Malaysian J Pathol 2016; 38(1) : 25 – 32

ORIGINAL ARTICLE
Assessment of pathological response to neoadjuvant
chemotherapy in locally advanced breast cancer using the
Miller-Payne system and TUNEL
Christina SHINTIA MD, Hardjolukito ENDANG MD and Kartini DIANI MD

Anatomical Pathology Department, Faculty of Medical Universitas Indonesia

Abstract

Background: Responses to neoadjuvant (before surgery) chemotherapy in locally advanced breast

cancer (LABC) consist of clinical and pathological responses. Evaluating chemotherapy response
is essential to predict survival rate and guide future chemotherapy. Until now, the evaluation of
pathological response mainly involves quantitative assessment and is often inconsistent with clinical
response. We explored the evaluation of pathological responses by both quantitative and qualitative
methods, i.e. by evaluating the cellularity of tumour cells and the percentage of apoptosis. Materials
and method: A cross-sectional analytical retrospective study was conducted on tissue of LABC
diagnosed between 2010 and 2014 at the Department of Anatomical Pathology, Faculty of Medicine,
Universitas Indonesia Cipto Mangunkusumo Hospital and Division of Surgical Oncology, Cipto
Mangunkusumo Hospital. Biopsy and resection specimens were compared to evaluate reduction in
cellularity, which were subsequently categorized into stages of Miller-Payne (MP) classification.
The resection specimens were stained with TUNEL and the percentage of apoptosis was calculated.
Reduction in cellularity between biopsy and mastectomy specimens with TUNEL staining is
evaluated as a modification of the MP method. Results: We found no association between clinical
responses with percentage of apoptosis, MP pathological responses and modified MP. There was
a correlation between the dead cell evaluated by MP and by modified MP (p=0.000). Conclusion:
Modified MP increases the degree or grading of pathological responses, but it does not improve
the correlation with clinical response.

Keywords: locally advanced breast cancer, apoptosis, Miller-Payne, neoadjuvant therapy

INTRODUCTION Solid Tumor (RECIST). Both criteria provide

evaluation based on assessment of reduced
Breast cancer is the most common cancer in
tumour size.3 There are some systems to evaluate
Indonesian women based on the 2010 Indonesian
the pathological responses, such as the systems
National Cancer Registry, contributing to 18.6%
of Miller-Payne (MP), Chevallier and NSABP
of cancers in Indonesian women. The American
B-18, Pinder, Sataloff and Smith.4,5 In the MP
College of Surgeons reported in the National
system, the responses are evaluated based on
Cancer Data Base that 9% of female breast
reduction in tumour cellularity between biopsy
cancer in America are in stage III, which is in
and mastectomy specimens.2,5-7 The system
contrast many other countries (Latin America,
includes the following classification, i.e. Grade 1:
Asia, Africa) where more than half of breast
no change, no significant reduction in malignant
cancer patients are at stage III and IV.1,2
cells; Grade 2: a minor loss of tumour cells
Responses to neoadjuvant (before surgery)
(≤ 30%); Grade 3: reduction in tumour cells
chemotherapy are categorised to clinical and
between 30% and 90%; Grade 4: disappearance of
pathological responses. Evaluating chemotherapy
tumour cells > 90%; Grade 5: no malignant cells
response is essential to predict survival rate
identifiable, DCIS may be present. Grade 1-4
and to guide future chemotherapy. Clinical
are categorized as partial pathological response
responses can be evaluated by two criteria, i.e.
(pPR) and grade 5 as pathological complete
the WHO and Response Evaluation Criteria In
response (pCR).2
Address for correspondence: Shintia Christina, jl. Pisangan Baru No 6. Rt 002, Rw 011, Jakarta Timur, Indonesia. Tel: +62-856-8856-905,
Fax: +62-215817490. E-mail: [email protected]

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Malaysian J Pathol April 2016

Complete clinical regression does not cellularity of tumour cells and the percentage
have direct correlation with pCR, therefore, of apoptosis.9
the evaluation of specimens obtained from
mastectomy is the gold standard for determining MATERIALS AND METHODS
response to therapy. 6 The evaluation of
A cross-sectional analytical study was performed.
pathological response following neoadjuvant
The study was conducted at the Department of
chemotherapy has now become an essential
Anatomical Pathology, Cipto Mangunkusumo
independent prognostic factor.4,5 Thus far, the
Hospital Universitas Indonesia between
evaluation of residual tumour after neoadjuvant
December 2014 and March 2015. The selected
chemotherapy is only based on decreased/
sample was all cases of locally advanced
reduced tumour cellularity. Tumour regression
breast cancer which had undergone biopsy and
following neoadjuvant therapy may also be
mastectomy at Cipto Mangunkusumo Hosptial
caused by apoptosis.8,9 Morphological changes
and had been treated with neoadjuvant therapy
in cells undergo apoptosis are condensation of
between 2010 and 2014.
chromatin, nuclear fragmentation, cell shrinkage
After completing neoadjuvant chemotherapy,
and blebbing of plasma membrane.10-12 Residual
the clinical response was evaluated based on
tumour cells can still be seen. However, the
WHO criteria (complete, partial, stable and
determination of apoptosis in haematoxylin-eosin
progressive).19
(HE) stained cells during the first stage of DNA
Pathological response was evaluated by two
fragmentation can be uncertain.13,14
observers (SHI and ESR) independently. It was
Chemotherapy initiates apoptosis through
assessed based on the MP system by comparing
extrinsic and intrinsic pathways, in which
the tumour cellularity between biopsy and
both pathways activate caspase. 11 Caspase
mastectomy. The percentage difference was
activation is a marker of ongoing cell damage,
calculated and response categorized according
an important marker of the cell’s entry point into
to the MP grading system described earlier.2,5-7
the apoptotic signaling pathway and it can be
A modified MP (MMP) evaluation was
detected by immunohistochemistry. Caspase-3
performed by calculating the percentage of
has an important role as the most distal effector
apoptosis using TUNEL in the resected tissue
of caspase in the apoptosis pathway. Moreover,
divided by percentage of tumour cellularity in
it binds to the apoptosis inhibitors and therefore,
biopsy tissues and added with the number of
can prevent the development of apoptosis.12,15-17
death cells as scored by the MP system (Table 1).
TUNEL (Terminal deoxynucleotidyltransferase-
For cases with MP grade 5, AE1/AE3 staining
mediated deoxyuridine triphosphate in situ nick
was used when necessary to confirm whether
end labeling) is a technique used to detect the
the apoptotic cells were epithelial (tumour) cells
early stage of DNA fragmentation, which is a
instead of other cells.
hallmark of apoptosis. The assay is very sensitive
for detecting early DNA fragmentation before
Statistics
apoptosis can be identified morphologically.12,13,18
The correlation between clinical response
Clinical responses are often inconsistent with
and apoptosis percentage was analyzed using
pathological responses, especially since the
Mann-Whitney test. Fisher test was undertaken
first stage of DNA fragmentation is difficult to
to determine whether there was a difference
evaluate with certainty. In this study we evaluated
in clinical response between MP and MMP.
pathological responses by both quantitative
Spearman correlation test was performed to
and qualitative methods, i.e. by evaluating the

TABLE 1: Criteria for Modified Miller-Payne Classification

Biopsy (b) Resection+ TUNEL (rT) Δ(b-rT)

Grade 1 100 100% 0

Grade 2 100 70-99% 1%-≤30%
Grade 3 100 10-69% 31%-90%
Grade 4 100 1-9% 91%-99%
Grade 5 100 0 100%

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determine the correlation between clinical increased scores and 18 cases did not have any
reduction of tumour mass and the number of change (Tables 3 and 4).
tumour cell death.
Clinical responses
RESULTS Clinical responses were evaluated based on the
WHO criteria. We found partial responses in 33
There were 203 cases of breast cancer that had
cases (78.57%), stable response/non-responsive
received neoadjuvant chemotherapy in Cipto
cases in 6 subjects (14.28%) and progressive
Mangunkusumo Hospital between 2010 and
response in 3 cases (7.14%).
2014. 42 of these cases with tissue obtained
from both biopsy and mastectomy procedures,
Correlation between clinical responses and total
were retrieved from the archives of Department
cell death and apoptosis
of Anatomical Pathology, Faculty of Medicine
The results of statistics showed that there was no
Universitas Indonesia which serves the Cipto
correlation between clinical response and total
Mangunkusumo Hospital.
cell death and apoptosis (p=0.108).
Evaluation of pathological responses based on
Correlation between clinical responses and
the Miller-Payne system
pathological responses based on Miller-Payne
The results of pathological responses based on
grading system and the responses according to
the MP system are presented in Table 2. The
the modified Miller-Payne system
evaluation of MP pathological responses in
The Fisher’s exact statistical test showed that
our study did not reveal any cases of grade 4.
there was no correlation between clinical
Grade 1 cases showed reduction in cellularity
responses and MP grading (p=1.000), as well as
between 0-8.11%; while for grade 2 cases, there
with MMP grading (p=0.655). Clinical reduction
was reduced cellularity of 12.50-27.63%. Grade
of tumour mass was correlated to the number
3 cases demonstrated reduction in celularity
of dead tumour cells, which was analyzed using
between 31.51-86.88% and grade 5 cases showed
Spearman test to demonstrate the correlation. It
reduction of as much as 100%. For two grade
was not correlated to the MP system (p=0.177;
5 cases, AE1/3 staining were performed on 2
r=0.212) and there was a weak correlation to the
slides each and demonstrated that there was no
MMP system (p=0.609; r=0.081). To identify the
epithelial tumour left (Figures 1 & 2).
correlation between clinical response and the
number of dead tumour cells, Mann-Whitney
Evaluation of apoptosis percentage
test was undertaken. The evaluation on the
There was no significant difference of cellularity
number of dead tumour cells between MP and
in grade 1 cases between the specimens obtained
MMP systems did not reveal any correlation
from mastectomy and biopsies whereas in grade
(p=0.198; p=0.108).
2 cases, there was reduction in cellularity up
To consider whether there is a difference
to 20.57%. Grade 3 cases showed reduced
between number of dead cell evaluated by MP
cellularity between 31.98-85.46%, while the
and Modified MP system, the Wilcoxon test was
reduction for grade 4 and grade 5 cases were
performed and showed significant difference
between 92.30-95.74% and as much as 100%
(p=0.000).
respectively. The evaluation of pathological
responses by the MMP system had caused altered
scores in the grading system, i.e. 24 cases had

TABLE 2: Pathological response based on MP system

MP grading Σ Case Cellularity reduction

Grade 1 15 (35.71%) 0-8.11%

Grade 2 8 (19.05%) 12.50-27.63%
Grade 3 17 (40.48%) 31.51-86.88%
Grade 4 0 0
Grade 5 2 (4.76%) 100%

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Malaysian J Pathol April 2016

A B

C D

E F

G H
FIG. 1: MP responses. (A) MP grade 1, biopsy. (B) MP grade 1, mastectomy (400x) (C) MP grade
2, biopsy. (D) MP grade 2, mastectomy (400x) (E) MP grade 3, biopsy. (F) MP grade 3,
mastectomy (400x). (G) MP grade 5, biopsy. (H) MP grade 5, mastectomy (400x).

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FIG.2: AE1/AE3 staining in two cases with grade 5 MP. (A) MP grade 5 response- a complete
histological response with no residual tumour cells (HE, 100x). (B) AE1/3 staining (100x).
(C) MP grade 5 response with foam cells and no residual tumour cells (HE,400x). (D) AE1/3
staining (400x).

DISCUSSION that the incidence of ductal type (70-75%) is

more common than the lobular type (5-15%).7,20
Most cases included in our study were stage III
Clinical responses in Cipto Mangunkusumo
B breast cancer (97.62%) and there was a case
Hospital were evaluated based on WHO criteria.
in stage IIIA (2.38%). The majority of cases,
33 cases had partial clinical responses, 6
i.e. 41 (97.62%) were at T4. The average age
stable responses and 3 progressive responses.
of patients was 49 years. We found that the
Some reports suggest that partial clinical and
most common histological type was invasive
pathological responses are the dominant groups
carcinoma (no special type), comprising 80%
(60-80%) while complete responses have a lower
of cases (32 out of 42 cases). The findings are
prevalence (3-30%).21
consistent with published literature, which report

TABLE 3: Comparison of grade using the MP and Modified MP systems

∑ cases Mean percentage death cell ∑ cases total apoptotic cells

MP with MP MMP

Grade 1 15 -71,94 5 0
Grade 2 8 20,38 1 20,57
Grade 3 17 54,85 27 63,25
Grade 4 0 - 7 94,56
Grade 5 2 100 2 100

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Malaysian J Pathol April 2016

TABLE 4: Change in pathological response grade using the MP and modified MP systems

Grade MP Grade MP modification Number of cases

A. No change
Grade 1 Grade 1 5
Grade 2 Grade 2 0
Grade 3 Grade 3 11
Grade 4 Grade 4 0
Grade 5 Grade 5 2
B.  Change
Grade 1 Grade 2 1
Grade 2 Grade 3 7
Grade 3 Grade 4 6
Grade 1 Grade 3 9
Grade 2 Grade 4 1

Pathological responses evaluated using the There was no statistical correlation found
MP system were compared to the MMP system between clinical responses and apoptosis, which
and showed increased grading of pathological may be because most clinical responses were
response indicating better responses with MMP partial responses. Figure 4 shows that there
system. About 24 cases showed increase scores is a tendency of positive correlation, i.e. the
and 18 cases did not have any changes. There better the clinical responses, the more cells
was a significant difference in the number of undergoing apoptosis. Chemotherapy induces
dead cells detected before TUNEL staining (MP) cell death not only through the mechanism of
and after staining (Modified MP) (Figure 3). apoptosis, but also through other non-apoptotic
The results indicate that the detection of cells mechanisms such as necrosis, autophagy and
that have undergone apoptosis may reveal the mitotic catastrophe; however, apoptosis is the
true pathological responses. These findings are major mechanism that causes cell death.8,9,16,22
consistent with published literature suggesting There was no significant correlation between
that the mechanism of cell death in tumour clinical responses and pathological responses
following the administration of chemotherapy in our study because (1) most cases had partial
is actually through apoptosis.8,9 clinical and pathological responses, (2) there is

A B

FIG. 3: (A) Tumour cells in a mastectomy with no obvious apoptosis on routine stain (H&E, 400x). (B) The same
specimen. Apoptotic cells (brown nuclear staining) revealed with TUNEL (400x)

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Apoptoc percentage

Stable & Paral

progressive
Clinical response
FIG. 4: Correlation between clinical response and apoptotic percentage

a wide range of each grade in the MP system response compared to measuring the tumour
as well as in clinical responses and (3) different size.
methods for evaluating clinical and pathological We found two cases with partial clinical
responses may cause undetected correlation responses, but had complete pathological
between MP/Modified MP grading and the response and 1 case with increased tumour
clinical responses.23 Changes in tissues obtained volume who had progressive clinical response
from mastectomy (tumour bed) following the and grade 3 MP system (the number of dead cell
administration of neoadjuvant chemotherapy are of 75.43%) and grade 4 Modified MP with the
stromal hyalinization, oedema, fibroelastosis, number of total dead cell and apoptosis as many
large sheets of stromal foamy histiocytes, as 92.30%. Changes in the stroma of mastectomy
lymphocytes aggregates and haemosiderophages. specimens due to chemotherapy, such as stromal
Area of tumour necrosis may develop nodules, fibrosis, fibromyxoid, fibroelastotic stroma and
which consist of collections of histiocytes and microcalcification, may also cause bias in clinical
cholesterol clefts.24 Changes in non-neoplastic response evaluation.2,4,21,26 This phenomenon was
tissues obtained from mastectomy following found in two cases in our study.
the neoadjuvant treatment may result in no
correlation between clinical and pathological Conclusion
responses. Apoptosis does not correlate with clinical
Our study did not show any correlation, either response. Modified MP increases the degree or
between clinical response and MP, or between grading of pathological responses, but it does not
clinical response and Modified MP, as well improve the correlation with clinical response.
as between clinical reduction of tumour mass Pathological responses to neoadjuvant therapy
and the number of cell death (measured both as one of prognostic factors should be confirmed
by MP and Modified MP techniques). These by histopathology examination using modified
findings are consistent with the results of some MP since the reduction of tumour size is not
studies suggesting that clinical responses do representative of the number of cell death. There
not demonstrate the pathological responses; are reactions in the non-neoplastic elements that 10
therefore pathological examination on specimens may contribute to the discrepancy.
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