1 3 4 5 6 7 8 9 10 11 12 12 13 14 We believe that screen-detected DCIS is more often associated with suspicious microcalcifications representing high-grade DCIS, which has been detected before it has had the chance to progress to invasive cancer. Therefore, it is hypothesized that screen-detected DCIS is biologically more aggressive than interval DCIS. To compare screen-detected DCIS with interval DCIS in such a retrospective study, the clinicopathological and biological characteristics of both groups were evaluated for differences. Screen-detected DCIS was classified as DCIS detected by screening mammography, when the examination from two years earlier failed to reveal an abnormality. Interval DCIS was classified as DCIS detected within the two-year interval between two subsequent screening rounds, when the earlier examination failed to reveal an abnormality. Age, tumor size, and pathological grade were studied for their known relation with local recurrence. Finally, the expression of established prognostic biomarkers in breast cancer was studied by immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), Her2/neu, p53, and cyclin D1. PATIENTS AND METHODS Patients and Tumors The Dutch screening program for breast cancer has been gradually implemented in the North Netherlands since 1991. It offered biennial mammography to women 50–69 years old, and since 1999 women 70–74 years old have also been included. Women received mammography in the cranio-caudal and medio-latero-oblique direction for each breast. Two radiologists evaluated the mammograms by a double independent reading. From January 1992 to December 2001, 128 consecutive patients were treated for pure DCIS at our institution. To identify patients for inclusion in the study, all women who had actually attended the screening program at least two subsequent rounds with a two-year interval at the time of diagnosis were considered as attenders. Patients who had skipped one or more screening rounds previous to the diagnosis and patients who had not been attending the program at all were considered nonattenders. Patients’ records were checked to obtain this information, and if there was no information regarding the participation of the screening program at the time of diagnosis the general practitioner was consulted. Out of the 128 consecutive patients, 102 attenders and 26 nonattenders could be identified. For immunohistochemistry, patients were selected on the availability of sufficient paraffin-embedded tissue. Thirteen out of the 26 nonattenders and 74 out of the 102 attenders remained, respectively, for evaluation of Her2/neu overexpression, estrogen receptor (ER) expression, progesterone receptor (PR) expression, p53 expression, and cyclin D1 expression using tissue microarray analysis as part of a project protocol that had been approved by the medical ethics committee. The patients in the study-group (n = 74) were divided into two groups. Patients with DCIS that had been detected by screening mammography were classified as screen-detected patients, when the examination two years earlier failed to reveal an abnormality (n = 54). Patients with DCIS that had been detected within the two-year interval between two subsequent screening rounds were classified as interval patients, when the earlier examination failed to reveal an abnormality (n = 20). Mammography and Pathological Assessment 15 16 Tissue Microarray Construction 17 Immunohistochemistry 1 TABLE 1. Antigen retrieval methods and antibodies Antibody Clone Supplier Dilution Antigen retrieval Secondary antibody Supplier Tertiary antibody Supplier ER 6F11 Ventana a Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwave RAMBIO Dako SARBIO Dako PR 1A6 Ventana a Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwave RAMBIO Dako SARBIO Dako Her-2/Neu CB11 Ventana a Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwave RAMBIO Dako SARBIO Dako p53 BP-53-12-1 Biogenix 1:800 Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwave RAMBIO Dako SARBIO Dako Cyclin D1 SP4 Neomarkers 1:50 Tris/HCL 0.1M (pH 9.5) 30’ 98°C microwave RAMBIO Dako SARBIO Dako a ER, estrogen receptor; PR, progesterone receptor;; RAMBIO, rabbit anti-mouse biotin; SARBIO, swine anti-rabbit biotin. Evaluation of Immunohistochemical Staining TM 18 Statistical Analysis Differences in clinicopathological characteristics between screen-detected and interval patients with DCIS in the study group, and between the study group and the excluded group of patients were analyzed by chi-square analysis. Differences in clinicopathological and biological characteristics between the study group and the nonattenders were also analyzed by chi-square analysis. Differences in age were tested by using the Mann-Whitney U test. Univariate analyses, investigating differences in pathological and biological features, was performed by logistic regression, using screen detection as a dependent variable. Multivariate analyses were performed with a logistic-regression model. The elimination of variables in a stepwise manner identified the statistically significant pathological and biological parameters. A p value of ≤.050 was considered as significant. All calculations were performed with SPSS 12.01 (SPSS inc., Chicago, IL). RESULTS 2 TABLE 2. Clinicopathological characteristics of the patients in the study group and differences between screen-detected and interval patients Clinicopathological characteristics Screen-detected n = 54 Interval n = 20 a Age (mean), years 58.9 60.7 .187 Family history of breast cancer   .055   Yes 8 (14.8) 7 (35)    No 46 (85.2) 13 (65)  Signs   Palpable mass 5 (9.3) 6 (30) <.001   Nipple discharge 2 (3.7) 9 (45)    Mastodynia 1 (1.9) 0 (0)    No objective signs 46 (85.1) 5 (25)  Mammography   .002   Microcalcifications 46 (85.2) 10 (50)    Mass 2 (3.7) 6 (30)    Combination mc’s and mass 6 (11.1) 4 (20)  Microcalcifications    <.001   Linear branching 22 (44.9) 4 (28.6)    Coarse granular 27 (55.1) 4 (28.6)    Fine granular 0 (0) 6 (42.9)  Mammographic size   .183   ≤2 cm 21 (42.6) 12 (60)    >2 cm 33 (57.4) 8 (40)    BCS 23 (42.6) 8 (40) .841   Mastectomy 31 (57.4) 12 (60)  Tumor size   .787   <16mm 19 (35.2) 6 (30)    16–40mm 17 (31.5) 8 (40)  >40 mm 18 (33.3) 6 (30)  Grade (EPWG)   .229   1 4 (7.5) 4 (20)    2 29 (53.7) 11 (55)    3 21 (38.9) 5 (25)  Grade (Van Nuys)   .025   1 4 (7.4) 6 (30)    2 24 (44.4) 9 (45)    3 26 (53.1) 5 (25)  Mann-Whitney U test. Values between parentheses are percentages. BCS, breast conserving surgery; EPWG, European Pathologist Working Group. a 3 TABLE 3. Comparison of clinicopathological characteristics between the study group and the group of patients that were excluded because of insufficient paraffin-embedded tissue Clinicopathological characteristics Study group n = 74 Excluded n = 28 a Age (mean) 59.6 61.5 .381 Microcalcifications (n = 86)   .063   Linear branching 26 (41.3) 5 (25)    Coarse granular 31 (49.2) 9 (45)    Fine granular 6 (9.5) 6 (30)  Tumor size   .458   <16mm 25 (33.8) 7 (25)    16–40mm 25 (33.8) 9 (32.1)    >40 mm 24 (32.6) 12 (42.9)  Grade (EPWG)   .184   1 8 (10.8) 6 (21.4)    2 40 (54.1) 10 (35.7)    3 26 (35.1) 12 (42.9)  Grade (Van Nuys)   <.001   1 10 (13.5) 15 (53.6)    2 33 (44.6) 6 (21.4)    3 31 (41.9) 7 (25)  Mann-Whitney U test. Values between parentheses are percentages. EPWG, European Pathologist Working Group. a There were no differences in clinicopathological and biological characteristics between the study group and the nonattenders (n = 13), except the fact that nonattenders were younger than the patients in the study group (55.9 years versus 59.6 years; p = .042). 4 TABLE 4. Univariate analysis of pathological and biological characteristics in screen-detected versus interval DCIS in the study group Pathological and biological features Screen-detected n = 54 Interval n = 20 OR by screen-detected 95% CI p-value Tumor size   <16mm 19 (35.2) 6 (30) 0.8 0.2 0.744   16–40mm 17 (31.5) 8 (40) 1.1 0.2 0.827   >40 mm 18 (33.3) 6 (30) 1   Grade (EPWG)   1 4 (7.5) 4 (20) 3.800 0.5 0.377   2 29 (53.7) 11 (55) 1.703 0.7 0.124   3 21 (38.9) 5 (25) 1   Grade (Van Nuys)   1 4 (7.4) 6 (30) 7.3 1.6    2 24 (44.4) 9 (45) 1.6 0.5 0.416   3 26 (53.1) 5 (25) 1 1.6 0.010 Her2/neu (n = 68)   Positive 29 (60.4) 2 (10) 6.5 1.3–31.0 0.020   Negative 19 (39.6) 18 (90) 1   ER(n = 62)   Positive 33 (75) 16 (88.9) 0.4     Negative 11 (25) 2 (11.1) 1 0.1–1.9 0.236 PR (n = 60)   Positive 18 (42.9) 13 (65) 0.3     Negative 24 (57.1) 5 (35) 1 0.1–1.0 0.042 p53 (n = 60)   Positive 10 (24.4) 4 (21.1) 0.8     Negative 31 (75.6) 15 (78.9) 1 0.2–3.1 0.776 Cyclin D1 (n = 60)   Positive 29 (69.1) 13 (72.2) 0.9 0.3–2.9 0.806   Negative 13 (30.9) 5 (27.8) 1   Univariate analysis using logistic regression. Figures in parentheses are percentages. OR, odds ratio; EPWG, European Pathologist Working Group; ER, estrogen receptor; PR, progesterone receptor. 95% CI, 95% confidence interval. DISCUSSION 4 19 20 21 22 23 24 25 12 26 27 26 27 DCIS lesions from patients in the study group were compared to DCIS lesions from patients who had not attended the screening program. Out of the 26 nonattenders, there were only 13 patients from whom sufficient paraffin-embedded tissue was available. Using chi-square analysis there were no differences in pathological and biological characteristics between the two groups. The difference in age could be explained by the fact that the nonattenders group also contained patients under 50-years of age. From these analyses it seems that DCIS in nonattenders is not pathologically and biologically more aggressive than DCIS in attenders of the screening program. However, because of the very small number of patients in the nonattenders group no hard conclusions can be drawn. 28 30 24 25 Although the statistical methods used were univariate and multivariate analysis, the numbers in both groups are small, which explains the broad 95% CI. The small numbers are due to the selection of patients in this study for study period, attendance of screening rounds, and availability of sufficient paraffin-embedded tissue. Clearly, further studies with larger populations are needed to elucidate the relative significance of the Her2/neu overexpression in women with screen-detected DCIS. Although 28 out of 102 patients were excluded because of lack of sufficient paraffin-embedded tissue, there is no reason to assume that this exclusion results in a significant selection bias. Apart from pathological grade according to Van Nuys, there were no differences in clinicopathological characteristics between the study group and the group of excluded patients. The group of excluded patients displayed a relatively large amount of low-grade DCIS according to Van Nuys (53.6%, p < .001), which is mainly derived from the interval group (n = 10, data not shown). If all patients would have been included the relation of pathological grade and probably Her2/neu expression with screen-detected patients would be even more significant. The inclusion of patients that actually took part in the screening program was necessary to analyze differences between screen-detected and interval DCIS, which, to our knowledge, has not been performed previously. 12 24 25 31 Ultimately, the question is how we should interpret these findings. The authors think the results from this study represent no evidence to alter patient management and screening recommendations. Instead, they should rather be regarded as support of current clinical practice in DCIS of the breast. They confirm that every DCIS should be treated until we are able to identify DCIS that will progress to invasive cancer if left untreated. In conclusion, since the advent of screening, the increased incidence of DCIS has raised concerns about the possibility of overdiagnosis of DCIS. This study has shown that screen-detected DCIS has a more aggressive tumor profile than interval DCIS. Therefore, screen-detected DCIS should not be regarded as an overdiagnosis per se, and every woman diagnosed with DCIS by mammographic screening should be treated properly according to existing guidelines or standards of care.