Materials and Methods

The study population comprised 228 women. One hundred eighty-eight women were recruited consecutively from the out-patient surgery, Department of Obstetrics and Gynecology, Falun Hospital (Uppsala, Sweden), and who attended for laser conization of the cervix uteri because of CIN or ASCUS. After informed consent, a serum sample was collected and frozen immediately in –70°C. The paraffin-embedded cone was stored at the Department of Pathology.

In addition, 40 apparently healthy volunteers of fertile ages were recruited because the number of participants with normal epithelium was expected to be fewer in those women who were undergoing conization because of abnormal Papanicolaou test results. Colposcopically directed punch biopsy specimens were taken at the outpatient clinic of the Department of Obstetrics and Gynecology and were paraffin-embedded. A serum sample was frozen as described earlier. A structured questionnaire included birth date, age, last menstruation, cycle day, history of abnormal Papanicolaou smear test, contraceptive use (if any), present or past smoking, cigarettes per day and duration, and climacteric status.

Serum cotinine was analyzed at Advanced Bioanalytical Services Laboratories, London, UK. One of the authors (T.T.) microscopically reviewed sections from the original paraffin blocks, and the most representative area was marked for tissue microarray (TMA). Two-millimeter punch biopsy specimens were taken from the blocks that corresponded to the marked area and joined into TMA paraffin blocks that contained 24-30 punch biopsy specimens. Each TMA block also included 1 control and 1 empty square to avoid diagnostic mistakes.

Immunohistochemistry was performed as previously described. In brief, glass slides were deparaffinized in xylene (2 × 15 minutes), dehydrated through graded alcohols, and endogenous peroxidase was blocked (with H ₂ o ₂ in 70% ethanol). Antigen retrieval was performed with target retrieval solution (pH 6.0 or pH 9.0; Labvision/NEOMARKERS, Thermo Fisher Scientific, Fremont, CA) in a decloaking chamber (Biocare Medical, Walnut Creek, CA) for 4 minutes at 125°C. Thereafter, the slides were immunostained in the automated staining instrument, in which primary antibodies and secondary reagent were each incubated for 30 minutes at room temperature. Finally, the slides were incubated with diaminobenzidine as chromogen for 10 minutes and counterstained with Mayers hematoxylin (Sigma-Aldrich, St. Louis, MO) for 15 minutes. Slides were washed in distilled water for 10 minutes, dehydrated through graded alcohols to xylene, and mounted in Pertex organic mounting medium (Histolab, Gothenburg, Sweden).

Occasionally, it was not possible to evaluate expression of a specific tumor marker in a specific subject. Because it was not systematic, the whole study population was included, and the number of evaluations thus differed with a few diagnoses between the tumor markers.

Details about the 11 antibodies that were chosen for the study are given in Table 1 . One external senior pathologist, who was blinded for clinical details, evaluated all biopsy specimens. Frequency of stained cells and intensity of staining were diagnosed. A 4-grade semiquantitative score was used for frequency, for which 0 was the absence of biomarker expression, 1 was the expression in 1-19% of cancer cells, 2 was the expression in 20-49% of cancer cells, and 3 was ≥50% of cancer cells with expression of the tumor marker. Intensity of staining was graded in 4 steps: absent, mild, moderate, and strong. In the analyses in general, there was a good correlation between frequency and intensity, and the best discriminatory evaluation was used for presentation in the Tables.

For the analyses, the best explanatory cut-off level was used when the results of tumor marker expression, in relation to smoking habits, were dichotomized. When there was no evidence of any correlation between tumor marker expression and smoking habits, dichotomization was made so that an equal number of patients, if possible, were included in the 2 groups. For statistical analyses, the JMP statistical package (SAS Institute Inc, Cary, NC) was used. The t test was used for significance testing in correlations between serum cotinine and tumor marker expression. A χ ² test (likelihood ratio) was used for correlations between smoking habits and tumor marker expression, and logistic regression was used for estimating odds ratios and 95% confidence intervals. The study was approved by the Research Ethical Committee, Uppsala University.

Results

Mean and median age of the study population was 36.6 years and 34.0 years, respectively. Smokers averaged 34.9 years of age, and nonsmokers averaged 37.7 years of age ( P = .06). Eighty-three of the women (36.9%) were smokers, which we defined as daily smoking and serum cotinine levels above 12 ng/mL; 142 women (63.1%) were nonsmokers. There were no findings of measurable serum cotinine in the latter group.

The histopathologic diagnoses were distributed on 87 normal squamous epithelium (38.2%), 31 ASCUS (13.6%), 49 CIN I (21.5%), and 61 CIN II-III (26.8%). None of the 40 healthy volunteers had CIN.

Smoking was associated with a significantly lower p53 expression in CIN, but not in those women with normal epithelium, compared with nonsmokers ( Table 2 ). There was also a lower interleukin-10 expression in smokers with CIN than in nonsmokers.

TABLE 2

Smoking habits and correlation to tumor marker expression in cervical intraepithelial neoplasia and normal epithelium

Variable	Normal epithelium				Cervical intraepithelial neoplasia
	Smokers, n (%)	Nonsmokers, n (%)	Odds ratio (95% CI)	P value	Smokers, n (%)	Nonsmokers, n (%)	Odds ratio (95% CI)	P value
p53 frequency 2-3 vs 0-1 a	3 (10.0)	5 (11.6)	0.84 (0.16–3.74)	.83	7 (17.1)	28 (35.0)	0.38 (0.14–0.93)	.03
Interleukin-10 frequency 1-3 vs 0 a	8 (25.8)	11 (25.6)	1.01 (0.34–2.90)	.98	15 (34.9)	47 (58.0)	0.39 (0.18–0.82)	.01
Cyclooxygenase-2 frequency 3 vs 0-2 a	11 (31.4)	10 (21.3)	1.70 (0.62–4.68)	.30	10 (22.2)	10 (11.6)	2.17 (0.82–5.76)	.12
E-cadherin frequency 3 vs 0-2 a	18 (56.3)	26 (61.9)	0.79 (0.31–2.02)	.62	32 (84.2)	69 (90.8)	0.54 (0.17–1.80)	.31
Fragile histidine triad frequency 2-3 vs 0-1 a	1 (3.2)	2 (4.9)	0.65 (0.03–7.09)	.73	9 (22.5)	26 (34.7)	0.55 (0.22–1.29)	.17
Retinoblastoma protein strong vs none-moderate intensity b	16 (51.6)	16 (37.2)	1.80 (0.71–4.66)	.22	16 (38.1)	21 (27.6)	1.61 (0.72–3.59)	.24
Cytokeratin 10 frequency 1-3 vs 0 a	12 (38.7)	14 (29.8)	1.49 (0.57–3.89)	.41	15 (34.1)	19 (22.6)	1.77 (0.78–3.97)	.19
Epidermal growth factor receptor frequency 3 vs 0-2 a	10 (32.3)	11 (25.0)	1.43 (0.51–3.98)	.49	26 (63.4)	56 (65.9)	0.90 (0.41–1.98)	.79
p16 frequency 1-3 vs 0 a	8 (25.0)	5 (10.6)	2.80 (0.84–10.19)	.09	37 (88.1)	69 (82.1)	1.61 (0.57–5.26)	.38
CD4+ frequency 2-3 vs 0-1 a	7 (20.6)	10 (21.7)	0.93 (0.30–2.75)	.90	23 (50.0)	52 (58.4)	0.71 (0.35–1.46)	.35
Ki-67 intensity strong vs none-moderate a	28 (93.3)	38 (88.4)	1.84 (0.37–13.51)	.47	36 (87.8)	63 (76.8)	2.17 (0.79–6.99)	.14

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