Ethics
The study was reviewed and approved by the research ethics committee, Uppsala University, Uppsala, Sweden.
Patients characteristics
Patients with esophageal carcinoma, admitted to the Department of Oncology, University Hospital, Uppsala, Sweden, during 1990–2000 were identified. These patients formed a consecutive series. A total of 126 patients fulfilled the inclusion criteria but 26 patients were lost due to inadequate (n = 9) or absence of histological material (n = 17). The present study is based on a retrospective examination of esophageal carcinoma diagnostic biopsy samples from clinical cases, all retrieved tumor samples were re-evaluated before analysis.
The following parameters were studied: age, gender, weight loss, performance status according to the Eastern Cooperative Oncology Group (ECOG) [12] at first admittance, smoking habits, alcohol habits and differentiation of the tumor. Anatomical localization of the tumor were grouped into an upper part (15–24 cm), a middle part (25–34 cm) and a lower part of the esophagus (35–46 cm. The tumor status were characterized into localized (Primary tumor with or without local node metastases) or advanced disease (With distant metastases).
Treatment strategy was recorded as:
1. Preoperative radiation treatment, total dose of 40 Gy in 2 Gy fractions. This irradiation was given concomitant with chemotherapy (Cisplatin 100 mg/m2 and 5-FU 750 mg/m2, three cycles with an interval of three weeks of which two were given concomitantly).
2. Curatively intended radiation treatment, total doses 60–64 Gy.
3. Palliative treatment included radiation treatment, total dose 36 Gy in 3 Gy fractions. These patients also received brachytherapy and in some cases, also palliative chemotherapy.
4. Palliative treatment only including chemotherapy.
HPV plasmids
Plasmids containing HPV 16, 18, 31, 33, 39, 45, 52, 58 and 67 were supplied by T. Matsukura (National Institute of Health, Japan), A. Lörincz (Digene Corporation) or G. Orth (Institut Pasteur, Paris) or prepared by cloning from PCR products of clinical samples. The plasmids were used both as positive controls and to estimate the sensitivity of the assay.
Deparaffination procedure
The biopsies were fixated by treatment in buffered formalin followed by paraffin embedding. Paraffin blocks from the primary tumor were cut in 10-μm sections and 4 sections/patient were collected in the same microcentrifuge tube. Samples were de-waxed in 1 ml xylene for 5 min. in room temperature and centrifuged at 14,000 rpm for 5 min. The supernatant was removed. This step was then repeated 5 times. Samples were gently vortexed between each step. 1 ml 95% ethanol was then added for 5 min. in room temperature and this was repeated with 70% ethanol. The samples were centrifuged 14.000 rpm for 5 min. between the ethanol changes. The samples were then dried in a 37°C heated block with open lids for 20 min. to remove residual ethanol.
Proteinase K digestion
200–400 μl of dilution buffer was added to each tube (0.2 M Tris-HCL pH 8.0, 1% sodium dodecyl sulfate, 10 mM EDTA, 1 mg/ml proteinase K). The volume of the dilution buffer was dependent of the amount of tissue available. Samples were subsequently incubated at 50°C overnight and thereafter heated to 94°C for 10 min. to inactivate the proteinase K.
Protein precipitation
50 μl of saturated NaCl solution (approximately 6 M) was added to each tube and the samples gently vortexed for 5 min. The tubes were then centrifuged at 14.000 rpm at room temperature for 5 min. After centrifugation, white pellets were visible in the bottom of the tubes and supernatants were transferred to a new tube.
DNA precipitation
DNA was precipitated with 2.5 volumes of 95% ethanol in -20°C for 1 hour and then pelleted by centrifugation at 14.000 rpm in room temperature for 30 min. The pellet was then washed with 70% ethanol followed by centrifugation for 5 min. DNA pellets were air dried and finally re-suspended in 100 μl TE buffer (100 mM Tris-HCL, pH8.0, 1 mM EDTA).
Real-time PCR
The detection of the HPV was performed using a real-time PCR based method previously described [13]. Briefly, the real-time PCR assay detects and quantifies HPV 16, 18, 31, 33, 39, 45, 52, 58 and 67. The assay is based on three parallel real-time PCRs from each patient sample: a) Reaction 1 detects and quantifies HPV types 16, 31, 18 and 45 (HPV 18 and 45 detected and quantified together) using three different fluorophores, b) Reaction 2 detects and quantifies HPV types 33, 39, 52, 58 and 67 (HPV 33, 52, 58 and 67 detected and quantified together), again using three different fluorophores, and c) Reaction 3 detects and quantifies the amount of a human single copy gene (HMBS, Homo sapiens hydroxymethylbilane synthase, GenBank accession number M95623.1). Reaction 1 includes a total of seven PCR primers and three probes, Reaction 2 a total of five PCR primers and two probes and Reaction 3, two PCR primers and a single probe. All probe and primer sequences have been described [13]. By relating the HPV copy number to the number of nuclear gene equivalents (from Reaction 3) a measure of HPV load is obtained.
The system has a dynamic range from 102 to 107 HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1–10 percent of the main type. The system has been evaluated with respect to technical specificity and sensitivity, reproducibility, reagents stability, sample preparation protocol and applied to the analysis of clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies.
The PCR amplification was performed in a 25 μl volume containing 1× PCR buffer gold (Applied Biosystems, Foster City, CA, USA), 300 μM 6-carboxy-X-rodamine (Molecular Probes inc, Eugene OR, USA), 3.5 mM MgCl2, 200 nM each of dATP, dCTP, dGTP and 400 nM dUTP (Pharmacia Biotech, Uppsala, Sweden), 0.625 U AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA), 3 μg BSA (Sigma Chemical Co., St. Louis, MO, USA) and 200 nM of each primer and probe, and 3 μl DNA extract. Amplification and detection was performed using a 7700 Sequence Detection System (Applied Biosystems Inc., Foster City, CA, USA). The amplification ramp included an initial hold step of 10 min. at 95°C followed by a two-step cycle consisting of 15 sec. at 95°C and 1 min. at 57°C, repeated 40 times. Each real-time PCR run included reactions with no template controls containing all PCR components but without template DNA to ensure that the reagents mix were free of contaminants.
Statistics
The survival rates were estimated with the Kaplan-Meier product limit method, whereas univariate analysis was performed with log-rank test. Cox regression analysis was performed to investigate if certain continuous factors had a significant effect on survival. Spearman's rank order correlation was utilized for tests of associations between factors. A 5% significance level was used throughout the study.
