Cancer Epidemiology Biomarkers & Prevention Vol. 10, 153-154, February 2001
© 2001 American Association for Cancer Research
No Increased Ki67 Expression in Ductal Carcinoma in Situ Associated with Invasive Breast Cancer1
Ashraful Hoque,
David G. Menter,
Aysegul A. Sahin,
Nour Sneige and
Scott M. Lippman2
Departments of Clinical Cancer Prevention [A. H., D. G. M., S. M. L.] and Pathology [A. A. S., N. S.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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Introduction
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Several studies indicate that 
50% of patients with
positive biopsies for
DCIS3
of the breast may develop invasive breast cancer within 1–24 years of
diagnosis (1)
. The biological mechanisms involved in the
progression of DCIS to invasive cancer are not fully understood.
Although Ki67 labeling indices increased in invasive breast cancer
(2
, 3)
, there are limited data on this marker in DCIS.
Recently, Imamura et al. (4)
reported that
Ki67-associated proliferative activity of intraductal components
(indicated by MIB1, an antibody that recognizes Ki67) is a significant
prognostic determinant of disease-free survival in invasive ductal
breast carcinoma. Allred et al. (5)
found a
differential expression of growth factor receptor HER-2/neu
in DCIS with invasive disease versus DCIS without. On the
basis of the above data, we conducted a case-control study of Ki67 in
DCIS, with or without invasive breast cancer, testing the hypothesis
that DCIS associated with invasive cancer would have a higher Ki67
expression than would DCIS alone.
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Materials and Methods
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Paraffin-embedded archival breast tissue specimens and
surgical pathology reports of 100 consecutively examined controls (pure
DCIS) and 100 consecutively examined cases (DCIS with invasive
component) were identified from the pathology database of The
University of Texas M. D. Anderson Cancer Center during the period of
1986–1999. H&E-stained slides of each DCIS case were reviewed and
graded by a pathologist (A. A. S.) using standard grading criteria,
as described previously (6)
. Cases and controls were
frequency-matched according to nuclear grade. Sociodemographic
characteristics and clinical variables were abstracted from patients’
medical records. We determined Ki67 labeling indices by staining for
KiS5 (Dako, Carpinteria, CA) binding to Ki67. KiS5 is an antibody
similar to MIB1 (4)
in its ability to recognize Ki67
protein. On each slide, two or three fields with the strongest Ki67
staining were digitized (at x200) via computerized image analysis
techniques and quantified to determine the Ki67 labeling index of the
lesion (i.e., the mean percentage of stained area within the
two or three fields). Student’s t test was used to compare
mean Ki67 indices of cases and controls. The 
2
test was used to compare proportions of Ki67 scores among DCIS lesions
graded 1, 2, or 3. Odds ratios and 95% confidence intervals were
computed using logistic regression models. A probability value <0.05
was considered statistically significant. The sample size of 100 cases
and 100 matched controls gave the study an 80% power to detect an odds
ratio of 2.22 of increased Ki67 expression (
= 0.05, two-sided
test).
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Results
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The mean Ki67 labeling indices of DCIS associated with
invasive breast cancer and DCIS alone were similar (Fig. 1)
, and the odds ratio for Ki67 was 0.92 (95% confidence interval,
0.79–1.06), which remained unaltered after adjusting for necrosis. We
did find a correlation, however, between Ki67 labeling indices and
nuclear grades among both cases and controls; higher nuclear grade
correlated with higher Ki67 labeling index. This trend was stronger for
DCIS alone (P < 0.0001) than for DCIS associated with
invasive cancer (P = 0.08).
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Fig. 1. An example of immunostaining (via DAB enzymatic signal enhancement) of
Ki67 in a DCIS sample (at x200). Ki67 staining was observed as a
black signal on a gray background
(top panel). A single DCIS-containing acinus within this
sample was outlined with a digital stylus (represented by the
dotted lines) to exclude noncellular material (middle
panel). Segmentation of the nuclei (small outlined areas
within the acinus) was performed and quantified, followed by the
numbering of each nucleus (middle panel).
Columns, means (M) of Ki67 labeling
indices in DCIS alone (2.26 ± 0.22) and DCIS associated with invasive
breast cancer (1.91 ± 0.18; P = 0.22; bottom
panel); bars, SE.
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Discussion
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We did not observe a statistically significant difference in Ki67
labeling indices between DCIS associated with invasive breast cancer
and DCIS alone. In 157 specimens of invasive breast cancer with a DCIS
component, Imamura et al. (4)
found higher
median MIB1 labeling indices (indicative of higher Ki67 expression) in
invasive foci than in intraductal components. To extend this finding,
we had hypothesized that DCIS associated with invasive cancer would
have a higher Ki67 expression than would DCIS alone. A unique aspect of
our study was in restricting Ki67 evaluations to only the DCIS
component of cases or controls.
De Potter et al. (7)
suggested that increased
cell proliferation and enhanced cell motility associated with
overexpressed c-erbB-2 may contribute to the development and
progression of DCIS. As with our Ki67 findings, Allred et
al. (5)
reported that the incidence of
HER-2/neu expression is lower in DCIS with invasive breast
cancer than in DCIS alone. These findings suggest that the expression
of c-erbB-2 and/or Ki67 may be down-regulated in a
significant proportion of DCIS lesions as they progress to invasive
cancer or that a subset of invasive breast cancers may develop de
novo by mechanisms independent of c-erbB-2 and/or
Ki67.
Two limitations of our study involved the statistical or biomarker
study design. The sample size of our study did not provide the power to
detect Ki67 odds ratios <2.22 (see "Materials and Methods" above).
Although cases and controls were matched by nuclear grade and adjusted
for necrosis, we did not assess and so could not adjust for other
potentially important cellular/molecular prognostic markers
(1, 2, 3
, 5
, 8
, 9)
. Ongoing studies will help further define
the role of Ki67 in DCIS (8
, 9)
, including its
associations with c-erbB-2 and other cellular/molecular
markers.
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Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by NIH-National Cancer
Institute Grants CN-65004, CA16672, CA7730, and CA56452. S. M. L.
holds the Margaret and Ben Love Professorship in Clinical Cancer
Care. 
2 To whom requests for reprints should be
addressed, at Department of Clinical Cancer Prevention, Box 236, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe
Boulevard, Houston, Texas 77030. Phone: (713) 745-3672; Fax:
(713) 794-4679; E-mail: slippman{at}mdanderson.org
3 The abbreviation used is: DCIS, ductal carcinoma
in situ.
Received 9/ 8/00;
revised 11/16/00;
accepted 11/27/00.
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References
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Allred D. G., Berardo M. D., Prosser J., O’Connell P. Biologic and genetic features of in situ breast cancer. Silverstein M. J. eds. . Ductal Carcinoma in Situ of the Breast, : 37-49, Williams & Wilkins Baltimore, MD 1997.
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Introduction
Materials and Methods
