2015, Volume 31, Number 3, Page(s) 188-193
Significance of Morphometric Parameters in the Categorization of Breast Lesions on Cytology
Hemant YADAV, Meenu GILL, Divya SRIVASTAVA, Veena GUPTA, Sumiti GUPTA, Rajeev SEN
Department of Pathology, PGIMS Rohtak, HARYANA, INDIA
Keywords: Breast, Cytology, Morphometry
The aim of this study was to assess the utility of
the morphometric parameters in cytological aspirates in the
categorization of breast lesions.
Material and Method: The study was carried out on 50 females
presenting with a breast lump for fine needle aspiration cytology.
Air-dried smears from the aspirate were stained with Leishman and
Giemsa stains while alcohol-fixed preparations were stained with the
PAP stain. They were subsequently categorized into benign, borderline
and malignant categories on light microscopy. Morphometry was
performed on the stained smears. The morphometric parameters
studied were mean nuclear area, mean cytoplasmic area, perimeter
and nuclear/cytoplasm ratio.
Results: Morphometric parameters revealed a progressive and
statistically significant increase in values from benign to borderline
to malignant cases. On comparing benign with borderline and
malignant, all the four parameters were found to be statistically
significant with a p-value of less than 0.05 while on comparing
borderline with malignant, two of the parameters, i.e. mean nuclear
area and mean cytoplasmic area, were statistically significant.
Conclusion: This study introduces morphometry as a highly objective
tool to supplement the entirely subjective fine needle aspiration
cytology in the crucial differentiation of benign from malignant
lesions and especially the borderline cases in the gray zone comprising
of atypical ductal hyperplasia and ductal carcinoma in situ.
Breast lesions account are one of the largest group
of conditions necessitating pathological, radiological
and surgical intervention. A palpable mass is the most
common symptom of underlying malignancy and must be
distinguished from the normal nodularity of the breast. The
most common palpable lesions are invasive carcinomas,
fibroadenomas and cysts1
Fine needle aspiration cytology (FNAC) is the first
diagnostic modality employed for the diagnosis of breast
masses. However, cytology has its own disadvantages such as
interobserver and intra-observer variability. This “gray zone”
in cytology is estimated to constitute 8.9% of cases. This
encompasses three categories - technical limitations (4.5%),
inexperience of the cytopathologist (2.4%) and overlap of
cytological features of benign vs. malignant (2%)2.
Alterations in nuclear structure are the morphologic
hallmarks of cancer diagnosis. A large number of
parameters have been studied by morphometry, but nuclear
parameters such as area, perimeter, diameter or axes have
consistently been found to be significant.
The progression pattern of nuclear morphometric parameters
have shown gradually increasing values from benign
to atypical, ductal carcinoma in situ (DCIS) and further
to invasive carcinoma and carcinoma with lymph node
In this era of automation, this study introduces morphometry
as a highly objective tool to supplement the entirely
subjective FNAC in the crucial differentiation of benign
from malignant lesions.
This prospective study was conducted on patients presenting
with breast masses in the Department of Pathology after
obtaining ethical clearance from P. G. Board of studies of
A total of 50 cases were included in the study. FNAC was
performed with a 23-gauge needle on patients presenting
with a breast lump. Air-dried smears were made by direct
drying while one of the cytological smears was fixed in 95%
ethanol. Air-dried smears were subsequently stained with
the Leishman and Giemsa stains while alcohol-fixed preparations
were stained with the Papanicolaou (PAP) stain.
Stained slides were examined on the light microscope with
subsequent categorisation into benign, borderline and
malignant categories (Figure 1A, 2A, 3A). All the cases
were finally confirmed on histopathology.
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|Figure 1: A) Benign breast lesion; (Leishman; x200), B) Morphometric study (PAP; x200).
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|Figure 2: A) Cellular smear from borderline lesion revealing atypical cells with enlarged pleomorphic and hyperchromatic nuclei along
with benign bimodal population (Leishman; x200); B) Morphometric study (PAP; x200).
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|Figure 3: A) Malignant breast lesion (Leishman; x200), B) Morphometric study (PAP; x200).
The PAP-stained smears were subjected to quantitative
morphometric studies by image analysis software. Images
provided by a charged video camera coupled with the
Olympus BX 51 microscope at a magnification of 200X
were stored on host the computer based on a Pentium 4
processor with the Microsoft Windows Vista operating
system through a digital frame grabber and processing
was done by the image analyzer software Microsoft Image-
Pro Plus version 6.3. The area on the slide to be imaged
was visually selected. Random cell clusters with minimum
overlapping of nuclei were selected, excluding any damaged
cell cluster. Measurements were made with 20X objective
magnification which when added to the 10X video ocular
resulted in an image magnification of 200X on the monitor
The cells with a clearly identifiable nuclear border were
selected in each microscopic field (Figure 1B, 2B,3B). A total
of a hundred nuclei were measured in each case. Nuclear
features were measured by outlining their digitalized
images on the monitor screen with the help of a computer
mouse. Mean nuclear area and perimeter were measured
by outlining the nuclear borders and clicking ‘Measure’.
Cytoplasmic area in the cells with clearly visible cell
boundaries was measured by outlining the cell borders. If
this was not possible, the cytoplasm of adjacent cells with
the nuclei in focus was divided between the cells.
Nuclear to Cytoplasmic Ratio
The nuclear to cytoplasmic ratio (N/C) was calculated by
using an Excel chart.
Morphometric parameters calculated by the software were
compared among different categories using the ANOVA
statistical test. The post hoc statistical test was used for
comparison between two categories or grades. All the
statistical tests were applied via SPSS software.
On the basis of FNAC smears, the cases were divided into
benign (11 cases), borderline (9) and malignant (30) based
on their cytological appearance.
Out of the 11 benign cases reported on cytology, 8 were
diagnosed as fibroadenoma and the remaining 3 as benign
mammary lesion with cystic change. Nine cases with a
diagnosis of proliferative mammary lesion with atypia were
grouped under the borderline category. Thirty cases with
a cytological diagnosis of breast carcinoma were placed
under the malignant category.
Most of the benign lesions in our study were seen in the
age group of 21-30 years. Borderline lesions were most
commonly seen in the 30-40 years age group whereas
malignant lesions had their maximum incidence in 6th
decade of life.
As shown in Table I, a comparative analysis of morphometric
parameters in various breast lesions revealed a progressive
increase in the MNA, MCA, N/C ratio & perimeter from the
benign to carcinoma spectrum. These results were found to
be statistically significant in all parameters (p<0.05).
Table II shows comparison of the morphometric parameters
between the study groups. On comparing benign with
borderline and malignant all the four parameters were
found to be statistically significant with a p-value of less
than 0.05 while on comparing borderline with malignant,
two of the parameters, i.e. MNA and MCA, were statistically
significant with a p-value of 0.024 and 0.043, respectively.
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|Table II: Statistical significance of morphometric values between study groups (post hoc test)
Finally on cyto-histopathological correlation, all the
benign and malignant cases were confirmed the same
on histopathology as that of cytological diagnosis but
discordance (33.33%) was seen in borderline lesions. Out
of nine cases diagnosed as borderline on cytology, five were
reported as atypical ductal hyperplasia, one as DCIS and
the other three as fibroadenoma.
A mass in the breast, whether benign or malignant, is a
cause of anxiety for the patient and her family members.
Though histopathological diagnosis is a universally
accepted confirmatory mode of diagnosis and follow
up, fine needle aspiration cytology of breast lumps is an important part of triple assessment (clinical examination,
imaging, and FNAC) of palpable breast lumps3
is the minimally invasive and first diagnostic modality
employed for the diagnosis of breast masses. It is a useful,
simple, quick, highly reproducible, and cost-effective
method for the early diagnosis of breast disorders4
recent years, FNAC cytology is increasingly being used for
the pre-operative diagnosis of breast cancer.
Alterations in cyto-nuclear features are the morphologic
hallmarks of cancer diagnosis5. The cyto-nuclear changes
can be quantified by a more reproducible computer-based
method known as morphometry.
The present study was planned to assess the significance
of nuclear morphometry in differentiating benign and
malignant breast lesions, especially in those cases where
diagnostic dilemmas are encountered.
As we proceed from benign to borderline to malignant
cases, there is progressive increase in MNA, perimeter,
MCA and N/C ratio. The same results were reported by
Narasimha et al.6 Abdalla et al.7 and Kalhan et al.8
evaluated MNA in benign and malignant cases and found a
more than 2-fold increase in the values.
MNA is the most studied parameter in nuclear morphometry
in the published literatüre7,9,10-13. In our study, an
MNA of 45.45±3.88 μm2 was observed in benign cases while malignant ones had an MNA of 115.10±9.01 μm2,
indicating a significant increase in MNA from the benign to
malignant spectrum which is in concordance with studies
done by Narasimha et al.6, Abdalla et al.7, and Kalhan
et al.8. An intermediate MNA value of 107.03±6.60 was
observed in borderline cases which is comparable to study
by Narasimha et al.6 (Table III).
The differences in the observed values of different morphometric
parameters in various studies may be due to the
application of different morphometric methods. However,
a strictly standardized and uniform technique along
with regular calibration of computerized morphometric
analysers may augment the precision and accuracy,
enhancing the reproducibility of results.
On comparing morphometric parameters among the three
categories, the present study showed significant results on comparing various morphometric variables of benign with
borderline and malignant cases. However, comparison of
borderline with malignant category revealed a statistically
significant role of mean nuclear area and mean cytoplasmic
area while other parameters such as perimeter and N/C
were found to be insignificant statistically. Abdalla et
al.7 compared MNA and perimeter between benign
and malignant and the results were in concordance with
the present study. In the study by Narasimha et al.6,
the results were also significant in respect to MNA and
perimeter while comparing the three categories (Table IV).
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|Table IV: Comparison of statistical significance of morphometric parameters between different groups
In conclusion, computerized image analysis using detailed
nuclear morphometry and other parameters acts as a
highly objective tool supplementing the entirely subjective
cytological interpretation in the successful discrimination
of various breast lesions.
Further studies on a large number of cases are needed to
elucidate the role of morphometry in delineating other
breast lesions like ADH and DCIS.
CONFLICT OF INTEREST
The authors declare no conflict of interests.
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