Study population and specimens
Seventy-four patients with CRC at the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) were enrolled in the study between February 2017 and December 2017. A total of 148 (74 × 2) paired CRC fresh-frozen and adjacent normal tissue specimens (at least 5 cm from the reception margin) were obtained during surgical resection. Both tissue statuses were confirmed by histopathologic diagnosis performed by two pathologists. The study was approved by the institutional review board of the Second Affiliated Hospital of Wenzhou Medical University. Written informed consent was obtained from each patient. Small pieces (each approximately 500 mg) of tumoral and paired adjacent normal tissues were resected, mixed with 500 µL RNA-Later, stored at 4°C for 1 h, and stored at -20°C for 24 h; the samples were stored over the long-term at -80°C. Moreover, 4 tissue microarrays (TMAs) with 717 biopsies was obtained from Professor Chang (Changhai Hospital, The Navy Military Medical University, Shanghai, China). Of these tissues, those from 669 patients had undergone curative surgery and the other 48 were from paracancerous tissues. The basic information of patients with CRC is shown in Supplementary Table 1.
Polymerase chain reaction (PCR) detection
DNA was extracted from 100 mg of these tissues by QIAamp DNA mini kit (DP304, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA was quantified using NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, MA, USA), and then stored at -20°C until use.
A previous study showed that the UL47, UL56, and UL77 genes of HCMV can be detected simultaneously in five different tumors [13]. Therefore, conservative regions of these three genes were selected to detect HCMV infection in this study to minimize the differences in detection caused by gene mutation. PCR was carried out using specific primers for the UL47, UL55, and UL77 genes of HCMV [13] (Table 1). MRC-5 cells (Medical Research Council cell strain 5, MRC-5, ATCC, Manassas, VA, USA) transfected with HCMV were used as a positive control, and the negative control consisted of sterile double-distilled water. PCR amplification was performed in a T100TM Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR samples contained 200 ng DNA temple, 1× Taq MasterMix (Tiangen Biotech Co., Ltd., Beijing, China), and 0.3 μL of each specific forward and reverse primers. The total volume was adjusted to 25 μL with double-distilled water. After initial denaturation at 95°C for 5 min, 35 cycles of DNA amplification were performed (95°C for 30 s, annealing for 58°C 30 s, 72°C for 30 s), followed by terminal extension at 72°C for 10 min. Finally, 5 μL of PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. Samples for which a band was detected in the correct position on the agarose gel and showing the correct sequence were regarded as HCMV(+).
RNA-seq and GEP analysis
Five HCMV(+) CRC tissues detected by PCR were selected for RNA-seq. GEPs were pre-processed with Cutadapter and FastQC to remove jointed reads and low-quality reads. Tophat (v.2.0.0) software was used to compare the sequences with the human and HCMV genomes. The GEPs of HCMV were determined using Integrated Genomics Viewer and Partek® Genomics Suite™ (version 6.5 beta, Partek, Inc., St. Louis, MO, USA) (Figure 1). Gene expression was calculated as follows:
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FPKM
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Total exon fragments
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Mapped reads (million) × exon length (kb)
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Miri Shnayder [14] considered more than two HCMV reads in the sample as positive for viral gene expression. Another study considered at least 10 reads as viral-positive infection [15]. Considering that in most cases, only a few sequences were identical to those of specific viruses because of the large differences between the human and viral genomes, we determined that at least one viral fragment (read) in a sample was considered as positive for HCMV infection.
Immunohistochemistry (IHC) detection of pIEA
Four TMAs containing 717 biopsy samples were deparaffinized in xylene and rehydrated through a graded alcohol series. Endogenous peroxidase activity was blocked with a 0.3% solution of hydrogen peroxide and methanol. Subsequently, anti-pIEA antigens (1:50 ZM-0078, ZSGB-BIO, Beijing, China; 1:200, sc69834, Santa Cruz Biotechnology, Dallas, TX USA) with 10 mM citrate buffer (pH 6.0) were retrieved at 98°C for 30 min with 1 mM EDTA buffer (pH 7.5). TMAs were then cooled and placed in PBS solution. After blocking in 20% normal goat serum for 30 min in a humidifier chamber, the TMAs were blotted and covered with antibodies and incubated for 2 h at room temperature (22°C). The arrays were then washed with PBS and incubated for 30 min with EnVisionTM+Dual Link System-HRP (Dako, Carpinteria, CA, USA). After rinsing 3 times with PBS for 3 min each time, the slides were incubated with DAB reagent (Dako) for 3–5 min and evaluated under a light microscope. The TMAs were then rinsed, counter-stained with hematoxylin, and observed under a Leica microscope (DM4000b; Wetzlar, Germany).
The IHC results were independently assessed by eight researchers including two pathologists blinded to the clinical data. pIEA staining in either cells of tissues was considered as pIEA-positive expression. Scores for pIEA expression were evaluated from the extent of staining as follows: 0 (0–1%), 1 (2–24%), 2 (25–50%), and 3 (51–100%). The number of pIEA(+) cells and total adenocarcinoma cells in each block was accurately counted to determine the staining extent. Grade 0 was regarded as negative, Grades 1 and 2 were identified as low expression (pIEA(+)), and Grade 3 was identified as high expression (pIEA(++)).
Clinical features and survival analysis
Patients with CRC with intact IHC data were included in survival analysis. Continuous clinicopathological data such as patients’ age were classified as dichotomous variables. Our primary outcome of interest was disease-free survival (DFS) and overall survival (OS). DFS was defined as the number of months from the date of undergoing surgery to the date of first relapse. Patients who experienced second primary tumors of other histotypes were counted as censored in DFS analysis. OS was measured in months from the date of undergoing surgery to the date that the patient died. Kaplan–Meier’s analysis with log-rank test and univariate Cox regression analysis was performed to determine the contribution of different levels of pIEA expression to survival.
Statistical analysis
Continuous variables were analyzed using Student’s t-test or non-parametric U test. Categorical variables were analyzed using Chi square test or Fisher’s exact test. Statistical analysis and graphing were performed with SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA), GraphPad Prism 7.0 (GraphPad, Inc., La Jolla, CA, USA), and R 3.5.1 (http://www.r-project.org/). A P value <0.05 was considered as statistically significant.
