Methylation status at HYAL2 predicts overall and progression-free survival of colon cancer patients under 5-FU chemotherapy

Genomics 106 (2015) 348–354

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Methylation status at HYAL2 predicts overall and progression-free survival of colon cancer patients under 5-FU chemotherapy Katrin Pfütze a,b,⁎, Axel Benner c, Michael Hoffmeister d, Lina Jansen d, Rongxi Yang a,b, Hendrik Bläker e, Esther Herpel e,f, Alexis Ulrich g,h, Cornelia M. Ulrich i, Jenny Chang-Claude j, Hermann Brenner d,k, Barbara Burwinkel a,b a

Helmholtz-University Group Molecular Epidemiology, German Cancer Research Center (DKFZ), Germany Molecular Biology of Breast Cancer, Department of Obstetrics and Gynecology, University of Heidelberg, Germany c Division of Biostatistics, German Cancer Research Center (DKFZ), Germany d Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Germany e Department of General Pathology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany f NCT Tissue Bank, National Center for Tumor Diseases (NCT), Heidelberg, Germany g Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany h Division of Molecular oncology, National Center for Tumor Diseases (NCT), Germany i Division of Preventive Oncology, National Center for Tumor Diseases (NCT)/German Cancer Research Center (DKFZ), Germany j Division of Cancer Epidemiology, Unit of Genetic Epidemiology, German Cancer Research Center (DKFZ), Germany k German Cancer Research Center (DKTK)Germany b

a r t i c l e

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Article history: Received 21 July 2015 Received in revised form 5 October 2015 Accepted 6 October 2015 Available online 21 October 2015 Keywords: DNA methylation HYAL2 Predictive marker Prognostic marker Colorectal cancer Chemotherapy 5-FU

a b s t r a c t DNA methylation variations in gene promoter regions are well documented tumor-specific alterations in human malignancies including colon cancer, which may influence tumor behavior and clinical outcome. As a subset of colon cancer patients does not benefit from adjuvant chemotherapy, predictive biomarkers are desirable. Here, we describe that DNA methylation levels at CpG loci of hyaluronoglucosaminidase 2 (HYLA2) could be used to identify stage II and III colon cancer patients who are most likely to benefit from 5-flourouracil (5-FU) chemotherapy with respect to overall survival and progression-free survival. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Colorectal cancer (CRC1) is the third most frequently diagnosed cancer in both women and men worldwide, with over one million diagnosed cases and ∼ 600,000 deaths in 2008, accounting for 9.8% of all cancer diagnoses and 8.1% of cancer deaths, respectively [1,2]. CRC tumors exhibit a significant heterogeneity even within the same pathologic stage in both prognosis and response to therapy [3]. Various treatment strategies were able to generally improve survival among CRC

⁎ Corresponding author at: Group Molecular Epidemiology, German Cancer Research Center (DKFZ), Molecular Biology of Breast Cancer, Department of Gynecology and Obstetrics, University of Heidelberg, German Cancer Research Center, Im Neuenheimer Feld 581, 69115 Heidelberg, Germany. E-mail address: [email protected] (K. Pfütze). 1 Colorectal cancer.

http://dx.doi.org/10.1016/j.ygeno.2015.10.002 0888-7543/© 2015 Elsevier Inc. All rights reserved.

patients over the past decades [2,4], however a subset of patients do not show benefit, especially if an adjuvant therapy is applied. For example, for patients diagnosed with stage II tumors the benefit of chemotherapy is highly debated [5,6,7]. To improve the prediction of disease outcome and the selection of patients for treatment, intensive research has been performed to identify prognostic and predictive biomarkers for CRC, including both genetic and epigenetic alterations [8,9,10]. Due to this critical relevance for the control of gene transcriptional activities [11] and the involvement of DNA methylation in cancer pathogenesis [12], DNA methylation levels are promising biomarker candidates. Recently, it has been shown that modified methylation signatures of IGFBP3 and EVL can predict the clinical prognosis of CRC patients [8]. Also, DNA methylation of selected extracellular matrix genes has been shown to be useful to stratify the stage II colon cancers for their risk of recurrence and thereby, suggest patients who would benefit from adjuvant chemotherapy [8]. Moreover, altered methylation signatures in

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tumor cells can be important predictors for treatment response either independently or by causing a specific CRC phenotype e.g. CpG island methylator phenotype (CIMP2) [9]. The HYAL2 gene encodes the hyaluronoglucosaminidase 2 protein (Hyal2), a lysosomal hyaluronidase which can cleave hyaluronan (HA) into lower molecular weight subunits. It is generally accepted that HA has different effects according to its size. For example, native high molecular weight HA can inhibit angiogenesis in vitro while smaller fragments containing of 3–10 disaccharide units stimulate angiogenesis in vitro [13]. These smaller fragments result from Hyal2 cleavage and have been reported to be associated with chronic inflammation and tumor angiogenesis [14,15,16]. In endothelial cells, small HA fragments were reported to induce proteolytic activity and cell proliferation which may support epithelial–mesenchymal transition and migration [13,17]. In the last decade, hyaluronidases and HA were studied in various cancer types and were often found to be higher expressed in malignant tumors compared to normal tissue, for example in bladder, prostate, head and neck, brain and colorectal cancers [18]. In CRC, especially in advanced stages, Hyal2 overexpression has been observed in the extracellular space when comparing extracts from fresh frozen tumor tissue and adjacent normal tissue of 34 CRC patients on RNA and protein level, leading to the conclusion that Hyal2 is involved in CRC progression [19]. Moreover, a study evaluating the amount of immunohistologically stained HA-positive cells in 202 formalin-fixed paraffin-embedded (FFPE3) primary CRC tumors had shown that a high proportion of HA-positive cells predicted poor survival [20]. However, to our knowledge no study has been conducted evaluating the methylation level of HYAL2 in CRC tumors. As archived FFPE tumor tissue is a common source for clinical analysis regarding staging and rating of patients prognosis, we analyzed the methylation levels in the promoter region of HYAL2 corresponding to a CpG island shore [21] in FFPE tumor tissues. This CpG island shore was recently identified to be a better prognostic marker in breast cancer than the CpG islands itself [22,23]. The association of these levels with overall (OS4) and progression-free survival (PFS5) of patients that were diagnosed with stages II and III colon cancer were evaluated in the here presented study. The survival data were assessed for differences between patients who had received 5-fluorouracil (5-FU6) chemotherapy and those who did not.

2. Material and methods 2.1. Study populations and ethics statement Paraffin-embedded tumor tissue specimens were obtained from 232 patients with primary colon cancer (IDC-10 position C18) participating in the German DACHS-Study (Darmkrebs: Chancen der Verhütung durch Screening) who were diagnosed and treated between 2003 and 2007 [24,25]. The study was approved by the ethical committees of the Medical Faculty of the University of Heidelberg and of the Medical Chambers of Baden-Württemberg and Rhineland-Palatinate. Written informed consent is obtained from each participant. Patients were included in the present study, when they were diagnosed with stage II or III colon cancer, did not receive neoadjuvant therapy, and either received adjuvant chemotherapy consisting of 5-FU and folic acid or no chemotherapy. DNA methylation analyses were performed in samples from 232 patients. The clinical features of these patients are presented in Table 1. Survival analyses were performed either including all patients or patients with microsatellite stable (MSS7) tumors only to minimize 2 3 4 5 6 7

CpG island methylator phenotype. Formalin-fixed paraffin-embedded. Overall survival. Progression-free survival. 5-fluorouracil. Microsatellite stable.

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Table 1 Sample cohort: To evaluate the differences in methylation levels at HYAL2 at three CpG loci and investigate the potential prognostic and predictive impact of DNA methylation levels on OS and PFS, 232 primary colon cancer patients were included. The clinical data of the complete cohort are presented. Stage at diagnosis

Gender Age (years) MS status

Chemotherapy Smoking behavior (until diagnosis)

Follow-up time (Months)

Male Female Median Range MSS MSI NA Yes No Never Former smoker Current smoker NA Median Range

II

III

58 70 71 41–94 87 21 20 18 110 73 41 14 0 58.6 0.2–77.7

44 60 70 37–87 80 12 12 80 24 56 37 10 1 49.1 1.4–78.0

MS, microsatellite; MSS, microsatellite stable; MSI, microsatellite instable; and NA, not available.

survival effects depending on an extreme tumor phenotype. Since tumors with a classified microsatellite instable (MSI8)-high phenotype were reported to show different benefit from 5-FU and since in the present study cohort only 33 MSI cases were included in the cohort, a separate survival analysis of MSI-high cases was not performed. To evaluate the potential correlation of DNA methylation levels and gene expression, data for 277 primary colorectal adenomacarcinomas was assessed from The Cancer Genome Atlas (TCGA) public data portal (https://tcga-data.nci.nih.gov/tcga/) November–October 2015. For all cases, normalized DNA methylation data generated with Illumina Infinium HumanMethylation450 platform (versions 1–15, level 3) as well as normalized RNASeq data produced on Illumina HiSeq 2000 sequencers (version 2, level 3) were collected and correlation of DNA methylation level and gene expression was analyzed. Thereby two independent analyses were performed: first, DNA methylation at cg26460678 (CpG 3 in the MassARRAY amplicon, Fig. 1), second the average methylation at cg12150256, cg13341668, cg05118960 and cg03721058 (present in CpG island-1, Fig. 1) was evaluated for their influence in gene expression regulation of HYAL2.

2.2. DNA isolation For DNA isolation needle biopsies with 0.6 mm in diameter from FFPE tumor tissue were prepared from the Institute of Pathology of the University of Heidelberg. Biopsies were taken from the middle of the tumor tissue, which was determined by a pathologist and premelted at 65 °C. Paraffin was removed by two xylene washing steps and samples were rehydrated by washing in different ethanol dilutions. The pellet was dried at 37 °C for 10–15 min and 180 μl tissue lysis buffer and 20 μl Proteinase K from the DNAeasy Blood & Tissue Kit (Qiagen) were applied. Each tumor cell fraction was incubated for 24 h in a shaking heat block at 56 °C. The DNA in the lysed emulsion was further purified using the DNeasy Blood & Tissue Kit according to the manufacturer's recommendation and recovered in 50 μl elution buffer. The DNA concentration was quantified using the NanoDrop 1000 spectrophotometer (PEQLAB Biotechnologie GmbH).

8

Microsatellite instable.

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Fig. 1. Schematic diagram of HYAL2 promoter region: CpG island-1 and CpG island-2 are located before the translation start site. The flanking CpG sites of the analyzed CpG loci are presented. The MassARRAY amplicon used for DNA methylation analysis covers three independent CpG loci in the CpG island shore, which have been reported recently to be the most promising prognostic biomarkers in breast cancer [22,23]. All chromosomal positions refer to genome build NCBI36/hg18.

2.3. Methylation analysis using MALDI-TOF mass spectrometry MALDI-TOF mass spectrometry (Sequenom) described by Breitling et al. [26] was used for DNA methylation quantification. DNA was bisulfite converted using the EZ-96 DNA Methylation-Gold Kit according to the manufacturer's recommendation. In short, 500 ng DNA in a total volume of 20 μl and 130 μl of CT-Conversion Reagent were incubated at 98 °C for 10 min and 64 °C for 2 h. The bisulfite-treated DNA was cleaned up by adding 400 μl M-Binding Buffer to a Silicon-A Binding Plate to which the mixture was added. After a washing step with 400 μl M-Wash Buffer and a centrifugation for 5 min at 3500 rpm, 200 μl M-Desulphonation Buffer were applied and incubated for 18 min. Two further washing steps were performed and DNA was eluted in 50 μl M-Elution Buffer. Subsequently, an amplicon (Chr3:50335612–50335723, Gene build NCBI36/hg18) containing three independent CpG sides (Fig. 1) was amplified using a bisulfite-specific primer pair as follows: forward: 5′-aggaagagagTGTTTAAGAAGGGAATTAGTTTTGG-3′; reverse: 5′cagtaatacgactcactatagggagaaggctCTAACACATTATCCTATCACACAAA-3′. Uppercase letters indicate the sequence-specific regions, whereas the nonspecific tags are shown in lowercase letters. Amplification reactions were carried out in a total volume of 6 μl containing 10 ng bisulfite treated DNA, 0.25 μM of each primer, 1 μM dNTP mixture and 0.01 units of Hot Star TaqDNA polymerase (Qiagen). To increase sensitivity and specificity an alternative touchdown PCR protocol was performed: 95 °C for 2 min, 94 °C for 30 s, 59 °C for 30 s and 72 °C for 60 s for the first 5 cycles and annealing temperature was set to 57 °C and 55 °C for the next 5 cycles, respectively. After that, conditions changed to 94 °C for 30 s, 53 °C for 30 s and 72 °C for 60 s for 30 cycles and a final amplification step at 72 °C for 5 min. According to the manufacturer's recommendation (SEQUENOM EpiTyper Assay) PCR products were SAP treated and a T-cleavage was performed. In short, to the PCR product 1.5 units SAP was added, followed by incubation at 37 °C for 20 min and inactivation of the enzyme at 85 °C for 5 min. For T-cleavage reaction, 5 μl T-cleavage reaction mix containing 0.64 times T7 Polymerase Buffer, 0.22 μl T Cleavage Mix, 100 mM DTT, 0.4 μl T7 RNA & DNA Polymerase and 0.09 mg/ml RNase A were added to 2 μl of the SAP-treatment product. After incubating the reaction for 3 h at 37 °C, free ions were removed from the solution by adding 6 mg Resin followed by 30 min rotation incubation at room temperature. A volume of 5–15 nl of each sample was dispensed to a 384 SpectroCHIP using Nanodispenser 1.0 (SEQUENOM). The mass

spectra were obtained from a MassARRAY Compact MALDI-TOF (SEQUENOM) and visualized with MassARRAY EpiTyper v1.1 software. 2.4. Statistical analysis Statistical analyses were performed with the statistical software environment R (R version 2.13.2 (2011-02-25)). Beta regression of methylation levels with respect to treatment and survival classes (“good prognosis” vs. “poor prognosis”) was performed using the R package betareg, version 2.3-0. Multivariate models considered possible confounding variables age, gender, stage, and smoking behavior. Survival analyses with respect to overall survival (OS) and progression-free survival (PFS) of colon cancer patients were done using concordance regression models as described by Dunkler et al. [27] and implemented in the R package concreg, version 0.4. Multivariate regression models again considered additional covariates age, gender, smoking behavior and stage. The follow-up time was estimated using the reverse Kaplan– Meier estimate of potential follow-up [28]. 3. Results Using the categorized methylation levels, we performed survival analysis to evaluate the prognostic impact of DNA methylation levels at the analyzed region of HYAL2 gene and its usage as prognostic biomarker. First, we dichotomized the methylation values such that we first compared patients with methylation levels lower than 10% versus those with higher methylation levels and second those with methylation levels higher than 90% versus those with lower methylation levels. With this cut off, extreme methylation levels were compared. If less than 5% of the cases met the criteria for one of these groups, the analyses could not be performed. To assess differences of survival due to colon cancer phenotype, we analyzed either all patients together or included only patients with MSS primary tumors. In univariate as well as in the multivariate concordance regression analysis adjusting for age, gender, treatment, smoking behavior and stage, no significant prognostic association could be observed between methylation levels at one of the analyzed CpG loci and either colon cancer-specific OS or PFS (data not shown). In addition to the usability of differentially methylated CpG loci in HYAL2 as prognostic marker, their potential usage as predictive biomarker was elucidated. Therefore, the interactions between methylation level

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and 5-FU chemotherapy combined with folic acid was tested in survival models using again concordance regression models based on the dichotomized methylation values. First, patients who exhibited tumors with methylation levels b 10% were compared to those with methylation levels N10%. If less than 5% of the cases meet the criteria for one of these groups, the analyses could not be performed and were announced as not applicable (NA). Here, a highly significant interaction between treatment and methylation levels at CpG 1 was observed if all colon cancer phenotypes were analyzed as well as if only patients with MSS primary tumors were analyzed, while no association could be observed for CpG2 and CpG3 (Table 2). In addition, patients who exhibited tumors with methylation levels N 90% were compared to those with methylation levels b 90%. No significant interaction could be observed (data not shown). In order to investigate the nature of significant interactions and to examine whether the survival of a patient is correlated directly with the methylation level of the certain CpG locus, the applied chemotherapy alone or a combination of both, a subgroup analysis was performed (Table 3). Among patients who received chemotherapy, those with low methylation levels at CpG 1 had a borderline significant better OS and PFS (HR = 0.51; 95% CI: 0.25–1.04; p = 0.064 and HR = 0.46; 95% CI: 0.22–0.95; p = 0.035, respectively) than patients with higher methylation. In addition, patients with low methylation levels showed a drastically improved OS and PFS (HR = 0.17; 95% CI: 0.05–0.55; p = 0.003 and HR = 0.2; 95% CI: 0.06–0.63; p = 0.006, respectively) if they received a chemotherapy compared to those without a chemotherapy, while this improvement could not be observed in patients with higher methylation levels. The association of the methylation level at CpG 1 and OS and PFS was found in both analyses: when tumors with all phenotypes or only MSS tumors were included, suggesting a general informative value of HYAL2 methylation status on OS and PFS under therapy. For illustration, Kaplan–Meier overall and progression-free survival estimates of colon cancer patients were computed using the methylation level cut off of less than 10% methylation versus higher than 10% methylation (Fig. 2A and B). Patients, who received adjuvant chemotherapy and show low methylation levels at CpG 1 show more benefit from the given chemotherapy compared to those with higher methylation levels distinguishable by an improved OS and PFS. In contrast, patients without chemotherapy showed poor OS and PFS, if the primary tumor exhibited low methylation levels compared to those patients with higher methylation levels. In addition, patients with low methylation levels showed better OS and PFS if they receive a 5-FU chemotherapy compared to those without chemotherapy. Again, if patients had higher methylation levels the effect was the opposite.

Table 2 Differentially methylated CpG loci in HYAL2 as potential predictive biomarker: Concordance regression analysis indicates interactions between low methylation levels (b10%) and overall or progression-free survival and 5-FU chemotherapy combined with folic acid (treatment). p adj for interaction between [methylation b 10%] and treatment for all tumors CpG locus

N (methylation b10%/N10%)

OS

PFS

HYAL2_CpG 1 HYAL2_CpG 2 HYAL2_CpG 3

61/141 14/188 2/200

0.005 0.09 NA

0.004 0.74 NA

p adj for interaction between [methylation b 10%] and treatment for tumors classified as MSS CpG locus HYAL2_CpG 1 HYAL2_CpG 2 HYAL2_CpG 3

N (methylation b10%/N10%) 47/103 11/139 2/148

OS 0.01 0.42 NA

PFS 0.01 0.58 NA

MSS, microsatellite stable; OS, overall survival; PFS, progression-free survival; NA, not applicable; p-values b 0.05 are highlighted in red; p-values are adjusted for age, gender, stage at diagnosis, therapy and smoking behavior. No significant interaction could be observed for high methylation levels (N90%) versus lower methylation levels and survival under therapy (see supplementary Table 2).

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To evaluate whether DNA methylation itself can serve as a biomarker or the effect is more dependent on gene expression, the correlation of DNA methylation level and gene expression was analyzed. Data from 277 colorectal adenomacarcinomas in the TCGA public data portal revealed no correlation between DNA methylation and HYAL2 expression neither for cg26460678 (CpG 3 in the MassARRAY amplicon, Fig. 1) nor the average methylation at cg12150256, cg13341668, cg05118960 and cg03721058 (present in CpG island-1, Fig. 1) (data not shown). Interestingly, DNA methylation level at cg26460678, so the loci present in the CpG island shore showed much higher methylation levels than the CpG island itself (see supplementary data). As the CpG loci (cg26460678) present on the Illumina Infinium HumanMethylation450 was not the most promising CpG loci identified in this study and clinical data did not include chemotherapy data, we did not analyze the predictive potential of DNA methylation with the data set assed from TCGA. 4. Discussion Early detection and chemotherapeutic strategies have improved over the last decades leading to prolonged OS and PFS of CRC patients. However, a more personalized therapy would improve patient's response rates and quality of life by avoiding unnecessary treatment [4]. Therefore, biomarkers independent of pathological staging are needed. The ever-growing number of genes that show epigenetic alterations in cancer emphasizes the crucial role of these epigenetic alterations, and particularly of DNA methylation, for future diagnosis, prognosis and prediction of response to therapies [29]. DNA methylation markers have been suggested to improve either the prediction of patient survival or therapy response, although none of them is in clinical usage yet [9,30, 31,32,33,34]. As some patients, especially CRC patients diagnosed with stage II generally do not receive chemotherapy since their survival benefit could not be supported in various studies [5,6,7], markers which provide information regarding treatment benefit are needed. DNA methylation alterations in various genes of colon cancer tumors were reported to influence chemosensitivity to 5-FU [35,36]. Recently, the DNA methylation signatures of selected extracellular matrix genes were identified to help to stratify high-risk stage II colon cancer patients and thereby suggest those for therapy [8]. To our knowledge, the present study is the first report that suggests HYAL2 methylation status in primary tumors of CRC as a predictive biomarker. We show that low methylation levels at a specific CpG locus (chr3:50335646) in the CpG island shore are significantly associated with improved OS and PFS under chemotherapeutic conditions in patients with MSS phenotype tumors and when all tumor phenotypes were included. Stages II and III colon cancer patients with low methylation levels at this locus seem to benefit more from 5-FU adjuvant chemotherapy compared to patients with higher methylation levels. Therefore, methylation levels at this CpG may be useful to suggest patients for therapy. In this study, we investigated the DNA methylation levels at three independent CpG loci in the promoter region of HYAL2. Although the analyzed region of HYAL2 does not harbor a CpG island, the surrounding sequence is termed a CpG island shore. Methylation signatures at CpG island shores are known to be strongly related to gene expression and are commonly observed in CRC [21]. Correlation of methylation levels with RNA or protein expression could not be assessed in our study population, as archival FFPE tumor tissue was analyzed, as it is known, that protein as well as RNA based biomarkers identified in FFPE tumor tissue may be problematic due to fixation procedure and low RNA stability [37, 38]. Furthermore, methylation of HYAL2 has been reported as potential mechanism of expression regulation [39]. However, we were not able to support that fact by the analysis DNA methylation and RNASeq data of 277 colorectal adenocarcinomas assessed from TCGA. Nonetheless, the study is limited in some points. Although we analyzed about 200 colon cancer patients in total, the survival analysis is based on subgroups with smaller samples sizes, as given in Table 3. Especially tumors

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Table 3 Subgroup analysis for CpG1 in HYAL2 methylation as predictive biomarker: Concordance regression analysis indicate the nature of interaction between the methylation level at CpG1 and overall or progression-free survival and 5-FU chemotherapy combined with folic acid. All phenotypes N (b10%/N10%) or

OS

PFS

Comparison

Subgroup

N (w/o 5-FU/with 5-FU)

HR [95% CI]

p adj

HR [95% CI]

p adj

Between patients low methylation Levels at CpG1 vs. higher methylation levels Between patients with 5-FU Chemotherapy vs. patients without chemotherapy

W/o 5-FU With 5-FU Methylation b10% Methylation N10%

36/79 32/55 36/32 79/55

2.14 [0.90–5.07] 0.51 [0.25–1.04] 0.17 [0.05–0.55] 1.09 [0.45–2.63]

0.08 0.06 0.003 0.85

1.78 [0.78–4.06] 0.46 [0.22–0.95] 0.20 [0.06–0.63] 1.59 [0.70–3.60]

0.17 0.04 0.01 0.26

MSS only N (b10%/N10%) or

OS

PFS

Comparison

Subgroup

N (w/o 5-FU/with 5-FU)

HR [95% CI]

p adj

HR [95% CI]

p adj

Between patients low methylation Levels at CpG1 vs. higher methylation levels Between patients with 5-FU Chemotherapy vs. patients without chemotherapy

W/o 5-FU With 5-FU Methylation b10% Methylation N10%

29/49 25/47 29/25 49/47

2.33 [0.75–7.30] 0.62 [0.28–1.35] 0.23 [0.05–0.99] 1.24 [0.46–3.32]

0.15 0.23 0.05 0.67

2.05 [0.69–6.10] 0.57 [0.26–1.27] 0.26 [0.06–1.04] 1.66 [0.64–4.34]

0.2 0.17 0.06 0.3

MSS, microsatellite stable; OS, overall survival; PFS, progression-free survival; HR, average hazard ratio; CI, confidence interval; w/o, without; HRs and p-values b 0.05 are highlighted in red; all results are adjusted for age, gender, stage at diagnosis, and smoking behavior.

classified as MSI-high could not be analyzed independently due to their small sample number. Since it has been shown that CRC patients with MSI-high primary tumors respond differently to 5-FU based chemotherapeutic agents than those with other phenotypes [40,41], the analysis was performed either including all patients or only patients with primary tumors classified as MSS. Nevertheless, a conclusion could be drawn for colon patients in general. Further analysis including either more MSI tumors or exclusively this phenotype should be conducted to validate the potential usage of HYAL2 methylation as predictive marker for colon cancer patients in general and especially for the MSI phenotype. Furthermore, the potential of HYAL2 methylation to predict prognosis

should be investigated for rectal cancer. As CpG loci cg26460678 present on the Illumina Infinium HumanMethylation450 was not the most promising CpG loci identified in this study and clinical data did not include all necessary data, we did not analyze the predictive potential of DNA methylation with the data set assed from TCGA. The present study revealed that under chemotherapeutic conditions, low methylation levels were associated with favorable OS and PFS of patients. In contrast, we observed a borderline significant association with worse OS if patients with lower methylation of HYAL2 received no chemotherapy compared to those with higher methylation levels. The HYAL2 gene is located on chromosome 3p21.3, a well characterized

Fig. 2. Kaplan–Meier estimates of OS and PFS of stage II and III colon cancer patients with low or high methylation levels with and without 5-FU chemotherapy. Survival estimates were computed according to the methylation status at CpG 1 in HYAL2 (chr3:50335646). (A) Overall survival: patients, who received adjuvant chemotherapy and show low methylation levels at CpG 1 (dashed red line) show more benefit from the given chemotherapy compared to those with higher methylation levels (solid red line) distinguishable by an improved OS. In contrast, patients without chemotherapy and a primary tumor exhibiting low methylation levels (blue solid line) compared to those patients with higher methylation levels (dashed blue line) showed bad OS. In addition, patients with low methylation levels showed better OS if they receive 5-FU chemotherapy (dashed red line) compared to those without chemotherapy (dashed blue line). Again, if patients had higher methylation levels the effect was the opposite. (B) Progression-free survival: As observed for OS, under chemotherapeutic conditions the patients with low methylation levels (dashed red line) showed improved PFS compared to patients with higher methylation levels (solid red line). If no chemotherapy was applied, the effect was opposite (dashed and solid blue lines). In addition, patients with low methylation levels showed better PFS if they receive 5-FU chemotherapy (dashed red line) compared to those without chemotherapy (dashed blue line), while the effect was opposite for patients with higher methylation levels.

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region harboring several candidate tumor suppressor genes (i.e., RASSF1, SEMA3B, HYAL3, HYAL1, TUSC2, RASSF1, ZMYND10, NPRL2, TMEM115, and CACNA2D2) that is frequently deleted in diverse cancer types, including non-small cell lung cancer [42], head and neck squamous cell carcinoma [43], and breast cancer [44]. However, a deletion was not reported in CRC. If a copy number variation at this location occurs, a change in DNA methylation levels could be observed as well. Therefore, copy number variation analysis should be performed in future analysis. As it has been reported that HYAL2 is epigenetically silenced via DNA hypermethylation [39], low methylation levels can subsequently result in high expression levels of Hyal2 [19].This may result in an intensive cleavage of hyaluronan (HA) and a subsequent increase of its lower molecular weight units which are associated with chronic inflammation and tumor angiogenesis, thereby facilitating the growth of solid tumors [14,15,16]. It has been reported, that Hyal2 is involved in chemokinesis and motility of cells [45,46] and may support tumor growth and proliferation, promote migration and metastasis, and protect tumor cells against immune surveillance [47]. Nevertheless, we observed no significant association between methylation levels of HYAL2 and prognosis of CRC patients in general. Considering the function of Hyal2, the possibly improved perfusion of the primary tumor due to increased angiogenesis [14,15,16] may improve the drug delivery to the cancer cells and thereby improve the therapy effects. Further increased proliferation due to Hyal2 [47] may improve therapy effects, as generally strong proliferating cells are a better target for chemotherapeutic drugs than less proliferating ones [48]. An additional relationship between Hyal2 and 5-FU chemotherapy has been suggested by a study in which the transient overexpression of WOX1 and Hyal2 sensitized murine L929 fibroblasts to TGF-beta1induced apoptosis finally leading to inhibition NF-κB activity [49]. This pathway is comparable to the function of 5-FU which induces apoptosis via direct inhibition of NF-kB [50,51]. In conclusion, our data suggest the usage of the methylation level of HYAL2 at CpG1 (chr3:50335646) as a predictive biomarker for colon cancer patients diagnosed with stage II or III independent of their tumor phenotypes. Patients with methylation levels lower than 10% seem to benefit more from adjuvant 5-FU chemotherapy than those with higher methylation levels and could therefore be suggested for therapy. Further large studies with quantification of methylation levels are necessary to confirm the results observed in this study. If confirmed, this marker could contribute to a more personalized therapy resulting in an improved survival and/or quality of life of patients and may reduce treatment costs. Conflicts of interest All authors declare no conflict of interest. Acknowledgments This work was supported by grants from the German Research Council (Deutsche Forschungsgemeinschaft, grant numbers BR 1704/ 6-1, BR 1704/6-3, BR 1704/6-4 and CH 117/1-1), and the German Federal Ministry of Education and Research (grant numbers 01KH0404 and 01ER0814). The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygeno.2015.10.002. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics, 2008, Ca Cancer J. Clin. 58 (2008) 71–96.

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