Journal of Cancer Therapy, 2012, 3, 841-845
http://dx.doi.org/10.4236/jct.2012.325107 Published Online October 2012 (http://www.SciRP.org/journal/jct)
841
The Reliability of Assessment of Ki-67 Expression on Core
Needle Biopsy and the Surgical Specimens of Invasive
Breast Cancer: Comparison of Local Pathologists’
Assessment and Central Review*#
Yoshio Mizuno1, Tsuneo Natori2, Naoko Takeda1,3, Junichi Yamada4, Hiroaki Abe4, Yuko Inoue3,
Hiroshi Seto5, Kazuhiko Sato1
1Department of Breast Oncology, Tokyo-West Tokushukai Hospital, Tokyo, Japan; 2Clinical Testing Business, Testing Division,
Special Reference Laboratories, Inc., Tokyo, Japan; 3Inoue Ladies Clinic, Tokyo, Japan; 4Department of Clinical Pathology, To-
kyo-West Tokushukai Hospital, Tokyo, Japan; 5Seto Hospital, Saitama, Japan.
Received August 28th, 2012; revised September 30th, 2012; accepted October 9th, 2012
ABSTRACT
Purpose: The aim of this study was to assess the reliability of Ki-67 expression on core needle biopsy (CNB) and the
surgical specimens of invasive breast cancer. We examined the concordance rate of Ki-67 expressions, hormone recep-
tors, and human epidermal growth factor receptor 2 (HER2) status from a CNB with from a surgery in invasive breast
cancer. Methods: A retrospective study was conducted on a clinical database of patients who underwent surgery for
early breast cancer. Of these, 193 patients who underwent CNB before the surgery were enrolled. A cut-off value of
20% was used for Ki-67-positive criteria. Expression of estrogen receptor (ER), progesterone receptor (PgR), and
HER2 were examined and compared with that of Ki-67. To evaluate discordance between the pathologists’ earlier as-
sessments, we re-examined Ki-67 expression among the Ki-67 discordant group in a central laboratory. Results: The
concordance rate for Ki-67 expression between the two specimen types was 77.7%, which was significantly lower than
that for ER, PgR, and HER2 expression (95.9%, 88.1%, and 91.6%, respectively). The concordance rate for re-exam-
ined Ki-67 expression among the Ki-67 discordant group improved to 93.8% and was not significantly different from
that for the other receptors. Conclusion: The concordance rate for Ki-67 expression between biopsy and surgical
specimens was significantly lower than that for ER, PgR, and HER2 expressions, but re-examination of Ki-67 expres-
sion in a central laboratory revealed no significant difference among the receptors, suggesting the need for standard
pathological assessment of Ki-67 expression for clinical use as a predictive marker of breast cancer.
Keywords: Ki-67; CNB; Concordance Rate; Standard Assessment
1. Introduction
In recent years, focus has been placed on the usefulness
of preoperative endocrine therapy for postmenopausal
hormone-receptor-positive breast cancer, patients with
the aim of improving breast conservation rates and se-
lecting patients with high susceptibility to endocrine
therapy [1-3].
Certain biomarkers provide clinically useful prognos-
tic and predictive information in preoperative endocrine
therapy. To identify alternative post-treatment factors
that predict recurrence-free survival after preoperative
endocrine therapy, Dowsett et al. examined changes in
the Ki-67 index before and after 2 weeks of preoperative
endocrine therapy, and found that patients with higher
Ki67 expression after 2 weeks of preoperative endocrine
therapy had significantly lower recurrence-free survival
[4-5]. In multivariable analysis, Ellis et al. recently
demonstrated that among 228 postmenopausal women
with ER-positive breast cancer who received preopera-
tive endocrine therapy, 4 factors—pathological tumor
size, node status, ER status, and the Ki-67 index—were
determined to predict long-term outcomes after comple-
tion of preoperative endocrine therapy [6].
Based on this, considering pretreatment histopa-
thological findings obtained using core needle biopsy
(CNB) and biological markers are important for deter-
mining a therapeutic strategy. In addition, variations in
the pre- and post-treatment using the Ki-67 index are
reported to be related to preoperative hormone therapy as
*This study was not funded by any public or private grant.
#All authors declare no conflict of interest.

The Reliability of Assessment of Ki-67 Expression on Core Needle Biopsy and the Surgical Specimens
of Invasive Breast Cancer: Comparison of Local Pathologists’ Assessment and Central Review
842
a predictor of prognosis and effect [4-8]. Variations in
the Ki-67 index are evaluated using CNB specimens be-
fore treatment and using surgical specimens after treat-
ment. On the other hand, it is important to consider that
there may be discrepancies in the Ki-67 index between
the CNB and surgical specimens, even though preopera-
tive treatment is not performed.
Consequently, to ascertain the necessity for standard-
izing Ki-67 index measurement methods in preoperative
hormone therapy, we evaluated the Ki-67 index concor-
dance rates between preoperative CNB and surgical
specimens. Furthermore, we re-examined the discordance
cases in the Ki-67 index through central review, in addi-
tion to comparing the concordance rates in the expression
of other biological markers.
2. Patients and Materials
We retrospectively analyzed data from patients who un-
derwent primary surgery for early breast cancer at To-
kyo-West Tokushukai Hospital from August, 2008 to
October, 2011. Of these, 193 consecutive patients who
received CNB before surgery were enrolled. We retro-
spectively analyzed data from patients who underwent
primary surgery for early breast cancer at Tokyo-West
Tokushukai Hospital from August, 2008 to October,
2011. Of these, 193 consecutive patients who received
core needle biopsies (CNB) before surgery were enrolled.
The inclusion criteria included 1) diagnosis of breast
cancer confirmed by CNB and 2) no evidence of metas-
tases at initial assessment, and the exclusion criteria in-
cluded 1) evidence of metastases at initial assessment; 2)
inoperable tumors (stage T4, N2, or N3); 3) patients with
neoadjuvant chemotherapy, and 4) patients with ductal
carcinoma in situ. For CNB, a 16- or 18-gauge automated
needle device with a 22-mm throw biopsy gun was used.
Three or more CNB specimens were obtained per patient.
CNB specimens were placed in 20% formalin for >6
hours to <48 hours. The original tumors had been fixed
in buffered formalin and embedded in paraffin. One rep-
resentative tissue block for each tumor was selected for
routine evaluation of estrogen receptor (ER), progester-
one receptor (PgR), human epidermal growth factor re-
ceptor 2 (HER2), and Ki-67 by immunohistochemical
analysis. The clone MIB-1 (Dako, Denmark) was used
for immunohistochemical analysis of Ki-67.
According to our institutional ethics committee’s poli-
cies, general consent is taken from all the patients who
undergo medical care.
3. Methods
All cases were evaluated by registered local pathologists.
Although many different systems for grading of patho-
logical responses by local pathologists have been pro-
posed, no standard method has been adopted. The con-
cordance rates for assessment of ER, PgR, HER2, and
Ki-67 by local pathologists were reviewed, and in cases
of non-matching Ki-67, the tumor diameters (approxi-
mately indicative of tumor heterogeneity) and operative
method (approximately indicative of formalin fixation
condition) were studied. Next, the non-matching cases
from August, 2008 to October, 2011 were reassessed by
central review. The central review was performed by
scanning magnification to count at least 1000 cells in the
most densely labeled areas. For all non-matching cases,
the percentage of tumor cells with any nuclear staining
was recorded.
4. Statistical Analysis
Statistical significance for concordance rates between the
2 types of specimens was evaluated by Wilcoxon t-test.
To evaluate the consequence of formalin and genetic
heterogeneity, parameters, such as the operative method
and tumor size, were analyzed by χ2 analysis.
5. Results
The mean patient age was 56.3 years (median, 55.5 years;
range, 30 - 91 years).
Seventy-three patients ultimately underwent mastec-
tomy and the remainder underwent breast-conserving
surgery.
For ER, 152 cases showed positive and 33 cases
showed negative findings for both CNB and surgical
specimens; 3 cases showed positive findings for CNB
specimens and negative findings for surgical specimens;
and 5 cases showed negative findings for CNB speci-
mens and positive findings for surgical specimens. For
PgR, 111 cases showed positive findings for CNB and
surgical specimens, 59 cases showed negative findings
for CNB and surgical specimens, 12 cases showed posi-
tive findings for CNB specimens and negative findings
for surgical specimens, and 11 cases showed negative
findings for CNB specimens and positive findings for
surgical specimens. For HER2 expression, 27 cases
showed positive findings for CNB and surgical speci-
mens; 137 cases showed negative findings for CNB and
surgical specimens; 11 cases showed positive findings
for CNB specimens and negative findings for surgical
specimens; and 4 cases showed negative findings for
CNB specimens and positive findings for surgical speci-
mens. For Ki-67, 85 cases showed positive findings for
CNB and surgical specimens; 65 cases showed negative
findings for CNB and surgical specimens; 28 cases
showed positive findings for CNB specimens and nega-
tive findings for surgical specimens; and 15 cases

The Reliability of Assessment of Ki-67 Expression on Core Needle Biopsy and the Surgical Specimens
of Invasive Breast Cancer: Comparison of Local Pathologists’ Assessment and Central Review
843
showed negative findings for CNB specimens and posi-
tive findings for surgical specimens. Concordance be-
tween CNB and surgical specimens for marker expres-
sion is shown in Tables 1-4. In our series, the concor-
dance rate for Ki-67 expression between the two speci-
men types was 77.7%, significantly lower than that for
ER, PgR, and HER2 expression (95.9%, 88.1%, and
91.6%, respectively) (Table 5).
Table 1. Concordance rate between CNB and surgical speci-
mens for ER status.
Positive surgical
specimen
Negative surgical
specimen
Positive (CNB) 152 3
Negative (CNB) 5 33
Concordance rate for ER: 95.9%.
Table 2. Concordance rate between CNB and surgical speci-
mens for PgR status.
Positive surgical
specimen
Negative surgical
specimen
Positive (CNB) 111 12
Negative (CNB) 11 59
Concordance rate for PgR: 88.1%.
Table 3 Concordance rate between CNB and surgical speci-
men for HER2 status.
Positive surgical
specimen
Negative surgical
specimen
Positive (CNB) 27 11
Negative (CNB) 4 137
Concordance rate for HER2: 91.6%.
Table 4. Concordance rate between CNB and surgical speci-
men for Ki-67 expression.
Positive
(surgical specimen)
Negative
(surgical specimen)
Positive (CNB) 85 28
Negative (CNB) 15 65
Concordance rate for Ki-67: 77.7%.
Table 5. Comparison of concordance rates between ER,
PgR, and HER2 with that of Ki-67.
Concordance rate
Comparison of concordance
rates with Ki-67
ER 95.9% P < 0.01
PgR 88.1% P < 0.01
HER2 91.6% P < 0.01
Ki-67 77.7% -
Analysis of the operative methods used among the 151
concordant cases showed that breast-conserving surgery
was performed in 94 cases (62.3%) and mastectomy in
57 cases (37.7%). No significant difference in parameters,
such as operative method (mastectomy vs. breast-con-
serving surgery), were observed between the two patient
groups (Table 6).
Analysis of tumor size among the 151 concordant
cases showed that pT1 tumor occurred in 92 cases and
pT2 tumor in 59 cases. No significant difference in pa-
rameters, such as tumor size (pT1 vs. ≥ pT2) was ob-
served between the two patient groups (Table 7).
The central laboratory re-examination of Ki-67 ex-
pression among the Ki-67 discordant group cases showed
that 30 of the 43 non-matching cases should have been
placed in the concordant group. The concordance rate for
Ki-67 after re-examination by the central review im-
proved to 93.8% (Table 8).
6. Discussion
The Ki-67 index is a crucial factor as a predictor of
therapeutic effect and prognosis in preoperative hormone
therapy [9-12]. There are, however, certain identified
problems with regard to the Ki-67 index including stan-
dardization of measuring method, reproducibility of
Table 6. No significant difference between operative meth-
ods.
Breast-conserving Surgery MastectomyN
Concordance94 cases (62.3%) 57 (37.7%)151
Discordance26 (61.9%) 16 (38.1%)42
Table 7. No significant difference between tumor size <pT1
and ≥pT2.
<pT1 ≥pT2 n
Concordance 92 cases (60.9%) 59 (39.1%) 151
Discordance 29 (69.0%) 13 (31.0%) 42
Table 8. Comparison of concordance rates between ER,
PgR, and HER2 with that of Ki-67 (central review).
Concordance rate
Comparison of concordance rates
with Ki-67 (central review)
ER 95.9% P = 0.42
PgR 88.1% P = 0.02
HER291.6% P = 0.08
Ki-67 77.7% -

The Reliability of Assessment of Ki-67 Expression on Core Needle Biopsy and the Surgical Specimens
of Invasive Breast Cancer: Comparison of Local Pathologists’ Assessment and Central Review
844
measurement results, and establishment of cutoff values
[13-16].
Matthew et al. evaluated 209 breast cancer patients
using concordance rates between biological markers in
needle biopsy and surgical specimens. Their report indi-
cates that concordance rates of Ki-67, ER, PgR, HER2,
tumor grade, mitotic rate were 59%, 88%, 78%, 81%,
59%, and 61%, respectively; and the concordance rates
of Ki-67, PgR, HER2, tumor grade, and mitotic rate were
lower than that of ER [17].
The current investigation indicated that the concor-
dance rate of Ki-67 expression in CNB and surgical
specimens was 77.7%, significantly lower than those of
ER (95.9%), PgR (88.1%), and HER2 (91.6%). The fol-
lowing factors were thought to cause discordance: 1)
tumor heterogeneity, 2) differences in formalin fixation
conditions, and 3) a lack of standardization of the meas-
uring method by pathologists. Although the concordance
rates between tumor diameter and its equivalence to tu-
mor heterogeneity, as well as Ki-67 expression were
compared to determine the effect on tumor heterogeneity,
no significant correlation was observed. Further, al-
though the concordance rates between the operative
method and its equivalence to formalin fixation condi-
tions as well as Ki-67 expression were compared to con-
sider the effect on formalin fixation, no significant cor-
relation was observed. In order to consider the lack of
standardization of the measurement method by patholo-
gists, we reexamined the discordant cases in the Ki-67
index through central review.
In reexamination through central review, the concor-
dance rate of Ki-67 expression was 93.8%, which was
almost equivalent to that of ER, PgR, and HER2. Ki-67
measurements were examined microscopically by the
pathologists; the percentage of positive cells to total tu-
mor cells was calculated and this was reported as the
Ki-67 labeling index. Because the pathologists used vis-
ual judgment, there was some discordance reported be-
tween the pathologists in the percentage of positive cells.
Finally, because the concordance rate of Ki-67 expres-
sion was almost equivalent to that of ER, PgR, and
HER2 in our reexamination through central review, it
was suggested that standardization of the measurement
method by pathologists is important.
In order to ascertain the necessity of whether the
methods to measure the Ki-67 index should be standard-
ized in preoperative hormone therapy, we evaluated the
index concordance rates between preoperative CNB and
surgical specimens, and reexamined the discordance
cases in the Ki-67 index through central review. In CNB
and surgical specimens, the concordance rate of Ki-67
was lower than that of other biological markers. However,
in the reexamination through central review, the concor-
dance rate of Ki-67 was almost the same as that of other
biological markers. Therefore, for the clinical application
of Ki-67, it is necessary to standardize the methods to
measure Ki-67 index.
7. Acknowledgements
The authors would like to thank Enago (www.enago.jp)
for the English language review. This study was pre-
sented in part at the 34th Annual San Antonio Breast
Cancer Symposium (poster).
REFERENCES
[1] J. S. Chawla, C. X. Ma and M. J. Ellis, “Neoadjuvant En-
docrine Therapy for Breast Cancer,” Surgical Oncology
Clinics of North America, Vol. 19, No. 3, 2010, pp. 627-
638. doi:10.1016/j.soc.2010.04.004
[2] J. A. Olson Jr., G. T. Budd, L. A. Carey, L. A. Harris, L. J.
Esserman, G. F. Fleming, P. K. Marcom, G. S. Leight Jr.,
T. Giuntoli, P. Commean, K. Bae, J. Luo and M. J. Ellis,
“Improved Surgical Outcomes for Breast Cancer Patients
Receiving Neoadjuvant Aromatase Inhibitor Therapy:
Results from a Multicenter Phase II Trial,” Journal of the
American College of Surgeons, Vol. 208, No. 5, 2009, pp.
906-914, Discussion 915-916.
doi:10.1016/j.jamcollsurg.2009.01.035
[3] Y. H. Chia, M. J. Ellis and C. X. Ma, “Neoadjuvant
En-docrine Therapy in Primary Breast Cancer: Indica-
tions and Use as a Research Tool,” British Journal of
Cancer, Vol. 103, No. 6, 2010, pp. 759-764.
doi:10.1038/sj.bjc.6605845
[4] M. Dowsett, S. R. Ebbs, J. M. Dixon, A. Skene, C.
Grif-fith, I. Boeddinghaus, J. Salter, S. Detre, M. Hills, S.
Ash-ley, S. Francis, G. Walsh and I. E. Smith, “Bio-
marker Changes during Neoadjuvantanastrozole, Ta-
moxifen, or the Combination: Influence of Hormonal
Status and HER-2 in Breast Cancer—A Study from the
IMPACT Trialists,” Journal of Clinical Oncology, Vol.
23, No. 11, 2005, pp. 2477-2492.
doi:10.1200/JCO.2005.07.559
[5] M. Dowsett, I. E. Smith, S. R. Ebbs, J. M. Dixon, A.
Skene, R. A’Hern, J. Salter, S. Detre, M. Hills and G.
Walsh, “IMPACT Trialists Group, “Prognostic Value of
Ki67 Expression after Short-Term Presurgical Endocrine
Therapy for Primary Breast Cancer,” Journal of the Na-
tional Cancer Institute, Vol. 99, No. 2, 2007, pp. 167-170.
doi:10.1093/jnci/djk020
[6] M. J. Ellis, Y. Tao, J. Luo, R. A’Hern, D. B. Evans, A. S.
Bhatnagar, R. H. A. Chaudri, A. von Kameke, W. R.
Miller, I. Smith, W. Eiermann and M. Dowsett, “Out-
come Prediction for Estrogen Receptor-Positive Breast
Cancer Based on Postneoadjuvant Endocrine Therapy
Tumor Characteristics,” Journal of the National Cancer
Institute, Vol. 100, No. 19, 2008, pp. 1380-1388.
doi:10.1093/jnci/djn309
[7] M. J. Ellis, V. J. Suman, J. Hoog, L. Lin, J. Snider, A.
Prat, J. S. Parker, J. Luo, K. DeSchryver, D. C. Allred, L.

The Reliability of Assessment of Ki-67 Expression on Core Needle Biopsy and the Surgical Specimens
of Invasive Breast Cancer: Comparison of Local Pathologists’ Assessment and Central Review
845
J. Esserman, G. W. Unzeitig, J. Margenthaler, G. V.
Ba-biera, P. K. Marcom, J. M. Guenther, M. A. Watson,
M. Leitch, K. Hunt and J. A. Olson, “Randomized Phase
II Neoadjuvant Comparison between Letrozole, Anastro-
zole, and Exemestane for Postmenopausal Women with
Estro-gen Receptor-Rich Stage 2 to 3 Breast Cancer:
Clinical and Biomarker Outcomes and Predictive Value
of the Baseline PAM50-Based Intrinsic Subtype—
ACOSOG Z1031,” Journal of Clinical Oncology, Vol. 29,
No. 17, 2011, pp. 2342-2349.
doi:10.1200/JCO.2010.31.6950
[8] M. J. Ellis and C. Ma, “Letrozole in the Neoadjuvant
Setting: The P024 Trial,” Breast Cancer Research and
Treatment, Vol. 105, Suppl. 1, 2007, pp. 33-43.
doi:10.1007/s10549-007-9701-x
[9] C X. Ma, C. G. Sanchez and M. J. Ellis, “Predicting
En-docrine Therapy Responsiveness in Breast Cancer,”
Oncology, Vol. 23, No. 2, 2009, pp. 133-142.
[10] M. Dowsett, I. E. Smith, S. R. Ebbs, J. M. Dixon, A.
Skene, C. Griffith, I. Boeddinghaus, J. Salter, S. Detre, M
Hills, S. Ashley, S. Francis, G. Walsh and R. A’Hern,
“Proliferation and Apoptosis as Markers of Benefit in
Neoadjuvant Endocrine Therapy of Breast Cancer,” Cli-
nical Cancer Research, Vol. 12, Part 2, 2006, pp. 1024s-
1030s. doi:10.1158/1078-0432.CCR-05-2127
[11] M. J. Ellis, W. R. Miller, Y. Tao, D. B. Evans, R. H. A.
Chaudri, Y. Miki, T. Suzuki and H. Sasano, “Aromatase
Expression and Outcomes in the P024 Neoadjuvant
En-docrine Therapy Trial,” Breast Cancer Research and
Treatment, Vol. 116, No. 2, 2009, pp. 371-378.
doi:10.1007/s10549-008-0161-8
[12] M. Toi, H. Sasano and M. J. Ellis, “Future Prospects of
Neoadjuvant Endocrine Therapy in Postmenopausal
Breast Cancer,” Japanese Journal of Cancer and Che-
motherapy, Vol. 36, No. 6, 2009, pp. 1035-1042.
[13] M. Dowsett, T. O. Nielsen, R. A’Hern, J. Bartlett, R. C.
Coombes, J. Cuzick, M. Ellis, N. L. Henry, J. C. Hugh, T.
Lively, L. McShane, S. Paik, F. Penault-Llorca, L.
Prud-kin, M. Regan, J. Salter, C. Sotiriou, I. E. Smith, G.
Viale, J. A. Zujewski and D. F. Hayes, “Assessment of
Ki67 in Breast Cancer: Recommendations from the In-
ternational Ki67 in Breast Cancer Working Group,”
Journal of the National Cancer Institute, Vol. 103, No. 22,
2011, pp. 1656-1664. doi:10.1093/jnci/djr393
[14] E. Luporsi, F. André, F. Spyratos, P. M. Martin, J. Jac-
quemier, F. Penault-Llorca, N. Tubiana-Mathieu, B. Si-
gal-Zafrani, L. Arnould, A. Gompel, C. Egele, B. Poulet,
K B. Clough, H. Crouet, A. Fourquet, J. P. Lefranc, C.
Mathelin, N. Rouyer, D. Serin, M. Spielmann, M. Haugh,
M. P. Chenard, E. Brain, P. de Cremoux and J. P. Bellocq,
“Ki-67: Level of Evidence and Methodological Con-
sid-erations for Its Role in the Clinical Management of
Breast Cancer: Analytical and Critical Review,” Breast
Cancer Research and Treatment, Vol. 132, No. 3, 2012,
pp. 895-915. doi:10.1007/s10549-011-1837-z
[15] R. Yerushalmi, R. Woods, P. M. Ravdin, M. M. Hayes
and K. A. Gelmon, “Ki67 in Breast Cancer: Prognostic
and Predictive Potential,” The Lancet Oncology, Vol. 11,
No. 2, 2010, pp. 174-183.
doi:10.1016/S1470-2045(09)70262-1
[16] M. T. Weigel and M. Dowsett, “Current and Emerging
Biomarkers in Breast Cancer: Prognosis and Prediction,”
Endocrine-Related Cancer, Vol. 17, 4, 2010, pp. R245-
R262. doi:10.1677/ERC-10-0136
[17] M. Ough, J. Velasco and T. J. Hieken, “A Comparative
Analysis of Core Needle Biopsy and Final Excision for
Breast Cancer: Histology and Marker Expression,” The
American Journal of Surgery, Vol. 201, No. 5, 2011, pp.
pp. 692-694. doi:10.1016/j.amjsurg.2010.02.015