR-hsa-27620 0.473 1.68 103 piR-hsa-32195 0.483 1.04 103 piR-hsa-32182 0.485 2.05 103 piR-hsa-11362 0.540 3.73 103 piR-hsa-27493 0.547 6.03 103

Table 2B. Two-phase validation of deregulated piRNAs identified by NGS Training phase Validation phase piRNA FCa P value AUCb (Sensc/Specd/Cutoff) piRNA FCa P value AUCb (Sensc/Specd/Cutoff) piR-hsa-28876 0.43 <0.0001 0.8065 (75.3; 70.0; 0.0278) piR-hsa-28876 0.60 <0.0001 0.7074 (66.0; 65.3; 0.0278) piR-hsa-5937 0.47 <0.0001 0.8060 (71.8; 72.5; 0.0655) piR-hsa-5937 0.55 <0.0001 0.7673 (73.6; 65.3; 0.0655) piR-hsa-23210 0.85 0.0220 0.6060 (67.5; 50.0; 0.0018) piRNA panel NA <0.0001 0.7649 (70.4; 71.4; –0.2339) piR-hsa-32159 0.93 0.0349 0.5973 (58.2; 50.6; 0.0110) piR-hsa-23209 2.83 0.3998 0.5550 (52.5; 52.5; 0.0038) Abbreviation: NA, not applicable. aFC ¼ fold change. bAUC ¼ area under the curve. cSens ¼ sensitivity. dSpec ¼ specificity. subsequent analysis. In total, 48 piRNA were found to be patients from the training phase of the study. As the Cq values of significantly deregulated in serum samples of colon cancer piR-26131 were higher than 35 and this piRNA was not detected patients compared with healthy donors (nonadjusted P < in all samples, it was eliminated from further analyses. Subse- 0.01). Furthermore, 97 and 87 piRNAs were detectable in colon quently, piR-28131 was chosen as the best endogenous control cancer patients and healthy donors, respectively, in more than for the normalization of data obtained by RT-qPCR. It was proved 50 copies per 1 million of reads. From these, 31 piRNAs were that its expression is highly stable in all analyzed sets of samples found to be significantly deregulated (23 downregulated, 8 (Supplementary Table S3). upregulated; Table 2A) in serum samples of colon cancer patients compared with healthy donors (nonadjusted P < 0.01; Supple- The training phase of the study mentary Fig. S1). According to the criteria described in the study Serum samples from 80 colon cancer patients and 80 healthy design (adjusted P < 0.005; at least 50 copies of piRNA in each donors were included in the training phase of the study. As < pooled library; logFC > 1.5), five piRNAs (piR-5937 – DQ575659, shown in Table 2B, serum levels of piR-5937 (P 0.0001; Fig. < ¼ piR-28876 – DQ598676, piR-23210 – DQ592932, piR-23209 – 2A), piR-28876 (P 0.0001; Fig. 2D), piR-23210 (P 0.0220), ¼ fi DQ592931, piR-32159 – DQ not available) were chosen for and piR-32159 (P 0.0349) were signi cantly lower in colon further evaluation in the training phase of the study. cancer serum samples compared with healthy controls. These results are in agreement with sequencing data. The deregulation Identification of an appropriate endogenous control of piR-23209 (P ¼ 0.3998) was not confirmed. In addition, the Based on the results of NGS (fold change, SD, at least 500 copies expression of piR-5937 and piR-28876 decreased significantly per 1 million of reads, expressed in all prepared libraries), 3 with advanced clinical stage (P < 0.0005; Fig. 2C and F), piRNAs (piR-28131 – DQ597916, piR-27622 - DQ597347, whereas there were no correlation between piRNAs expression piR-26131 – DQ595899) were chosen as potential endogenous and grade, location, and size of the tumor (P > 0.05). Further- controls for normalization of RT-qPCR data. First, expression of more, the ROC analysis was performed to determine the all 3 piRNAs was measured in randomly selected 40 serum sensitivity and specificity of individual piRNAs for colon samples of healthy donors and 40 serum samples of colon cancer cancer detection. In case of piR-5937 (cutoff: 0.0655; Fig. 2B)

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Figure 2. Diagnostic performance of piR-5937 and piR-28876 in the training phase of the study. A, The expression of piR-5937 is significantly downregulated in serum samples of colon cancer patients compared with healthy donors (P < 0.0001). B, ROC analyses based on the expression of piR-5937 (AUC ¼ 0.8060). C, The levels of piR-5937 decrease significantly with advanced clinical stage (P ¼ 0.0003). D, The expression of piR-28876 is significantly downregulated in serum samples of colon cancer patients compared with healthy donors (P < 0.0001). E, ROC analyses based on the expression of piR-28876 (AUC ¼ 0.8065). F, The levels of piR-28876 decrease significantly with advanced clinical stage (P < 0.0001). , P < 0.05; , P < 0.01; and , P < 0.001.

and piR-28876 (cutoff: 0.0278; Fig. 2E), the AUC values were ¼1.268 þ 15.246piR-5937 þ 3.811piR-28876 (cutoff: higher than 0.80, and the best results were reached for piR-28876 –0.2339). Unfortunately, subsequent ROC analysis did not prove (AUC ¼ 0.8065, sensitivity 75%, specificity 70%; Table 2B). that usage of this two-piRNA–based panel would provide better results than piR-5937 or piR-28876 separately (Table 2B). Thus, the The validation phase of the study Spearman correlation analysis was performed to assess the relation Based on the criteria mentioned in the study design (P < 0.01; Cq between these two piRNAs. The results confirmed positive corre- values < 35; detection rate > 80%), only two piRNAs (piR-5937 and lation in case of CRC patients (r ¼ 0.8154; P < 0.0001) as well as in piR-28876) were further analyzed in validation phase of the study case of healthy controls (r ¼ 0.7391; P < 0.0001). Finally, the that involved 179 serum samples of colon cancer patients and 100 expression of both piRNAs was measured in 20 paired serum serum samples of healthy donors. In addition, 20 paired samples of samples from colon cancer patients before and 1 month after the colon cancer patients before and 1 month after the surgery were surgery. As shown in Fig. 3C and F, the levels of analyzed piRNAs analyzed in the third phase of the study. As shown in Table 2B, both were significantly higher in postoperative samples (P < 0.05). piRNAs were significantly downregulated in serum samples of patients compared with healthy controls (P < 0.0001; Fig. 3A and Comparison of diagnostic potential of piR-28876/piR-5937 D). However,there was no association betweenpiRNAs' expression with CEA and CA19-9 and clinical stage of the disease. Similarly to the results obtained in The capacity of piR-28876/piR-5937 and of CEA/CA19-9 mar- the training phase of the study, the AUC values were higher than kers to detect colon cancer was tested. In total, 138 colon cancer 0.70 for both piRNAs when using ROC analysis (Fig. 3B and E). patients with known levels of CEA and CA19-9 were included in Moreover, in case that only the patients in stage I were included in this analysis. As shown in Fig. 4A–D, CEA enabled to identify 66 ROC analysis, the diagnostic potential of analyzed piRNAs was still colon cancer patients (48%; cutoff 5 ngmL 1), whereas CA19-9 satisfactory (piR-5937: AUC ¼ 0.8189, sensitivity ¼ 71.4%, spec- only 36 (26%; cutoff 27 UmL 1), when cutoff values routinely ificity ¼ 66.3%; piR-28876: AUC ¼ 0.7256, sensitivity ¼ 64.3%, used in our reference laboratory were applied. However, decreased specificity ¼ 65.3%). Furthermore, we wanted to know whether the expression of piR-5937 (cutoff: 0.0655) was observed in 98 of 138 combination of both piRNAs enables us more accurate detection of colon cancer patients (71%), whereas the decreased levels of piR- colon cancer patients. For this purpose, the diagnostic panel 28876 (cutoff: 0.0278) were noticed in 95 of 138 patients (69%). comprising of piR-5937 and piR-28876 was established, and The highest diagnostic sensitivity was reached in case of combi- diagnostic score was calculated according to the formula: DXscore nation of CEA, CA19-9, and both piRNAs (86%; Fig. 4E).

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Figure 3. Diagnostic performance of piR-5937 and piR-28876 in the validation phase of the study. A, The expression of piR-5937 is significantly downregulated in serum samples of colon cancer patients compared with healthy donors (P < 0.0001). B, ROC analyses based on the expression of piR-5937 (AUC ¼ 0.7673). C, The levels of piR-5937 increased significantly 1 month after the surgery of colon cancer patients (P ¼ 0.0458). D, The expression of piR-28876 is significantly downregulated in serum samples of colon cancer patients compared with healthy donors (P < 0.0001). E, ROC analyses based on the expression of piR-28876 (AUC ¼ 0.7074). F, The levels of piR-28876 increased significantly 1 month after the surgery of colon cancer patients (P ¼ 0.0010). , P < 0.05 and , P < 0.001.

Discussion healthy donors. In total, 97 and 87 piRNAs were detectable in colon cancer patients and healthy donors, respectively, in more Recent studies indicated that noncoding RNAs play an impor- than 50 copies per 1 million of reads and the top 10 piRNAs tant role in epigenetic regulation of cancers (27–29). Therefore, accounted for 93% of all detected piRNAs. The most abundant they may serve as novel biomarkers for patients with malignant piRNAs were piR-28131 (DQ597916), piR-1207 (DQ570956), diseases. As the tissue-based diagnosis remains invasive and time- and piR-28877 (DQ598677). Furthermore, NGS revealed 31 consuming, minimally invasive techniques such as blood-based piRNAs to be significantly deregulated in serum samples of colon tests are highly requested. Since 2008, circulating miRNAs are cancer patients compared with healthy donors. In 2016, Yuan and largely analyzed for their potential to serve as novel noninvasive colleagues (23) performed RNA sequencing analysis using plasma biomarkers in cancer patients (30–32). They were proved to be extracellular vesicles derived from healthy controls, colorectal present in various body fluids including serum, plasma, urine, or cancer patients, prostate cancer patients, and pancreatic cancer saliva (33). In addition, they are extremely stable and resistant to patients to thoroughly examine the extracellular RNA composi- degradation by ribonucleases (30); thus, they may be involved in tion and distribution in human plasma. In accordance with our cell-to-cell communication and other complex processes (34). data, they identified 118 different piRNAs, and the top 10 piRNAs Importantly, RNA sequencing revealed that not only miRNAs but accounted for 96% of them. Importantly, among the top three also the other types of noncoding RNAs including piRNAs are most abundant piRNAs were again piR-000765 (DQ570956) and stably present in human blood (21, 22). In 2015, Yang and piR-020326 (DQ597916); thus, it seems that these piRNAs could colleagues (9) firstly described the presence of piR-57125 play important roles in circulation. Interestingly, the most (DQ596014) in serum and plasma and proved that similarly to expressed piRNAs in both studies showed identical sequences miRNAs, this piRNA remains extremely stable regardless of repet- 0 0 and only differed at 5 or 3 ends by one base, suggesting that they itive freeze-thawing or long-term incubation at room tempera- are derived from the same precursor sequence. ture. Since that time, several other articles described the presence Currently, RT-qPCR is the most commonly used approach for of piRNAs in circulation and their deregulation in blood samples small noncoding RNA expression quantification. Nevertheless, in of cancer patients (8, 20, 35). case of piRNAs, there are no well-established reference controls To our knowledge, this is the first study analyzing the expres- that could be used for expression normalization. Therefore, based sion of circulating piRNAs in serum samples of colon cancer on the results of NGS, we have chosen three piRNAs and tested patients using NGS and subsequent RT-qPCR validation. Firstly, their suitability to serve as normalization controls for piRNA small RNA sequencing was performed to assess the expression expression. Finally, piR-28131 was identified as the best gene profile of piRNAs in serum samples of colon cancer patients and with high stability in all analyzed sets of samples. However,

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Figure 4. Diagnostic potential of piRNAs compared with CEA and CA19-9. A, Downregulated expression of piR-5937 (cutoff (dCt): 3.932) enables to diagnose colon cancer with higher sensitivity than CEA (cutoff: 5 ng mL1). B, Downregulated expression of piR-5937 (cutoff (dCt): 3.932) enables to diagnose colon cancer with higher sensitivity than CA19-9 (cutoff 27 U mL1). C, Downregulated expression of piR-28876 (cutoff (dCt): 5.169) enables to diagnose colon cancer with higher sensitivity than CEA (cutoff: 5 ng mL1). D, Downregulated expression of piR-28876 (cutoff (dCt): 5.169) enables to diagnose colon cancer with higher sensitivity than CA19-9 (cutoff 27 U mL1). E, Detected cases number using CEA, CA19-9, piR-5937, piR-28876, or their combination.

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Circulating PIWI-Interacting RNAs in Colon Cancer

further confirmation is needed. Interestingly, this piRNA is treatment. Nevertheless, it will be necessary to validate these data thought to be one of the tRNA-derived piRNAs as its sequence on larger and independent sets of patients. Further, we are aware overlaps with the 50 end of 10 Gly tRNAs (36). This could be one of of the fact that the use of a biomarker which is negatively the reasons of its high abundance in circulation. correlated with the disease is difficult to translate into clinical From the 31 piRNAs differentially expressed between serum use. Thus, we expect these piRNA biomarkers not to be used samples of colon cancer patients and healthy donors, five were separately but in combination with other biomarkers upregulated chosen for further validation (piR-5937, piR-28876, piR- in CRC serum samples to increase analytical performance and 23210, piR-23209, and piR-32159). During the training phase reduce the possibility of technical failure of the measurement. of the study, downregulated levels of piR-5937, piR-28876, We are also aware of potential limitations of this study, and it is piR-23210, and piR-32159 in serum samples of colon cancer evident that still many issues must be addressed in order to patients were confirmed. In addition, the expression of all four establish piRNAs as novel diagnostic tools. Firstly, expression piRNAs decreased significantly with advanced clinical stage. profiling was performed using pooled samples of colon cancer Because the blood is exposed to a wide variety of cell types, it is patients/healthy donors as an insufficient amount of RNA was difficult to identify a tissue or organ of origin of these piRNAs. isolated from individual blood serum samples. Thus, because the In addition, cell stress, external stimuli, or nutrition can affect quantity of free-circulating RNAs in body fluids can be very the presence of small RNAs in circulation (37). As mentioned variable and a subject to interindividual variation, a bias could previously, piR-28876 was found to be significantly down- be introduced into the results using this kind of an approach. regulated in tumor tissue of colorectal cancer patients com- Today, improved kits for small RNA library preparation are pared with adjacent healthy tissue (18). Thus, it seems that this available tagging efficiently in samples with low piRNA abun- piRNA could potentially act as the tumor suppressor. Never- dance and enabling to use as little as 1 ng of total RNA (39). theless, it will be necessary to confirm this assumption by Secondly, all patients were of the same ethnicity, and samples detailed in vitro and in vivo studies. Concerning piR-23210, its were enrolled in a single center. On the other hand, the dynamics levels were overexpressed in the metastatic tissue of colorectal of piRNAs prior and after the surgery was analyzed only in case of cancer patients compared with the control benign tissue (18). several patients. In addition, these samples were obtained from a Interestingly, piR-5937 overlaps significantly with the 50 end of different hospital with various times of sample storage, and 8 Glu tRNAs and differs by only one base from piR-5938, which although the methodology of serum samples collection and PCR is one of the most abundant piRNAs in human serum (36). measurement was the same in the whole course of the study, we However, nothing is known about the source and function of have observed a small shift in piRNAs' expression. To establish this piRNAs in circulation. Concerning the piR-23209, its final cutoff values for candidate piRNAs, our results need to be deregulated levels were previously found in plasma samples further validated in the multicenter prospective studies with of pancreatic cancer patients as well as in prostate cancer (23); independent cohorts of patients and laboratory facilities. however, the training phase of this study did not confirm In summary, growing availability of NGS technologies enables significant deregulation of this piRNA in colon cancer patients. the identification of novel classes of circulating noncoding RNAs. Finally, the validation phase of the study was carried out with Up today, only limited number of studies analyzed the expression the aim to further characterize the diagnostic potential of two profiles of piRNAs in human blood. Our data underline the selected piRNAs (piR-5937 and piR-28876) that enabled to enormous potential for circulating piRNAs to serve as novel differentiate colon cancer patients from healthy donors with the noninvasive biomarkers, and although their release mechanisms highest sensitivity and specificity. Similarly to the results obtained and biological significance require further study, their involve- during the training phase of the study, both piRNAs were signif- ment in colon cancer pathogenesis is evident. icantly downregulated in serum samples of colon cancer patients, and their diagnostic potential was high also in case that only the Disclosure of Potential Conflicts of Interest patients in clinical stage I were included. Unfortunately, the No potential conflicts of interest were disclosed. combined expression of both piRNAs did not provide better results than piR-5937 and piR-28876 separately as these markers Authors' Contributions were positively correlated. Pinsky and colleagues (38) proved that Conception and design: P. Vychytilova-Faltejskova, M. Svoboda, O. Slaby if the additional marker is positively correlated with the primary Development of methodology: P. Vychytilova-Faltejskova, M. Sachlova marker, then it is unlikely to increase the AUC, even when it has a Acquisition of data (provided animals, acquired and managed patients, good diagnostic ability on its own. Lastly, the comparison of both provided facilities, etc.): P. Vychytilova-Faltejskova, M. Sachlova, Z. Kosarova, piRNAs with currently used biomarkers CEA and CA19-9 was K. Slaba, Z. Kala, M. Svoboda, R. Vyzula performed. It was proved that although the elevated levels of CEA Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. Vychytilova-Faltejskova, L. Radova, M. Sachlova, are detected in less than 50% of colon cancer patients, down- K. Slaba, Z. Kala, R. Vyzula, W.C. Cho, O. Slaby regulation of piR-5937 and piR-28876 was observed in almost Writing, review, and/or revision of the manuscript: P. Vychytilova-Faltejskova, 70% of all tested samples. However, the highest diagnostic Z. Kala, M. Svoboda, I. Kiss, R. Vyzula, W.C. Cho, O. Slaby sensitivity was reached in case of the combination of all four Administrative, technical, or material support (i.e., reporting or organizing markers. In addition, the levels of both piRNAs significantly data, constructing databases): M. Sachlova, K. Slaba increased in serum samples of patients 1 month after the surgery, Study supervision: O. Slaby Other (made an analytic part): K. Stitkovcova which indicates that their levels are linked to the presence of the tumor in colon cancer patients. Considering these facts, it is Acknowledgments obvious that mentioned piRNAs could serve as promising bio- The work has been supported by Ministry of Health of the Czech Republic, markers for early colon cancer detection as well as potential novel grant no. 16-31765A (P. Vychytilova-Faltejskova, Z. Kala, I. Kiss, and O. Slaby) biomarkers for the monitoring of patients after the surgical and MH CZ-DRO (MMCI, 00209805, to O. Slaby and M. Svoboda), by the

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Ministry of Education, Youth and Sports of the Czech Republic under the project advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate CEITEC 2020 (LQ1601, O. Slaby). this fact.

The costs of publication of this article were defrayed in part by the Received March 22, 2018; revised May 1, 2018; accepted June 22, 2018; payment of page charges. This article must therefore be hereby marked published ﬁrst July 2, 2018.

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1028 Cancer Epidemiol Biomarkers Prev; 27(9) September 2018 Cancer Epidemiology, Biomarkers & Prevention

Circulating PIWI-Interacting RNAs piR-5937 and piR-28876 Are Promising Diagnostic Biomarkers of Colon Cancer

Petra Vychytilova-Faltejskova, Karolina Stitkovcova, Lenka Radova, et al.

Cancer Epidemiol Biomarkers Prev 2018;27:1019-1028. Published OnlineFirst July 5, 2018.

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