Oncocytology

 
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Original Article
 
Scoring system of fine needle aspiration cytology samples for the detection of non-high-grade ductal breast carcinoma
Ayumi Ryu1, Akemi Takenaka2, Shigenori Nagata3, Yasuhiko Tomita4
1Cytologist, Department of Cytology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka, Japan.
2Chief Cytologist, Department of Cytology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka, Japan.
3Staff, Department of Pathology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka, Japan.
3Chief, Department of Pathology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka, Japan.

Article ID: 100004OCYAR2013
doi:10.5348/ocy-2013-4-OA-1

Address correspondence to:
Tomita Yasuhiko
Department of Pathology, Osaka Medical Center for Cancer and Cardiovascular Diseases
1-3-3 Nakamichi, Higashinari, Osaka
Osaka 537-8511
Japan
Phone: 81-6-6972-1181
Fax: 81-6-6981-8336
Email: yasuhiko-tomita@umin.ac.jp

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How to cite this article:
Ryu A, Takenaka A, Nagata S, Tomita Y. Scoring system of fine needle aspiration cytology samples for the detection of non-high-grade ductal breast carcinoma. Oncocytology Journal 2013;3:1–7.


Abstract
Introduction: Although fine needle aspiration cytology (FNAC) is usually performed for breast lesions that are difficult to diagnose clinically and radiologically, in cases of carcinoma definitive diagnosis by FNAC is difficult in cases lacking obvious nuclear atypia, that are non-high-grade ductal carcinoma (NHGDC).
Aims: We aimed to clarify cytopathological characteristics useful for differential diagnosis between NHGDC and benign breast lesions.
Methods: We enrolled 62 patients who were difficult to diagnose or who were suspected of malignancy by routine FNAC examination. The definitive histopathological diagnosis was breast cancer in 42 patients and others in 21. The slides were re-examined to classify under the following 12 categories: dissociation of cells, cell size, cell uniformity, prominent nucleoli, nuclear margins, chromatin pattern, overlapping pattern of cells, presence of necrosis, presence of mucin, appearance of myoepithelial cells, appearance of bipolar bare nuclei, and appearance of foam cells.
Results: Univariate analysis revealed that chromatin pattern, nuclear margins, irregular overlapping of cells, appearance of myoepithelial cells, and appearance of bipolar bare nuclei were statistically significant. Multivariate analysis revealed that irregular overlapping of cells and appearance of myoepithelial cells were independent factors that were useful for differential diagnosis. Combination of these factors proved to be a useful marker in the differential diagnosis of breast lesions.
Conclusions: Application of the scoring system to future cases of breast lesions might clarify the usefulness of the system.

Key Words: Fine needle aspiration cytology, Non-high-grade ductal carcinoma, Multivariate analysis, Scoring system


Introduction

The recent increase in use of mammography and ultrasonography has enabled the detection of breast cancer at an early stage. [1] Cancers detectable only with these diagnostic imaging methods are usually ductal carcinoma in situ or small-sized invasive ductal carcinoma, with lower histological grades than those detected by conventional methods. [1] [2] Therefore, the chance of encountering cases that are difficult for differential diagnosis is increasing. At the same time, the chance of accidental detection of benign breast lesions such as mastopathy and papilloma by mammography and/or ultrasonography have rapidly increased. [3]

Histological examination of needle biopsy specimens or fine needle aspiration cytology (FNAC) is usually performed for cases that are difficult to diagnose clinically and radiologically. [4] As it is less invasive to the patients compared with histological examination, FNAC is being increasingly performed. [5] Cytopathological features of malignant and benign lesions have been previously reported; [6] nevertheless, the differential diagnosis between benign and malignant lesions is difficult in some cases. [7] [8] [9]

In the cytological examination of breast tumors, the following features are considered as general criteria for malignancy: presence of necrosis and dissociated cells, irregular overlapping of cells, absence of myoepithelial cells, and presence of mucin and intracytoplasmic lumina. [6] In addition, nuclear atypia such as enlarged nuclei, nuclear pleomorphism and irregular chromatin are the core factors for the diagnosis of malignancy. However, lack of nuclear atypia in cases of non-high-grade ductal carcinoma (NHGDC) might diminish the diagnostic utility of these criteria. [7] [8]

In the present study, cases that were difficult to diagnose definitively by FNAC were collected and their cytopathological characteristics were re-examined. The results were analyzed using multivariate analysis to determine sets of parameters on FNAC which will be useful in differential diagnosis and a scoring system using these factors was formulated.


Materials and Methods

This study included 5682 patients who underwent FNAC for examination of breast lesions at our institute from August 2005 to October 2010. After aspiration of the breast lesion, samples were spread on glass slides, fixed in 95% ethanol and stained according to Papanicolaou's method. Out of 5682 patients, 174 (3.1%) and 205 (3.6%) patients were considered difficult to diagnose or were suspected of malignancy, respectively. The cytology slides from these patients were retrospectively reviewed. Forty-two cases were defined as being suitable for definitive diagnosis, and 246 specimens were considered inadequate for diagnosis. Twenty-eight patients were diagnosed with tumors with special histological features, such as invasive lobular carcinoma, mucinous carcinoma, and stromal sarcoma, and they were excluded from the analysis. Finally, 63 cases were enrolled for the present study. The definitive histopathological diagnosis was breast cancer in 42 cases and others in 21 cases (Table 1).

The slides were re-examined to classify the following 12 categories: dissociation of cells, cell size, cell uniformity, prominent nucleoli, nuclear margins, chromatin pattern, overlapping pattern of cells, presence of necrosis, presence of mucin, appearance of cells, appearance of bipolar bare nuclei and appearance of foam cells (Table 2). The former six categories were determined according to the criteria by Robinson et al. [10] (Figure 1). Overlapping pattern of cells was determined as follows: cellular structure was determined as regulated (Figure 2A), or deregulated (Figure 2B), then overlapping pattern was determined as score 1 (mostly regulated), score 2 (combined regulated and deregulated), or score 3 (mostly deregulated). Representative cases with myoepithelial cells, bipolar bare nuclei and foam cells are shown in figure 3.

For nuclear characteristics, slides were carefully examined using a 100× objective lens. The results were statistically examined for their diagnostic utility using the χ2 test and Fisher's exact probability test. Then, a logistic regression model was applied to clarify independent factors.


Results

The relationship between cytological parameters is summarized in table 3 and the statistical significance of each parameter for the diagnosis in table 4. The following factors were shown to be statistically significant for detection of malignancy: chromatin pattern (p < 0.05: score 1 vs. score 2); nuclear margins (p < 0.01: score 1 vs. score 2); overlapping pattern of cells (p < 0.01: scores 1+2 vs. score 3); appearance of myoepithelial cells (p < 0.0001: score 1 vs. scores 2+3, scores 1+2 vs. score 3); and appearance of bipolar bare nuclei (p < 0.0001: score 1 vs. scores 2+3). Multivariate analysis revealed that three factors were independently significant: overlapping pattern of cells (scores 1+2 vs. score 3); and appearance of myoepithelial cells (score 1 vs. scores 2+3 and scores 1+2 vs. score 3) (table 4).

Using these three parameters that were significant in multivariate analysis, a grading system for FNAC samples was established. Each case was scored by appearance of myoepithelial cells (0–2) and overlapping pattern of cells (0 or –1), then all the scores were added together. There were 21 cases with a score of –1 (all malignant), 24 with 0 points (19 malignant and five benign), 14 cases with 1 point (two malignant and 12 benign), and four cases with 2 points (all benign) (figure 4).


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Table 1: AbdominalHistological diagnosis of the 62 breast lesions.



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Table 2: Criteria for cytological classification of breast lesions.



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Table 3: Univariate analysis of each cytological category for the differentiation between benign and malignant lesions.



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Table 4: Multivariate analysis of categories for the differentiation between benign and malignant lesions.



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Figure 1: Representative pattern of cytological characteristics: A, B) Dissociation of the cells (A: Score 1, 10x, B: Score 2, 10x), C, D) Size of the cells (C: Score 1, 100x, D: Score 2, 100x), E, F) Cellular uniformity (E: Score 1, 100x, F: Score 2, 100x), G, H) Nucleoli (G: Score 1, 100x, H: Score 2, 100x), I J) Nuclear margin (I: Score 1, 100x, J: Score 2, 100x), K, L) Pattern of chromatin (K: Score 1, 100x, L: Score 2, 100x). (All slides were stained by Papanicolaou's method.)



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Figure 2: Overlapping pattern of the cells: A) Regulated, 20x, B) Deregulated, 20x (Stained by Papanicolaou's method).



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Figure 3: Arrows shows, A) Appearance of myoepithelial cells 40x), B) Bare bipolar nuclei (10x), and C) Foam cells (10x). All slides stained by Papanicolaou's method.



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Figure 4: Stratification of breast lesion with the combination of appearance of myoepithelial cells (0, 1, 2) and overlapping pattern of the cells (0, -1).



Discussion

It is clear that the presence or absence of nuclear atypia is the core diagnostic factor in the differential diagnosis of breast FNAC samples. [6] [11] [12] However, the presence of breast cancer without obvious nuclear atypia, namely NHGDC, is a confusing event in the diagnostic process. [11] The present study was designed to detect factors useful in distinguishing NHGDC from its benign counterparts. We showed that combination of two factors was useful in the differentiation between malignant and benign lesions.

Lim et al. have found that no morphological criteria, such as cellularity, nuclear enlargement, and discohesion, were useful for differentiation between benign and malignant lesions in FNAC of breast tissue. [12] Kumarasinghe et al. have concluded that cell discohesion, bare atypical nuclei, bipolar bare nuclei, and necrosis are discriminating features between NHGDC in situ and benign breast lesions. [7] The present study showed that chromatin pattern, nuclear margins, overlapping pattern of cells, appearance of myoepithelial cells and appearance of bipolar bare nuclei were significant in distinguishing between malignant and benign lesions, although the discriminating utility of each factor was limited, which is consistent with previous reports. [7] [11] [12]

Nuclear atypia is a generic term for several characteristics that indicate malignancy, such as nuclear size, nucleus to cytoplasm size ratio, nuclear pleomorphism, nuclear membrane morphology, presence of nucleoli and pattern of chromatin. [13] [14] Nucleus to cytoplasm size ratio is not commonly used for diagnosis of breast lesions stained with Papanicolaou's method as it is usually increased in non-cancerous ducts because their cytoplasm is fragile and destroyed. All the specimens in the present study had small monomorphic nuclei and no difference was observed between the specimens. Some specimens had prominent nucleoli, irregular nuclear margins and granular chromatin. Among these, most specimens with irregular nuclear margins and granular chromatin were breast cancer and these two factors proved to be significant in differential diagnosis.

Structure of the cell cluster was examined in the following two categories: dissociation of the cells and overlapping pattern of the cells. We showed that overlapping pattern of the cells was useful in differential diagnosis. Lack of cell-to-cell cohesion is considered to be characteristic of malignant lesions, [6] but the present study demonstrated that a considerable percentage of benign lesions contain dissociated cells or small clusters and therefore the utility of cell cluster size in differential diagnosis is limited. However, deregulated overlapping was observed in most cases of breast cancer, thus it could be a useful marker.

Presence of necrosis and mucin are important indicators of malignancy; [6] however, these findings were not observed in the present series.

Coexistence of myoepithelial cells and bipolar bare nuclei are useful markers that are suggestive of benign breast lesions. [6] [12] However, two of 21 benign lesions did not have myoepithelial cells. Even in benign breast lesions, lack of biphasic (epithelial and myoepithelial) cell pattern was observed in the specimen when excess proliferation of epithelial cells compared with myoepithelial cells developed. Bipolar bare nuclei are round or oval cells with faint cytoplasm, are often found in fibrous adenoma and originate from myoepithelial cells. [15] It has been demonstrated that fibroblasts also appear as spindle cells with bare nuclei. [16] Fibroblasts are observed in benign and malignant breast lesions, especially breast cancer with invasion. [16] The former (benign lesions) show myoepithelial characteristics, confirmed by immunoreactivity to p63 but the latter (malignant lesions) do not. [15] Nevertheless, morphological differentiation of the two on slides stained with Papanicolaou's method is difficult. [16] In the present study, two cases of breast cancers had bipolar bare nuclei, both of which proved to be invasive ductal carcinoma. Foam cells are found both in benign and malignant lesions; however, their appearance is not so common in malignant lesions. In the present study, marked foam cell appearance was observed in four cases, three of which were benign. Because of the small number of cases, this factor was not a significant factor in univariate analysis.


Conclusions

In the present study, several factors proved useful in the differential diagnosis of breast lesions, but none of these were found to be necessary or sufficient for differential diagnosis. Breast lesions without obvious nuclear atypia, necrosis and mucin are difficult to diagnose; therefore, diagnosis should be made by observation of several independent characteristics. The present scoring system showed that appearance of myoepithelial cells and overlapping of cells were useful for diagnosis of breast lesions. Application of the scoring system to future breast lesions might clarify the usefulness of the system.


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Author Contributions:
Ayumi Ryu - Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Critical revision of the article
Akemi Takenaka - Drafting the article, Critical revision of the article
Shigenori Nagata - Drafting the article, Critical revision of the article
Yasuhiko Tomita - Drafting the article, Critical revision of the article, Final approval of the version to be published
Guarantor of submission:
The corresponding author is the guarantor of submission.
Source of support:
None
Conflict of interest:
Authors declare no conflict of interest.
Copyright:
© Ayumi Ryu et al. 2013; This article is distributed the terms of Creative Commons Attribution License which permits unrestricted use, distribution and reproduction in any means provided the original authors and original publisher are properly credited. (Please see Copyright Policy for more information.)



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