Sample collection
The sample size for this study was calculated considering the incidence of prostate cancer in Pakistan to be 5.3% per 100,000 people, with a marginal error, and a confidence interval of 5% and 95%, respectively [15, 16]. Using the above parameters, the sample size was calculated to be 73. However, we were able to procure a total of 99 FFPE tissue blocks from the years 2019 and 2020, with a confirmed diagnosis of prostate cancer. In addition, a total of 33 FFPE tissue blocks from the year 2020, with a confirmed diagnosis of benign prostatic hyperplasia (BPH) were also included. The samples/blocks were obtained from the Department of Pathology and Laboratory Medicine at the Aga Khan University (AKU), Karachi after obtaining informed consent from all subjects. The study was approved by the AKU Ethics Review Committee (AKU-ERC #: 2021-1460-18525). All methods were performed in accordance with the relevant guidelines and regulations.
Histopathological analysis of prostate cancer biopsy samples
The FFPE tissue blocks were used to prepare slides for hematoxylin and eosin staining using standard protocol [17]. The tumors were graded using the International Society of Urological Pathology (ISUP) 2014 / WHO 2016 prostate cancer grade group system [18]. The specimens were graded by documenting histopathological parameters, such as Gleason major, minor and total scores, perineural invasion, and infiltration of lymphocytes in the specimen. To characterize the presence of lymphocytes in the tissue, light microscopy was used and the presence of lymphocytes was categorized into zero, +1, +2, +3 for the presence of zero, 1-12, 13-21, and >22 lymphocytes respectively.
DNA extraction from prostate carcinoma and benign prostatic hyperplasia FFPE tissue blocks
For each given sample, four 10 µm thick FFPE sections were cut using a microtome and were stored at room temperature into two autoclaved 1.5 ml microcentrifuge tubes each for RNA and DNA extractions until further use. In the first step, the sample was deparaffinized. For this purpose, each sample was washed with 1000 µl of xylene and mixed by vortexing for 30 seconds, followed by incubation for 10 minutes on a shaker at room temperature. After incubation, the tubes were centrifuged at 15,000 rpm for 2 minutes, and subsequently, the xylene was removed. This step was repeated twice until all the paraffin was replaced by xylene. In the next step, xylene was removed from the tissues using 100% ethanol. For this purpose, 1.5 ml of 100% ethanol was added to each sample and mixed using vortex for 30 - 60 seconds. After this, the tubes were left on the shaker for 5 minutes and then centrifuged at 15,000 rpm for 2 minutes followed by removal of the liquid phase. This step was repeated twice until all the xylene was removed. Finally, the samples were gradually rehydrated using different concentrations of ethanol (95% and 70% respectively). For this purpose, 1.5 ml of 95% ethanol was added to the dehydrated samples and mixed using vortexing for 30 seconds, after which they were kept on a shaker for 10 minutes and then centrifuged at 15,000 rpm for 2 minutes. This step was repeated once using each concentration, and the final step was performed using deionized water. Finally, DNA was extracted from each sample using DNeasy® Blood & Tissue kit (Qiagen, USA), following the manufacturer's instructions. The DNA was stored at -80⁰C until further use.
Extraction of RNA by TRIzol-chloroform method and cDNA synthesis
RNA was extracted from the tissues using the TRIzol-chloroform method [19]. Briefly, following the tissue digestion, 700 µl of TRIzol® reagent, (Invitrogen, Thermo Fisher Scientific, Inc.) was added to each sample and the samples were allowed to incubate on ice for 5 minutes to allow the disassociation of nucleoprotein complexes. After the incubation, 200 µl of chloroform was added to each tube, and contents were mixed vigorously, followed by another incubation at 4⁰C for 10 -15 minutes, and centrifugation for 5 minutes at 12,000 rpm, to allow the phase separation in the mixture. Following this centrifugation, the upper aqueous phase, where RNA is concentrated, was transferred to a fresh autoclaved 1.5 ml microcentrifuge tube without disturbing the interphase, and chilled 1000 µl isopropyl alcohol was added and incubated for 10 minutes at room temperature to chelate the RNA from the aqueous phase. Following the incubation, the tubes were centrifuged for 10 minutes at 12,000 rpm to obtain a white pellet of pure RNA at the bottom of the tube. The pellet was washed with 1000 µl of 70% ethanol and left to air dry. The pellet, containing RNA, was finally resuspended in 50 µl of nuclease-free water. The RNA was stored at -80⁰C until further use.
To remove the genomic DNA contamination in the RNA samples, and before cDNA synthesis, total RNA was treated with DNase I. For this purpose, 1 µg the total RNA template was combined in a 0.2 ml tube with 1 µl of (10X) reaction buffer containing MgCl2, 1 µl of DNase-I, RNase-free 1U/1µl (Thermo Fisher Scientific, Cat. No. EN0521), and suitable volume of nuclease-free water for a final volume of up to 10µl. The prepared reaction was incubated for 30 minutes at 37⁰C in the Master cycler X50a (Eppendorf, Germany). To prevent the hydrolysis of RNA after the DNase-I treatment, 1µl of 50 mM EDTA was added and samples were allowed to incubate for 10 minutes at 65⁰C. The DNase-I treated total RNA, from the above step, was converted to cDNA using the OneScript® plus cDNA synthesis kit (ABM, Canada. Cat. No. G236) following manufacturer's instructions and stored at -20⁰C till further use.
Conventional PCR for the detection of EBV in prostate cancer and benign prostatic hyperplasia samples
To detect the presence of EBV in the prostate cancer samples (n= 99), conventional PCR was employed using the EBNA-2 gene primers, since EBNA-2 is constitutively expressed in EBV-infected cells [20]. For PCR reaction, 100-150 ng of DNA template was combined with the 4 µl of BesTaq™ master mix (2X) (ABM, Canada, Cat. no. G464), 1 pM of custom-made forward and reverse primers (Table 1) (Macrogen, USA) and nuclease-free water to a final volume of upto10µl. The above reaction was used in PCR with following cycling conditions: initial denaturation for 10 minutes at 95⁰C, followed by 36 cycles of denaturation for 15 seconds at 95⁰C, annealing for 1 minute at 60⁰C, and an extension for 30 seconds each at 72⁰C, followed by the final extension for 1 minute at 72⁰C. The amplicons from the reaction were analyzed on 1.8% agarose gel against a 50-bp ladder (Promega, USA) using ChemiDoc® imaging system (Bio-Rad Laboratories, USA). The amplicons showing bands at 96 bps were considered positive for EBNA-2.
Latency mapping of EBV in prostate cancer and benign prostatic hyperplasia tissue samples using quantitative Polymerase Chain Reaction (qPCR)
Following the identification of EBV-positive prostate cancer tissues, a quantitative real-time PCR was employed to analyze the expression of EBV latency-associated genes (EBNA-3B, EBNA-3A, EBNA-2, EBNA-1, LMP-2A, LMP-2, LMP-1, EBER-2, EBER-1, BZLF-1, AND BHRF-1) and determine the EBV-latency profile in prostate cancer samples. For this purpose, 2µl of cDNA sample was combined with a mixture containing 4 µl of BlasTaq™ (2X) qPCR master mix (ABM, Canada, Cat. No. G891), forward and reverse gene-specific primers (Table 1) (Macrogen, USA) and nuclease-free water to a final reaction volume of up to 10µl in a 0.2 ml tubes (Bio-Rad Laboratories, USA. Cat. No. TLS0851). The prepared reactions were subjected to the following thermal cycling conditions using Bio-Rad 1000 thermal cycler CFX96 (Bio-Rad laboratories, USA): initial denaturation for 10 minutes at 95⁰C, followed by 40 cycles for denaturation for 15 seconds at 95⁰C, annealing for 1 minute at 45⁰C, and a cyclic extension for 30 seconds at 72⁰C). A melt curve analysis was set up between 55⁰C to 95⁰C with an increment of 0.5⁰C every 5 seconds to plot the specificity of the products. Each sample was run in duplicates, while non-template controls were supplied with an additional 2µl of nuclease-free water instead of cDNA template.
Table 1
shows EBV latency-associated genes along with sequences of the forward and reverse primers.
Genes
|
Sequence (5’-3’)
|
EBNA-1
|
Fwd TACAGGACCTGGAAATGGCC
Rev TCTTTGAGGTCCACTGCCG
|
EBNA-2
|
Fwd GCTTAGCCAGTAACCCAGCACT
Rev TGCTTAGAAGGTTGTTGGCATG
|
EBNA-3A
|
Fwd CCCCTTAACTCAACCCATTAACC
Rev CGGCCCCTCCATTGGT
|
EBNA-3B
|
Fwd TGCCGCTGCAAGAGAGG
Rev AGGTCCGATTGCAACATGGA
|
LMP-1
|
Fwd CAGTCAGGCAAGCCTATGA
Rev CTGGTTCCGGTGGAGATGA
|
LMP-2
|
Fwd GGTTCTCCTGATTTGCTCTTCGT
Rev CGCGGAGGCTAGCAACA
|
LMP-2A
|
Fwd TCCCTAGAAATGGTGCCAATG
Rev GAAGAGCCAGAAGCAGATGGA
|
EBER-1
|
Fwd TGCTAGGGAGGAGACGTGTGT
Rev TGACCGAAGACGGCAGAAAG
|
EBER-2
|
Fwd AACGCTCAGTGCGGTGCTA
Rev GAATCCTGACTTGCAAATGCTCTA
|
BZLF-1
|
Fwd AAATTTAAGAGATCCTCGTGTAAAACATC
Rev CGCCTCCTGTTGAAGCAGAT
|
BHRF-1
|
Fwd GGCTTACCTCGGTTCCCTCTTA
Rev TCCCGTATACACAGGGCTAACAGT
|
Fwd = Forward primer; Rev = Reverse primer. |
Analysis of differences in the histopathologic features of the EBV-positive and EBV-negative prostate carcinoma tissues
To compare the difference in the mean Gleason scores (major, minor, and total) between EBV-positive and EBV-negative prostate cancer samples, independent samples t-test was used. Similarly, the Pearson Chi-Square test was applied to determine the association of perineural invasion, intratumoral lymphocytes, stromal lymphocytes, and benign tissue lymphocytic infiltration with EBV status (positive or -negative), while the Spearman correlation test was applied to study the relationship between different histopathological parameters and EBV status. For all the statistical tests used in this study, a p<0.05 was considered statistically significant. IBM-SPSS version 23.0 was used to analyze the data.