2151
Correlation between MIB-1 and Other Proliferation
Markers
Clinical Implications of the MIB-1 Cutoff Value
Frédérique Spyratos, Ph.D.1
Magali Ferrero-Poüs, Ph.D.1
Martine Trassard, M.D.2
Kamel Hacène, D.Sc.3
Edelmira Phillips, M.T.4
Michèle Tubiana-Hulin, M.D.5
Viviane Le Doussal, M.D.2
1
Laboratoire d’Oncobiologie, Centre René Hugue-
nin, Saint-Cloud, France.
2
Département d’Anatomie et Cytologie Patholog-
iques, Centre René Huguenin, Saint-Cloud, France.
3 MIB-1 positive tumor cells and SPF, TK, histologic grade, and the mitotic index.
Département de Statistiques Médicales, Centre
René Huguenin, Saint-Cloud, France.
4
Département de Biologie, Centre René Huguenin,
Saint-Cloud, France.
5
Département de Médecine, Centre René Hugue-
nin, Saint-Cloud, France.
Address for reprints: Frédérique Spyratos, Ph.D.,
Laboratoire d’Oncobiologie, Centre René Hugue-
nin, 35 rue Dailly, 92210 Saint-Cloud, France;
Fax: 33-1-47-11-15-68; E-mail: f.spyratos@
stcloud-huguenin.org
Received September 10, 2001; revision received
November 1, 2001; accepted November 20, 2001.
© 2002 American Cancer Society
BACKGROUND. Cell proliferation is a major determinant of the biologic behavior of
breast carcinoma. MIB-1 monoclonal antibody is a promising tool for determining
cell proliferation on routine histologic material. The objectives of this study were to
compare MIB-1 evaluation to other methods of measuring cell proliferation, with
a view to refining the cutoff used to classify tumors with low and high proliferation
rates in therapeutic trials.
METHODS. One hundred eighty-five invasive breast carcinomas were evaluated for
cell proliferation by determining monoclonal antibody MIB-1 staining, histologic
parameters (Scarff–Bloom–Richardson grade and mitotic index) on paraffin sec-
tions, S-phase fraction (SPF) by flow cytometry, and thymidine-kinase (TK) content
of frozen samples.
RESULTS. There was a high correlation (P ⫽ 0.0001) between the percentage of
Multivariate analyses including MIB-1 at 5 different cutoffs (10%, 15%, 17% [me-
dian], 20%, 25%) and the other proliferative markers showed that the optimal
MIB-1 cutoff was 25% and that the mitotic index was the proliferative variable that
best discriminated between low and high MIB-1 samples. A MIB-1 cutoff of 25%
adequately identified highly proliferative tumors. Conversely, with a MIB-1 cutoff
of 10%, few tumors with low proliferation were misclassified.
CONCLUSIONS. The choice of MIB-1 cutoff depends on the following clinical ob-
jective: if MIB-1 is used to exclude patients with slowly proliferating tumors from
chemotherapeutic protocols, a cutoff of 10% will help to avoid overtreatment. In
contrast, if MIB-1 is used to identify patients sensitive to chemotherapy protocols,
it is preferable to set the cutoff at 25%. The MIB-1 index should be combined with
some other routinely used proliferative markers, such as the mitotic index. Cancer
2002;94:2151–9. © 2002 American Cancer Society.
DOI 10.1002/cncr.10458
KEYWORDS: breast carcinoma, proliferation, MIB-1, cutoff.
C ell division kinetics is an important predictor of the clinical out-
come of breast carcinoma patients.1 Cellular proliferation can be
measured using a variety of methods. Mitotic indices have been
widely used as part of various tumor grading methods.2– 4 DNA syn-
thesis initially was measured in terms of tritiated thymidine incorpo-
ration5; proliferation subsequently was measured by using static
methods such as flow cytometric determination of the S-phase frac-
tion (SPF).6 Cell proliferation also can be measured by specifically
assaying enzymes involved in DNA synthesis, such as thymidine-
kinase (TK), an enzyme involved in the one-step salvage pathway of
pyrimidine synthesis.7 Immunohistochemical determination of pro-
2152 CANCER April 15, 2002 / Volume 94 / Number 8

liferation indices is an expanding area of research, TABLE 1

based on detection of antigens present during cell Characteristics of the Patients and Correlation between MIB-1 Values
and Clinical, Histologic, and Biologic Variables
proliferation;8,9 the widely used Ki-67 now has been
supplanted by MIB-1 antibody, which has similar MIB-1
epitope selectivity10 but recognizes the target in par-
affin embedded tissues.11There is no consensus on the Variable n (%) Mean (SD) Median (range) P value
best proliferation index, or on the optimal methodol-
Age (yrs) 0.689
ogy, reagents, or data interpretation. Flow cytometry ⱕ 50 46 (25) 22.50 (20.33) 20 (1–90)
is widely used, but MIB-1 assays are increasingly pop- ⬎ 50 139 (75) 20.91 (19.04) 15 (1–80)
ular because of their minimal tissue requirements and Tumor size (pT) (mm) 0.112
suitability to routinely fixed tissues. Results for SPF ⱕ 20 86 (47) 19.19 (18.93) 12.5 (1–78)
⬎ 20 98 (53) 22.87 (19.49) 20 (1–90)
and MIB-1 are described as “encouraging” in recently
Lymph node status (pN) 0.636
published recommendations but not yet adequately pN ⫽ 0 111 (64) 21.36 (20.43) 15 (1–90)
characterized for use in clinical practice; in particular, 1 ⱕ pN ⱕ 3 46 (26) 22.35 (17.37) 20.5 (1–80)
further data from clinical trials based on immunohis- pN ⬎ 3 18 (10) 22.44 (21.16) 17.5 (1–75)
SBR grade 0.0001
tochemical (IHC) methods are required.12,13
I 30 (17) 7.13 (6.97) 5 (1–25)
The objectives of this study were 1) to technically II 90 (50) 19.44 (17.50) 15 (1–75)
validate MIB-1 staining before its routine use in our III 59 (33) 32.10 (21.04) 30 (1–90)
institution, and 2) to compare MIB-1 analysis to other MSBR 0.0001
measures of cell proliferation, with a view to refining 1 87 (49) 11.62 (13.91) 8 (1–70)
2 92 (51) 30.95 (19.39) 30 (1–90)
the cutoff used in therapeutic trials to classify tumors Mitotic index 0.0001
with low and high proliferation rates. The other mea- 1 39 (22) 6.51 (6.50) 5 (1–25)
sures of cell proliferation considered here were the 2 58 (32) 16.05 (16.73) 10 (1–70)
standard and modified Scarff–Bloom–Richardson 3 82 (46) 32.60 (19.06) 30 (1–90)
Histologic type 0.552
(SBR and MSBR) histoprognostic grading systems; the
Invasive ductal 159 (86) 22.19 (20.16) 20 (1–90)
mitotic index (included in SBR and MSBR); S-phase Invasive lobular 15 (8) 16.93 (13.75) 15 (1–50)
measurement by flow cytometry; and TK assay; both Others 11 (6) 14.36 (9.74) 15 (1–35)
the latter used frozen tissue. Estrogen receptor status 0.0004
Negative 34 (18) 34.74 (25.47) 27.5 (1–90)
Positive 150 (82) 18.39 (16.29) 15 (1–75)
Progesterone receptor status 0.0009
MATERIALS AND METHODS Negative 56 (30) 29.62 (23.43) 24.5 (1–90)
The characteristics of the patients are described in Positive 128 (70) 17.82 (16.05) 13 (1–75)
Table 1. From September 1998 to July 2000, 185 pa- DNA ploidy index 0.0001
tients with invasive breast carcinomas of sufficient Diploid 74 (40) 13.65 (15.01) 10 (1–75)
Aneuploid 111 (60) 26.41 (20.24) 25 (1–90)
size (macroscopic specimens) were enrolled in this S-phase fraction 0.0001
study. Immediately after confirmation of invasive car- Low 53 (34) 13.47 (14.67) 5 (1–60)
cinoma on frozen sections, part of the tumor was Intermediate 51 (32) 16.51 (12.04) 15 (1–50)
frozen and kept in liquid nitrogen for flow cytometry High 54 (34) 31.85 (23.10) 29.5 (1–80)
Thymidine kinase 0.0001
and TK assay. All tumor specimens were embedded in
Low 59 (34) 12.27 (10.71) 10 (1–45)
paraffin and stained with hematoxylin and eosin. After Intermediate 59 (34) 22.80 (17.56) 20 (1–80)
histologic analysis, 159 tumors were classified as in- High 57 (32) 29.54 (24.37) 30 (1–90)
vasive ductal carcinomas, 15 as invasive lobular car-
SD: standard deviation; SBR: Scarff–Bloom–Richardson; MSBR: modified SBR.
cinomas, and 11 as other types. All the invasive carci-
nomas apart from six mucinous carcinomas were
graded using the SBR2 and MSBR methods,3 based on Immunohistochemistry
the sum of scores for two parameters of nuclear grad- Tumor samples were fixed for 12–24 hours in ready-
ing (mitotic index and nuclear pleomorphism). Mi- to-use AFA (Carlo Erba reagenti, Rodano, Italy), con-
totic activity was quantified by microscopic examina- taining absolute ethanol (75% v/v), formol 40% (2%
tion of 10 high-power fields. Lymph node status (pN) v/v)/acetic acid (5% v/v)/water (18% v/v) and then
was determined on axillary lymphadenectomy speci- routinely embedded in paraffin blocks. Immunoper-
mens. Median age at diagnosis was 60 years (range, oxidase staining for MIB-1 was performed with an
33–99 years). Median tumor size was 22 mm (range, automated avidin-biotin complex method in a Tech-
10 –53 mm). mat 500 device (Dako SA, Trappes, France). The IHC

assay was performed on 4-␮m sections cut from the
blocks, float-mounted on adhesive (silanized) glass
slides (Tespa-coated slides), and left at 50 °C overnight
on a slide warmer. The key features of the IHC assay
included antigen retrieval in boiling 0.1 M citrate
buffer (pH 6.0) in a pressure cooker (4 minutes);
blocking of endogenous peroxidase activity with 1%
hydrogen peroxide for 15 minutes; incubation with
mouse monoclonal antibody MIB-1 (Immunotech,
Marseille, France) at a dilution of 1:100 for 1 hour;
linking with a rabbit biotinylated antibody against
mouse immunoglobulin G at a dilution of 1:100 for 30
minutes; enzyme labeling with streptavidin-horserad-
ish peroxidase (Dako) at a dilution of 1:100 for 30
minutes; chromogen development with 0.03% hydro-
gen peroxide and 1 mg/ml diaminobenzidine; and
counterstaining with Mayer’s hematoxylin (1 minute
in a 1:5 dilution), without differentiation in acid alco-
hol. Appropriate positive and negative controls were
run with each batch.
MIB-1 Index
The MIB-1 index was determined by using a semi-
quantitative visual method. Tumor cells were consid-
ered positive for MIB-1 only when clear nuclear stain-
ing was seen. The percentage of positive neoplastic
nuclei was estimated after scanning the entire tumor
surface at low power (⫻10 objective), including areas
of highest and lowest positivity. After this first analysis,
a quantitative assessment was made at ⫻20 and/or
⫻40 magnification by counting a total of 200 –500
tumor cells within representative fields. All nuclei with
homogeneous granular staining, multiple speckled
staining or nucleolar staining were counted as posi-
tive, regardless of staining intensity. Cells with cyto-
plasmic staining were excluded. This evaluation was
performed independently by two observers (V.L.D.
and M.T.), and an interobserver reproducibility test
was performed on the entire series (n ⫽ 185).
Estrogen Receptor and Progesterone Receptor Assays
The hormone receptor status of the tumor was re-
corded at the time of surgery. The tumor was consid-
ered to be steroid receptor positive if estrogen recep-
tor (ER) and progesterone receptor (PR) values
exceeded 15 fmol/mg protein by enzyme immunoas-
say (Abbott Laboratories, North Chicago, IL).
Flow Cytometric DNA Analysis and S-Phase
Measurement
Cell preparation was standardized as follows: 1) quick
specimen thawing; 2) tumor imprints performed sys-
tematically, stained with May-Grunwald-Giemsa and
observed by a pathologist to check for the presence of
Clinical Implications of MIB-1 Cutoff/Spyratos et al. 2153
malignant cells; 3) mechanical dissociation in phos-
phate buffer saline; and 4) DNA staining according to
Vindelov’s method. Flow cytometry was performed on
a FACScalibur device (Becton Dickinson, Mountain
View, CA). The cell cycle analysis was performed with
the Modfit LT 2.0 program (Verity Software House,
Topsham, ME). The DNA-diploid peak was located on
DNA histograms by using an external standardization
procedure with normal human lymphocytes posi-
tioned at the fifth part of the red fluorescence scale.
DNA ploidy and the SPF were obtained after gating on
a dot plot (signal peak width vs. signal peak area),
selecting a representative amount of debris and ex-
cluding doublets.
Rules established during a previous interlaboratory
control procedure14 were applied when using the differ-
ent cell cycle software models and for objective interpre-
tation of DNA histograms. They included graphic aggre-
gate subtraction. The limits for the ploidy index were
hypodiploid less than 0.95; 0.95 less than or equal to
diploid less than or equal to 1.1; aneuploid greater than
1.10; 1.90 less than or equal to tetraploid less than or
equal to 2.05. Multiploid tumors were characterized by
DNA histograms showing two or more aneuploid G0G1
peaks. Automatic procedures were not used. We calcu-
lated SPF when the coefficient of variation of the G0G1
peaks was lower than 5%, debris represented less than
20% of all acquired events, and a minimal aneuploid
fraction was present. When a unimodal histogram was
obtained, the diploid option of the software programs
was used. If DNA content was abnormal, only the ane-
uploid SPF was taken into account (the diploid SPF was
not calculated). In every case, the rectangular option was
chosen for SPF calculation. The debris subtraction op-
tion was always used. SPF adjusted for ploidy was cate-
gorized as low, intermediate, or high according to the
33rd and 66th percentiles.
TK Assay
Cytosols prepared for hormone receptor assays at the
time of initial surgery were stored in liquid nitrogen
until analysis. The cytosolic protein concentration was
determined using the BCA assay (Pierce, Rockford, IL)
with the same standard for all samples.
Thymidine-kinase enzyme activity was deter-
mined using the Prolifigen TK-REA assay (Sangtec
Medical, Bromma, Sweden), which is optimized for
the TK1 isoenzyme. Measurements were performed as
previously described15 with the following modifica-
tions recommended by the EORTC Receptor and Bi-
omarker Study Group16: cytosols were diluted 1:20 in
heat-inactivated normal calf serum containing 90 mM
Hepes, 18 mM KCl, 6 mM dithiotreitol, 8 mM MgCl2,
and 4 mM ATP; TK levels were expressed in milliunits
2154 CANCER April 15, 2002 / Volume 94 / Number 8

TABLE 2 TABLE 3
Distribution of MIB-1, SPF, and TK Values Quantitative Correlation between MIB-1 and SPF

Variable n Mean (SD) Median Range SPF (%)

MIB-1 (%) 185 21.30 (19.33) 17 1–90 MIB-1 (%) n Mean SD Median
SPF (%) 158 4.96 (4.62) 3.29 0.19–26.10
TK (mU/mg protein) 175 453.06 (2383) 90.18 0–30581 ⱕ 10 66 2.88 2.81 2.10
11–20 27 3.48 1.81 3.64
SPF: S-phase fraction; SD: standard deviation; TK: thymidine-kinase. 21–30 27 5.55 3.86 6.05
31–40 17 7.06 4.40 7.78
41–50 10 10.67 5.00 9.65
per milligrams of protein. Thymidine-kinase values ⬎ 50 11 11.16 8.11 8.51
were categorized as low, intermediate, or high accord- Total 158 4.96 4.62 3.29
ing to the 33rd and 66th percentiles.
SPF: S-phase fraction; SD: standard deviation.
Statistical Methods
Statistical analysis was performed using the SAS sta-
tistical package (SAS Institute, Cary NC). TABLE 4
Descriptive statistics were calculated for all prolifer- Quantitative Correlation between MIB-1 and TK
ation markers. Correlations between MIB-1, SPF, and TK TK (mU/mg protein)
were identified with Spearman rank coefficient. Associ-
ations with other clinical or pathologic variables were MIB-1 (%) n Mean SD Median
sought using Mann–Whitney or Kruskal–Wallis tests.
ⱕ 10 72 296.45 898.39 64.49
The Cox logistic regression model was used to
11–20 28 112.01 138.26 73.30
determine which variables best discriminated samples 21–30 32 197.66 348.04 99.72
with low MIB-1 from those with high MIB-1, as de- 31–40 20 301.46 337.66 196.76
fined by five different cutoff values. Each model was 41–50 9 3815.48 10063.23 184.68
obtained by a stepwise selection procedure and was ⬎ 50 14 579.40 577.03 388.22
Total 175 453.06 2382.61 90.18
evaluated using two statistics, namely, the Harrel c-
index, i.e., the area under the receiver operating char- TK: thymidine-kinase; SD: standard deviation.
acteristics curve, because the response is binary (the
higher the statistic, the better the model); and the
Akaike information criterion (AIC) to compare the five
models, that with the lowest value being the preferred
higher in DNA aneuploid tumors than in diploid tu-
model.
mors. There was a close relation between MIB-1 val-
RESULTS ues and SPF (Spearman ␳ ⫽ 0.52; P ⫽ 0.0001). Regard-
Correlations between MIB-1 values and other clinical ing ploidy status, the correlation between MIB-1 and
and pathologic variables are shown in Table 1. High SPF was detected only in aneuploid tumors (␳⫽ 0.57; P
MIB-1 was associated with SBR histologic Grade III (P ⫽ 0.0001; ␳ value for diploid tumors ⫽ 0.22; P ⫽ 0.066).
⫽ 0.0001), high MSBR grade (P ⫽ 0.0001), a high There was a significant relation between MIB-1 values
mitotic index (P ⫽ 0.0001), ER negativity (P ⫽ 0.0004), and TK values (Spearman ␳ ⫽ 0.33; P ⫽ 0.0001).
and PR negativity (P ⫽ 0.0009). MIB-1 did not corre- The mean and median SPF values increased pro-
late with age, tumor size, lymph node status, or his- portionally to the MIB-1 score (Table 3), and there was
tologic type. The distribution of MIB-1, TK and SPF is no major variability among tumors with the same
shown in Table 2. The overall median MIB-1 value was MIB-1 scores.
17%. Calculation of SPF was possible for 158 (85%) of When TK values were used instead of SPF values
the 185 tumors submitted for flow cytometry. The (Table 4), these comparisons remained significant but
median SPF was 3.29%. TK was determined in 175 failed to show a monotonous increase in the mean
tumors (95%), and the median value was 90.18 and median TK values.
mU/mg protein.
MIB-1 Categorization and Cox Logistic Regression
Association between MIB-1 and Other Proliferative Analysis
Factors Five cutoffs (10%, 15%, 17% [median], 20%, and 25%)
High MIB-1 was associated with a high mitotic index were used to dichotomize MIB-1 values into those
(P ⫽ 0.0001). Median MIB-1 values were significantly reflecting low and high proliferation rates.

TABLE 5
Results of the Cox Logistic Regression Model
Discriminant
MIB-1 cutoff (%) variables P value
10 Mitotic index 0.0001
SPF 0.025
15 Mitotic index 0.0001
17 Mitotic index 0.0001
20 Mitotic index 0.0001
25b Mitotic index 0.0001
TK 0.0033
Ploidy index 0.0329
ROC: receiver operating characteristic; AUC: area under the curve; AIC: Akaike information criterion.
a
Harrel’s c-index was highest in the model in which the MIB-1 cutoff was set at 25% (the higher the
statistic, the better the model).
b
The model with the cutoff set at 25% had the lowest AIC statistic and thus was the preferred model.
Each MIB-1 cutoff value then was entered as a
dependent variable into a logistic regression model, to
determine which of the variables listed in Table 1 best
discriminated high from low MIB-1 samples. Each
model was obtained by a stepwise selection procedure
and was evaluated with multiple statistics (Table 5).
Whatever the cutoff used for MIB-1, the mitotic index
was always the most discriminant variable, followed
by SPF when the MIB-1 cutoff was set at 10%, or by TK
and the ploidy index when the MIB-1 cutoff was set at
25%.
The Harrel c-index was highest in the model in
which the MIB-1 cutoff was set at 25% (the higher the
statistic, the better the model). With the AIC statistic,
the model with the cutoff set at 25% had the lowest
value and thus was the preferred model.
Distribution of Other Proliferation Markers According to
the MIB-1 Cutoff
The distribution of the other proliferation markers is
shown in Table 6 according to the five MIB-1 cutoffs.
Among the tumors with MIB-1 staining below 10%,
19% (11 of 59) of the overall population and 17% (4 of
24) of the aneuploid tumors were classified as high
SPF; 16% (10 of 62) were MSBR Grade 2 and 11% (7 of
62) had a mitotic index of 3. Among the tumors with
MIB-1 staining above 10%, 78% (77 of 99) of the overall
population and 81% (51 of 63) of the aneuploid tumors
were classified as intermediate or high SPF; 70% (82 of
117) were MSBR Grade 2 and 91% (107 of 117) had a
mitotic index of 2 or 3.
Among the tumors with MIB-1 staining below
25%, 23% (22 of 101) of the overall population and 14%
(6 of 42) of the aneuploid tumors were classified as
high SPF; 33% (36 of 110) were MSBR Grade 2, and
26% (29 of 110) had a mitotic index of 3. Among the
Clinical Implications of MIB-1 Cutoff/Spyratos et al. 2155
tumors with MIB-1 staining above 25%, 77% (44 of 57)
of the overall population and 82% (37 of 45) of the
aneuploid tumors were classified as high SPF; 81% (56
Harrel’s c-indexa
(ROC AUC) AIC of 96) were MSBR Grade 2 and 99% (68 of 69) had a
mitotic index of 2 or 3.
0.838 136.467 Intermediate cutoffs (15%, 17%, or 20%) were less
discriminatory.
0.823 138.677
Most of the tumors considered to have low pro-
0.809 142.592
0.802 144.895 liferation when the MIB-1 cutoff was set at 10% also
0.850 129.692 were considered to have low proliferation with the
other methods. When the MIB-1 cutoff was set at 25%,
a significant number of tumors considered to have low
proliferation had high proliferation with the other
methods. Conversely, most tumors with MIB-1 values
above 25% also were considered to have high prolif-
eration with the other methods.
DISCUSSION
Several approaches have been developed to study tu-
mor proliferation, including a labeling index,5 SPF
measurement by flow cytometry,6,17–19 immunohisto-
chemical detection of proliferation-associated anti-
gens,20 –29 and cytosolic TK assay.7,15 Highly prolifera-
tive breast tumors are associated with shorter patient
survival, whatever the method used to measure pro-
liferation.5,7,18,20,22,24 A strong correlation generally is
observed between these methods, even though they
do not measure the same biologic entities. Studies
mainly based on flow cytometric DNA analysis have
suggested that highly proliferative tumors show in-
creased sensitivity to neoadjuvant30,31 and adjuvant
chemotherapy,17,19 regardless of the impact of such
treatment on patient survival. In contrast, the ratio-
nale of chemotherapy for slowly proliferating tumors
is controversial.6,7,14 Hence, proliferative markers have
the potential to distinguish patients with rapidly pro-
liferating tumors that are likely to respond to chemo-
therapy from patients with slowly proliferating tumors
who may not need aggressive treatment. However, if
they are to be used in clinical trials, these factors must
be determined with techniques that are accurate, re-
producible, and available in most laboratories. De-
spite the many studies focusing on proliferative mark-
ers in breast carcinoma, the American Society of
Clinical Oncology tumor markers expert panel consid-
ered that data on SPF and immunohistochemical in-
dices are insufficient to recommend their routine
use.13 However, in practice, monoclonal antibody
MIB-1 is probably the most widely used proliferative
marker. It reacts with an antigen that is only present in
the nucleus of proliferating cells, has similar epitope
sensitivity to Ki-67, and, contrary to the latter, can be
used with paraffin sections.
In this study, we technically validated the use of
2156 CANCER April 15, 2002 / Volume 94 / Number 8

TABLE 6
Distribution of Other Proliferation Markers According to the MIB-1 Cutoff

MIB-1 < 10%, MIB-1 < 15%, MIB-1 < 17%,a MIB-1 < 20%, MIB-1 < 25%, MIB-1 > 25%,
Variable n n (%) n (%) n (%) n (%) n (%) n (%)

SPF
Low 53 31 (52) 33 (48) 36 (44) 36 (44) 40 (40) 13 (23)
Intermediate 51 17 (29) 23 (33) 29 (36) 29 (35) 38 (38) 13 (23)
High 54 11 (19) 13 (19) 16 (20) 17 (21) 23 (22) 31 (54)
Aneuploid SPF
Low 28 16 (66) 16 (62) 17 (57) 17 (57) 20 (48) 8 (18)
Intermediate 28 4 (17) 6 (23) 9 (30) 9 (30) 16 (38) 12 (27)
High 31 4 (17) 4 (15) 4 (13) 4 (13) 6 (14) 25 (55)
MSBR
1 87 52 (84) 62 (80) 67 (75) 67 (74) 74 (67) 13 (19)
2 92 10 (16) 16 (20) 22 (25) 23 (26) 36 (33) 56 (81)
Mitotic index
1 39 29 (47) 33 (42) 35 (39) 35 (39) 38 (35) 1 (1)
2 58 26 (42) 35 (45) 38 (43) 38 (42) 43 (39) 15 (22)
3 82 7 (11) 10 (13) 16 (18) 17 (19) 29 (26) 53 (77)

SPF: S-phase factor; MSBR: modified Scarff–Bloom–Richardson.

a
17% is the median value in our series.

MIB-1 in breast carcinoma and assessed the clinical
implications of the choice of MIB-1 cutoff. MIB-1
scores were compared with the results of flow cyto-
metric SPF determination and TK assay. MIB-1 scores
also were compared with other classic factors measur-
ing the proliferation rate, namely, SBR and MSBR
grades and the mitotic index, which is a component of
histologic grading system. We used five different
MIB-1 cutoffs, including the median value in our se-
ries and values chosen in previous series, to define the
most appropriate cutoff for distinguishing between
tumors with low and high proliferation rates.
The distribution of MIB-1 values in this series
(mean and median) were consistent with those found
in previous studies (Table 7). Reported links between
MIB-1 and other known prognostic factors also were
confirmed here. MIB-1 values tended to be higher in
patients younger than 50 years than in older patients,
and the lack of statistical significance was probably
because there were few patients younger than 50 years
in our series. Some authors have found a similar cor-
relation,20,27,28 whereas others have not.22,24,26 We ob-
served no correlation between MIB-1 and tumor size,
as previously reported.22,26 When such a correlation is
observed, it is usually weak.21,24,27,28 As generally re-
ported,21,22,28 MIB-1 values were lower in lobular car-
cinomas than in ductal carcinomas, but the difference
was not significant in our series, probably because
there were few lobular carcinomas. The lack of asso-
ciation with lymph node status in our study is consis-
tent with previous reports.25,26 MIB-1 correlated neg-
atively with hormone receptor status, in keeping with
most other studies.24 –28
MIB-1 correlated with histologic tumor grade.
Other authors also have observed a significant rela-
tion between MIB-1 and tumor grade in breast car-
cinoma.21,22,25–29,32 MIB-1 also correlated strongly
with MSBR, the modified SBR grade defined by the
mitotic index and nuclear pleomorphism. Finally,
we confirmed that the mitotic index, a component
of the SBR grading system, correlates strongly with
MIB-1.20,25,26,28,29,32
Regarding links between MIB-1 and the other pro-
liferation markers, we confirmed the good correlation
between MIB-1 and SPF measured on frozen tu-
mors.23,25,26 We also confirmed that the overall corre-
lation between MIB-1 and SPF was primarily because
of aneuploid tumors.22 This may be explained by the
finding that flow cytometric DNA analysis tends to
underestimate SPF in diploid tumors.
We also observed a correlation between TK and
MIB-1, a relation that, to our knowledge, has not been
examined previously. A nonlinear relation was ob-
served between TK values and MIB-1, indicating that
TK activity represents a different aspect of prolifera-
tion, even though the clinical significance of TK was
close to that of SPF in a recent large multicenter
study.7
Thus, we confirm that MIB-1 is strongly related to
histologic and nuclear grade, the mitotic index, and
SPF (mainly in aneuploid tumors) and further show
that MIB-1 is related to TK, albeit to a lesser degree.
 Clinical Implications of MIB-1 Cutoff/Spyratos et al. 2157

TABLE 7
Distribution of MIB-1 Values in Studies of Paraffin Sections of Breast Tumors

No. of patients Cutoffs used

Authors (type of tumors) Antibody used MIB-1 distribution for MIB-1

Arber et al. (1997)32 48 (T1–T2, N0) MIB-1 Median, 16.4% in patients without recurrence Median
Median, 36.7% in patients with recurrence
Clahsen et al. (1999)20 441 (pN0) MIB-1 NS 20%
Dettmar et al. (1997)22 90 (pN0) MIB-1 Median, NS 25%
Mean, 18%
Range, 1–81%
Ellis et al. (1996)23 75 (postmenopausal) MIB-1 Median, 9% ns
Mean, NS
Range, 1–83.4%
Jansen et al. (1998)24 341 MIB-1 Median, 7% 7%
Mean, NS
Range, 0.71–11%
Keshgegian and Craan (1995)25 135 MIB-1 Median, 8.2% 10%
Mean, NS
Range, 0–50%
MacGrogan et al. (1997)26 112 MIB-1 Median, 27.5% Median
Mean, NS
Range, 1–95%
Rudolph et al. (1999)27 371 (pN0) Ki-S5 (⫽MIB-1) Median, 23.5% 25%
Mean, NS
Range, 3–93%
Thor et al. (1999)28 486 MIB-1 Median, 28.6% Median
Mean, 32.2%
Range, 0–99%

NS: not specified.

To refine the correlation studies, we used more
sophisticated statistical models. Logistic regression
analysis showed that the mitotic index was the prolif-
eration variable that best discriminated high from low
MIB-1 samples. This is consistent with the finding that
KI-67 antigen expression increases during progression
of the mitotic cycle, increasing during the latter half
the SPF and peaking in the G2 and M phases.9 The
mitotic index is a rapid and cost-effective tool for
estimating tumor cell proliferation, and reasonable
reproducibility can be achieved with a strictly stan-
dardized methodology.4 In clinical trials, MIB-1 could
be used in conjunction with the mitotic index to en-
sure correct tumor classification on the basis of pro-
liferative potential.
Regarding the choice of MIB-1 cutoff, several sta-
tistical tests applied to the logistic regression analyses
indicated that a cutoff of 25% was optimal. This value
is within the range (7– 36.5%) of values in most pub-
lished series of breast carcinoma, as shown in Table 7.
The median often is used as a cutoff, with no rational
justification, and there is no consensus on the best
cutoff for MIB-1, possibly owing to the lack of a pub-
lished quality insurance program comparable to those
used for HER-2 and hormone receptors.33,34 Another
possible explanation for the wide range of MIB-1 cut-
offs is the clinical heterogeneity of published series. To
our knowledge, no attempt has been made to use
other proliferation markers to check the accuracy of
MIB-1– based tumor categorization. We found that the
choice of MIB-1 cutoff influenced the accuracy of this
marker, as judged on the basis of MSBR grade, the
mitotic index, SPF, and TK. With a MIB-1 cutoff of
10%, few tumors with low proliferation were misclas-
sified. Conversely, a MIB-1 cutoff of 25% acceptably
identified highly proliferative tumors. There is no ob-
vious difference between the cutoff set at 10% or 25%
regarding comparison with SPF. The difference is
more pronounced when MIB-1 is compared with
MSBR (16% vs. 33%) or the mitotic index (11% vs.
26%). This is not surprising, because the associations
between MIB-1 and MSBR and between MIB-1 and
the mitotic index are stronger than that between
MIB-1 and SPF. Hence, the choice of MIB-1 cutoff will
depend on the following clinical objective: if MIB-1 is
used to exclude patients with slowly proliferating tu-
mors from chemotherapy protocols, a cutoff of 10%
will help to avoid overtreatment. In contrast, if MIB-1
2158 CANCER April 15, 2002 / Volume 94 / Number 8
is used to identify tumors sensitive to chemotherapy,
it seems preferable to set the cutoff at 25%. At all
events, MIB-1 should be combined with some other
routinely used proliferative markers, such as the mi-
totic index.
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