Sample acquisition and preparation. Colorectal tissues were obtained from 55 patients with colorectal cancer as paired tissue specimens by dissection of tumor and tumor-adjacent tissues. The first set of paired colorectal tissues, collected from 45 patients in the Baltimore area, was provided by Dr. Curtis Harris at NCI. The second set of paired colorectal tissues was collected from 10 patients in the Houston area by one of the authors (S.Y. X). In the second set of tissues, the tumor-adjacent tissues were dissected from an area approximately 20 cm away from the tumor lesions. Normal colorectal tissues dissected from 10 individuals who died accidentally were also provided by Dr. Curtis Harris at NCI and were used as negative controls. Of the 55 colorectal cancer patients, 53 patients had adenocarcinoma in various colon locations, 1 had rectal squamous carcinoma, and 1 had descending colon adenoma. Of the 55 patients, 32 were male and 23 female; 22 were Black, 30 White, 2 Asian, and 1 Hispanic; and their ages ranged from 35 to 82. Among the 10 controls without colorectal cancer, 4 were Black, 5 White, and 1 Asian, and they ranged in age from 21 to 75. Specific locations of the cancers and their adjacent tissues are described in .
Colorectal HPV infection in the patients with colorectal cancer
All specimens were frozen immediately following the sample dissections and were kept at -70°C until further analysis. About 100 mg of each specimen was randomly coded and screened in a blinded manner for the presence of HPV DNAs. Each colorectal tissue was homogenized in an Eppendorf tube in 1 ml TRIzol solution (Invitrogen, Carlsbad, CA) using an electric homogenizer (Omni International, Marietta, GA) with a separate disposable probe for each tissue. The isolated DNA was dissolved in ~200 μl of 8 mM NaOH and adjusted to pH 7.0 with 1 M HEPES. Various precautions were taken to minimize sample-to-sample cross-contamination, including limiting the tissue processing and DNA extraction to a maximum of ten samples per day.
HPV DNA detection.
HPV DNA was first amplified with pooled HPV L1 consensus primers, PGMY09/11 (20
). This pooled primer set contains five 5′ primers and thirteen 3′ primers and amplifies more than 25 types of anogenital HPVs. This was followed by nested PCR using an internal primer set, GP5/6 (21
). Each PCR reaction was carried out in a total volume of 50 μl containing 5 μl purified DNA, 1X AmpliTaq Gold PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl), 2.5 mM MgCl2
, 200 μM of each dNTP, 100 nM of pooled PGMY09/11 primers, and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin Elmer, Foster City, CA). PCR was carried out by activation of AmpliTaq Gold DNA polymerase for 9 min at 94°C and followed by 40 cycles of 30 sec each at 94°C, 55°C, and 72°C, with a final extension of 7 min at 72°C. One microliter of the first-run PCR reaction was used as a template for the nested PCR. The conditions for the nested PCR (40 cycles) were identical to the first-run PCR with the exception of the use of 100 nM of the GP5/6 primers and annealing at 40°C for 30 sec. The amplified products with the expected size (141 bp) () were gel purified and sequenced. The individual sequence was then used in a BLAST search against GenBank HPV sequences (NCBI).
Fig. 1 Schematic diagram of HPV16 genome as a representative for relative positions of primers and probes used in this study. The bracket line in the middle of the panel represents a linear form of the virus genome for better presentation of head-tail junctions, (more ...)
Detection of HPV16 E6 (16E6), HPV18 E6 (18E6), and HPV45 E6 (45E6) DNA was also carried out by nested PCR, using primers named by the location of their 5′ ends in each virus genome. The first-run PCR primer pairs for 16E6 were Pr80 (5′-CTGACTCGAG/TTTATGCACCAAAAGAGAAC-3′) and Pr625 (5′-GATCAGTTGTCTCTGGTTGC-3′); for 18E6, Pr79 (5′-CTGACTCGAG/AGATGTGAGAAACACACCAC-3′) and Pr749 (5′-CTCGTCGGGCTGGTAAATGT-3′); and for 45E6, Pr113 (5′-TGACGATCCAAAGCAACG-3′) and Pr563 (5′-CCTACGTCTGCGAAGTCT-3′). First-run PCR was followed by re-amplification using nested primer pairs for 16E6, Pr106 (5′-GTTTCAGGACCCACAGGAGC-3′) and Pr562 (5′-GTACTCACCCC/TGATTACAGCTGGGTTTC-3′); for 18E6, Pr 121 (5′-ATCCAACACGGCGACCCTAC-3′) and Pr528 (5′-AGCACGAATGGCACTGGC-3′); and for 45E6, Pr131 (5′-ACCCTACAAGCTACCAGAT-3′) and Pr545 (5′-TTCTTGCCGTGCCTGGTC-3′). The final PCR yielded a 456-bp product for 16E6 (), a 407-bp product for 18E6, and a 414-bp product for 45E6.
All 16E6-positive samples were also examined by nested PCR for the presence of an intact E2 ORF to evaluate the integration of HPV16 DNA into the host genome. This was performed using the HPV16E2 (16E2) primer set Pr3385 (5′-TATTAGGCAGCACTTGGC-3′) and Pr3840 (5′-AATCCAGTAGACACTGTAATAG-3′) for first amplification and another primer set, Pr3439 (5′-CTTGGGCACCGAAGAAACAC-3′) and Pr3790 (5′-TTGGTCACGTTGCCATTCAC-3′), for nested amplification. The final nested PCR yielded a 351-bp product for 16E2 ().
Under these PCR and nested PCR conditions, the detection efficiency for plasmid HPV DNA by nested PCR was limited to 1-10 fg of DNA (equivalent to ~130-1300 HPV genome copies), depending on the type of HPV and the primers used (data not shown).
. Dot-blot hybridization was performed only for the nested L1 PCR reaction and has been described elsewhere (23
). A biotinylated HPV16 L1 probe (nt 6664 to 6684) () was used for the hybridization at concentration of 1 pmol/ml hybridization solution.
Validation of PCR reactions. Human GAPDH DNA in each DNA sample was screened by PCR amplification using a sense primer, Pr6732 (5′-GTCATCAATGGAAATCCCATCACC-3′), in combination with an antisense primer, Pr7207 (5′-TAATACGACTCACTATAGGGA/CCGTTCAGCTCAGGGATG-3′). This primer set amplifies a 496-bp product and provides an indication of good DNA quality for each sample. Colorectal DNA samples from which GAPDH DNA could not be amplified were dropped from further study.
For HPV amplification from colorectal DNA samples, two water controls were also included for both first-run and nested PCR. If either of the two water controls yielded a false-positive in the nested PCR, the whole set of PCR and nested PCR reactions were started over. A sample was considered to be HPV-positive if PCR products of the expected sizes were detected for both L1 and E6 and were further confirmed by DNA sequencing.
In situ polymerase chain reaction. The randomly labeled colorectal tissues, which had been fixed in 10% buffered formalin for 16-18 hrs at room temperature, were embedded in paraffin and then cut at 7 μm. The sections were placed on silane-coated slides (Labsco, Louisville, KY) and stored at 4°C until use.
The sections were deparaffinized in xylene twice for 10 min each and rehydrated twice for 5 min in each graded ethanol before being put into distilled water. The sections were then digested with 0.8% pepsin (DAKO, Carpinteria, CA) in 0.2 N HCl for 5 min at 37°C and rinsed in DEPC water before being subjected to a hot-start PCR amplification using AmpliTaq Gold DNA polymerase (24
). The PCR amplification was performed on the slide in 50 μl of reaction solution containing 1X AmpliTaq Gold PCR buffer, 4 mM MgCl2
, 200 μM each dATP, dCTP, and dGTP, 60 μM dTTP, 40 pM digoxigenin-11-dUTP solution (DIG) (Roche, Indianapolis, IN), 400 nM of each primer 16E6 Pr106 (sense) and 16E6 Pr562 (antisense), 10 U of AmpliTaq Gold DNA polymerase, and 28 μl water, and covered with Hybaid SureSeal (Hybaid, Franklin, MA). The slide was placed in aluminum foil on the sample block of a thermal cycler that was filled with mineral oil. After first denaturation at 95 °C for 10 min, the section underwent amplification for 30 cycles (95 °C for 1 min, 72 °C for 2 min, and 55 °C for 2 min). After PCR amplification, the sections were washed in stringent wash solution (DAKO) at 50 °C for 60 min. Detection of DIG incorporated into the PCR product was performed with an alkaline phosphatase (AP)-conjugated DIG antibody (DAKO) and visualized in a chromogen solution containing NBT (4-nitroblue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3-indolyphosphate) (DAKO). Nuclear fast red was used for counter-staining. A positive reaction was defined as the presence of a purple-blue precipitate in cell nuclei.
Statistics. A two-tailed Fisher’s exact test was used for the analysis. A two-tailed McNemar’s exact test was used for the analysis of paired categorical data.