Cytomorphology of Fibrocystic Change, High-Risk Proliferative Breast Disease, and Premalignant Breast Lesions

Clin Lab Med 25 (2005) 713–731

Cytomorphology of Fibrocystic Change, High-Risk Proliferative Breast Disease, and Premalignant Breast Lesions Shahla Masood, MD University of Florida Health Science Center, Department of Pathology, 655 West Eighth Street, 1st Floor, Clinical Center, Jacksonville, FL 32209, USA

It has been known for many years that women who have undergone a breast biopsy for so-called ‘‘fibrocystic disease’’ have an increased risk of breast cancer [1,2]. So attempts have been made to classify the spectrum of fibrocystic disease into reproducible and prognostically relevant categories. These studies have focused on the relative risk of subsequent development of breast disease in patients who have fibrocystic disease, based on the histological features in their biopsies. These efforts have resulted in introducing a new terminology of nonproliferative breast disease, proliferative breast disease without atypia, and proliferative breast disease with atypia (atypical hyperplasia); and in defining the specific histologic changes associated with each category [3–6]. It is generally agreed that nonproliferative breast disease carries no increased risk. Proliferative breast disease without atypia has slightly increased risk (1.5–2 times), and proliferative breast disease with atypia has moderately increased risk (4–5 times) for subsequent development of breast cancer. Patients who have carcinoma in situ have 8 to 10 times the risk of ultimate development of breast cancer [7,8]. This information has given a new significance to the recognition of the spectrum of morphological changes seen in breast disease. It is now possible to separate breast lesions into benign, premalignant, and malignant categories. The precise histological features of these breast lesions have been wellcharacterized [9–11]; however, despite the reliability of breast fine needle aspiration biopsy (FNAB) in separating benign from malignant lesions, the cytological features of premalignant lesions have not yet been well-defined. With interest in the use of FNAB for the diagnosis of breast lesions, and the increasing emphasis on breast cancer prevention, it is almost essential to

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differentiate low-risk benign changes from those known to be associated with a high risk of subsequent malignancy, because this information may have an important clinical implication [12,13]. Using the same diagnostic terminology for breast FNAB as is commonly used in diagnostic histopathology would also offer a significant advantage if it could be accurately applied. In a prospective study using mammographically guided fine needle aspirates in 100 nonpalpable breast lesions, the author’s group assessed the reliability of a cytological grading system to define the cytological features of proliferative and nonproliferative breast disease, and to differentiate between benign, premalignant, and malignant breast lesions. We developed a cytological grading system evaluating the aspirates for the cellular arrangement, the degrees of cellular pleomorphism and anisonucleosis, the presence of myoepithelial cells and nucleoli, and the status of the chromatin pattern. Values ranging from 1 to 4 were assigned to each criterion, and a score based on the sum of the individual values was calculated for each case. With scores ranging from a minimum of 6 to a maximum of 24, the cases were divided into nonproliferative breast disease without atypia (score 6–10), proliferative breast disease without atypia (score 11–14), proliferative breast disease with atypia (score 15–18), and cancer (score 19–24). Comparing the cytologic interpretation to the reported histologic diagnosis obtained from needle localization biopsies, we found a high degree of concordance between the results (Tables 1–3). We believe that by using strict cytological criteria, it may be possible to define the continuous spectrum of changes in breast lesions and separate hyperplasia from neoplasia. This grading system, now recognized as the Masood Cytology Index, is commonly used as a surrogate end point biomarker in chemoprevention trials [12,13]. Cytomorphology of high-risk proliferative breast disease and premalignant breast disease Nonproliferative breast disease The cell yield in these aspirates is variable and depends on the nature of the lesion. In noncystic lesions, the aspirate is scanty or moderate. Frequently the aspirate consists of clusters of monotonous, small, uniform-appearing epithelial cells arranged in monolayered sheets with a honeycomb pattern. Foam cells, apocrine cells, single naked cells, and fragments of stromal cells are frequently observed. The cells have regular nuclei with fine chromatin pattern. Nucleoli are not commonly seen. Myoepithelial cells are easily identified (Fig. 1). Proliferative breast disease without atypia Proliferative breast disease differs from nonproliferative breast disease in its higher cell yield and unique cellular arrangement. The cellularity is

Cellular arrangement

Cellular pleomorphism

Myoepithelial cells

Anisonucleosis

Nucleoli

Chromatin clumping

Scorey

Monolayer Nuclear overlapping Clustering Loss of cohesion

Absent Mild Moderate Conspicuous

Many Moderate Few Absent

Absent Mild Moderate Conspicuous

Absent Micronucleoli Micro and /or rare macronucleoli Predominatly micronucleoli

Absent Rare Occasional Frequent

1 2 3 4

y Total score: nonproliferative breast disease, 6–10; pro-liferative breast disease without atypia, 11–14; proliferative breast disease with atypia, 15–18; and carcinoma in situ and invasive cancer, 19–24. Reproduced with permission from: Masood S, Frykberg ER, McLellan GL, et al. Prospective evaluation of radiologically detected fine needle aspiration biopsy of nonpalpable breast lesions. Cancer 1990;66:1482.

CYTOMORPHOLOGY OF FIBROCYSTIC CHANGE

Table 1 Cytologic criteria/grading system for interpretation of mammographically guided fine needle aspiration biopsies

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Table 2 Cytologic findings compared with histologic diagnosis in 100 mammographically suspicious cases Histologic diagnosis

Cytology

No. Proliferative Carcinoma of Nonproliferative without Proliferative in situ Invasive cases breast disease atypia with atypia (LCIS, DCIS) cancer

Insufficient 9 cellular material Nonproliferative 34 breast disease Proliferative 17 without atypia Proliferative 23 with atypia Carcinoma 17 Total 100

7

2

-

-,-

-

29

4

-

1a,-

-

-,-

-

-

15

2

-

-

21

1a,1a

-

36

21

23

-,5 2,6

12

Abbreviations: DCIS, ductal carcinoma in situ; LCIS, lobular carcinoma in situ. a False-negative cytologic interpretations. From Masood S, Frykberg ER, McLellan GL, et al. Prospective evaluation of radiologically detected fine needle aspiration biopsy of nonpalpable breast lesions. Cancer 1990;66:1483; with permission.

moderate to high, depending on the degree of proliferative epithelial changes. There are increased numbers of tightly cohesive groups of ductal epithelial and myoepithelial cells, with some overriding of the nuclei, occasional loss of polarity, and some variability in the nuclear size. Micronucleoli may be seen. Cytological atypia is inconspicuous. Apocrine cells, histiocytes, and occasional naked nuclei are the accompanying cells in these aspirates (Fig. 2). Cytological differentiation between proliferative breast disease versus fibroadenoma or papillary lesions may occasionally be difficult. Using the criteria of cellularity, fronds, naked nuclei, and stromal fragments, Linsk and colleagues [14] and Bottles and coworkers [15] found overlapping cytological features between these two entities. The author’s group agrees with Table 3 Concordance between cytologic evaluation and histologic diagnosis in 100 mammographically guided fine needle aspirations Diagnosis

No. of cases

Concordance (%)

Nonproliferative breast disease Proliferative breast disease without atypia Proliferative breast disease with atypia Cancer

29/34 15/17 21/23 17/20

85 88 91 85

Modified from Masood S, Frykberg ER, McLellan GL, et al. Cytologic differentiation between proliferative and nonpro-liferative breast disease in mammographically guided fine-needle aspirates. Diagn Cytopathol 1991;7:583; with permission.

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Fig. 1. An example of nonproliferative breast disease (Masood Cytology Index of 6). (A) Direct smear representing low cellular aspirate showing benign-appearing ductal epithelial cells with associated stromal connective tissue (Pap stain
200). (B) Another view demonstrating monolayered collection of epithelial/myoepithelial cells with a cluster of apocrine metaplastic cells (Pap stain
400). (C) A higher view of apocrine neoplastic cells (Pap stain
600). (D) The corresponding histopathology (H & E stain
400).

Bottles and his associates that the presence of stroma defined as ‘‘clean, clearly demarcated fibrillary clusters of spindle cells’’ is the most important differentiating feature. The presence of abundant number of naked nuclei is in favor of fibroadenomas. Lack of hemosiderin-containing macrophages and absence of polymorphic population of tall columnar cells and ductal cells forming papillae are the features that may distinguish papillary breast lesions from proliferative breast disease. Proliferative breast disease with atypia (atypical hyperplasia) The cellular aspirates are frequently rich and are composed of multiple clustering of epithelial cells. The crowded clusters of cells show conspicuous loss of polarity and overriding of the nuclei. Nucleoli are present and display irregular and coarse chromatin pattern. Variation in nuclear size and cellular pleomorphism are also present. Within the crowded atypical epithelial cells, there is morphological evidence of myoepithelial cell differentiation (Fig. 3). Cytomorphology of malignant lesions Malignant lesions are characterized cytologically by a loosely arranged cellular pattern and isolated single cells. Rich cellularity, pleomorphism, and significant numbers of isolated single cells are frequent findings. Nuclear

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Fig. 2. An example of proliferative breast disease without atypia (Masood Cytology Index of 12). (A) Direct smear of a cellular aspirate (Pap stain
200). (B) Clusters of epithelial/myoepithelial cells showing crowding and overriding of nuclei (Pap stain
400). (C) Another view demonstrating the cell population of larger cells of epithelial origin intermixed with thinner, smaller, and darker myoepithelial cells (Pap stain
400). (D) The corresponding histopathology (H & E stain
200).

membrane abnormality, chromatin clumping, and macronucleoli are often present. Myoepithelial cells are absent. The cytological features are common in both carcinoma in situ and in invasive lesions, and cytological differentiation between these two entities is somewhat difficult. Atypical hyperplasia versus neoplasia As previously mentioned, the importance of recognizing atypical hyperplasia cannot be overemphasized, because of the substantial risk it represents [7,8]. This entity, however, presents a diagnostic challenge for the cytopathologist. The term ‘‘atypical hyperplasia’’ has been used to define noninfiltrating breast lesions with some, but not all, of the features of cancer. Thus atypical hyperplasia occupies an intermediate position between benign and malignant lesions. There may be a substantial degree of subjectivity and disagreement in making this diagnosis from histologic section. It is therefore reasonable to expect some difficulty in cytologically differentiating this from carcinoma or from benign changes without atypia; however, such differentiation is important in terms of the very different clinical management each of these diagnoses entails. The value of the author’s grading system lies in its potential for providing an objective and reproducible method for such diagnosis that can augment the pathologist’s judgment and presumably enhance the overall diagnostic accuracy.

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Fig. 3. An example of proliferating breast disease with atypia/atypical ductal hyperplasia (Masood Cytology Index of 16). (A) Crowded cluster of epithelial/myoepithelial cells with conspicuous overriding of the nuclei forming various sized slits and openings (Pap stain
400). (B) Another view demonstrated by presence of spindle cells of myoepithelial cells (Pap stain
400). (C) Another view showing loss of polarity and nuclear atypia with irregularly shaped openings (Pap stain
400). (D) The corresponding histopathology (H & E
600).

In the study conducted by the author’s group, nonproliferative breast disease scored 6 to 10, and in the majority of cases did not create any diagnostic difficulty. Monolayered cell arrangement and lack of cytological atypia were the predominant features seen in these lesions. Our missed diagnosis of lobular carcinoma in situ was most likely due to inaccurate localization and sampling, because lobular carcinoma in situ is often an incidental finding located outside the area of mammographic abnormality [16]. Furthermore, lobular carcinoma of the breast, particularly the noninvasive variety, is difficult to diagnose by cytologic examination because of its nonomorphic and well-differentiated appearance [17,18]. Proliferative breast disease was characterized by the presence of a cellular aspirate. Tightly packed, crowded appearances of clusters of epithelial cells with obvious overriding of the nuclei were the landmarks of this entity. There was a discrepancy between histology and cytology in two cases in which the degree of atypia in cytologic grading was underestimated. This probability should always be considered in cytologic diagnosis, and may be the result of sampling error. Proliferative breast disease with atypia presented with: (1) crowded threedimensional cell clusters, (2) moderate cellular pleomorphism and anisonucleosis, (3) occasional chromatin clumping and frequent micronucleoli, and (4) few myoepithelial cells. Two cases of carcinoma in situ found on open biopsy were missed by FNAB. In these cases, a review of cytologic samples

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revealed myoepithelial cells, suggesting that the sample did not, in fact, include the malignant lesion. In the study conducted by the author’s group, increased cellularity, loss of cellular cohesion, significant pleomorphism, anisonucleosis, and coarse chromatin pattern were the predominant features seen in malignant lesions. Macronucleoli, when present, were associated with malignancy, but their absence did not indicate benignity. The presence of myoepithelial cells in the smears was quite significant. Myoepithelial cells were absent in all cases of malignancy; however, they were found in nonproliferative as well as proliferative breast disease and atypical hyperplasia. Micronucleoli were commonly seen in benign and malignant lesions, and could not be used as a reliable indication of malignancy. There were no false-positive results in this study, which is generally considered to be the most important error to avoid, especially if definitive therapy is to be undertaken solely on the basis of cytology. Also, no invasive lesion was missed by cytologic diagnosis. Our group’s study further supports the findings of the others. Dziura and Bonfiglio in 1979 [19] described cell changes in ductal neoplasia samples obtained by means of Nordenstrom screw technique. Similarly, they found anisonucleosis, the presence of macronucleoli, chromatin clumping, marked nuclear overlap, and cellular disarray as cellular indicators of malignancy. Only limited predictability of the degree of hyperplasia was possible with their morphologic criteria, however. The study conducted by the author’s group differed from theirs in two ways. In their experimental design, samples were obtained from already excised lesions, and they used the screw technique of Nordenstrom. The author’s group used FNAB technique and obtained the samples on site. By far the most important aspect of our study is the ability of the cytological grading system to define the continuous changes in breast lesions and to separate hyperplasia from neoplasia. This is also the first study to advocate the use of the same diagnostic terminology in cytology as used in histology. This grading system should be further challenged in the interpretation of breast fine needle aspirates by other investigators to assess its reproducibility. In a follow-up study [20], the author’s group used this cytologic grading system in evaluation of palpable breast lesions in 156 consecutive FNAB. Follow-up histology was available in 146 cases. The interpretation of FNAB was performed by four pathologists in the author’s institution who have become familiar with the grading system through personalized training. Given the heterogeneity of sampling in palpable FNAB, particularly in larger size tumors, there was a diagnostic accuracy of 95%. Difficulty remained in differentiation between fibroadenoma and papillary lesions versus proliferative breast disease. Considering the pertinent differentiating features in these cases, there were still cases that were missed. There were six cases of in-situ and invasive carcinomas that were misinterpreted as atypical

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hyperplasia. Review of these cases demonstrated absence of cellular dyshesion commonly seen in the usual mammary neoplasia. Furthermore, these tumors were low grade. Similar limitations have been observed in a study conducted by Sneige and Staerkel [21]. They evaluated our grading system in a retrospective study of low-grade, in-situ lesions of the breast. They believe that our cytologic grading system should be complemented by incorporating architectural features such as cribriform or micropapillary patterns in differentiation between atypical hyperplasia and noncomedo-type carcinoma in situ. Silverman and colleagues [22] and Abnedroth and colleagues [23] believe that separation between various stages of proliferative breast disease is still problematic, and they refrain from making a definitive diagnosis of atypical hyperplasia and carcinoma in situ on fine needle aspirates. They believe, however, that in selected cases separation between ductal carcinoma in situ and atypical hyperplasia is possible. They recognize atypical hyperplasia when cells are arranged in flat cohesive sheets with distinct cell borders and myoepithelial cells. In contrast, ductal carcinoma in situ presents with single cells constituting more than 10% of atypical cells, cellular dyshesion, an inflammatory background, coarsely granular chromatin, and nuclear pleomorphism. Shiels and coworkers [24] studied the cytomorphology of 15 cases of ductal carcinoma in situ. The absence of myoepithelial cells, associated with a cellular smear, with conspicuous nuclear overriding, and with atypia are considered by these authors to be the most important cytological features differentiating ductal carcinoma in situ from atypical hyperplasia. Presently, most investigators advocate an intraoperative consultation or an excisional biopsy for any case diagnosed as atypical hyperplasia, or as suspicious or inconclusive for carcinoma [13,22,25]. Regardless of the experience and the promising reports in the literature defining various cytomorphology, practitioners need to recognize some of the limitations of FNAB to the study of breast disease. This may not necessarily be the result of inability to recognize different entities. The presence of heterogeneity of individual lesions is an important factor to be considered. As an example, in a study conducted by Lennington and colleagues in 1994 [26], the study authors reviewed 100 sequentially collected ductal carcinoma in situ cases from a consultation practice. Recognizing the bias of such a series toward exclusion of easily recognizable comedocarcinomas, the researchers studied the spectrum of mixed pattern lesions to identify variations and common features in the architectural arrangement of the various histologic patterns. Interestingly, atypical ductal hyperplasias were intermixed in 17 cases of ductal carcinoma in situ. Mixed patterns of comedo and noncomedo-type ductal carcinoma in situ were seen in 33 cases. In all the cases of combined atypical ductal hyperplasia and ductal carcinoma in situ, the more advanced patterns of ductal carcinoma in situ were seen in the central portion of the lesion, whereas the atypical ductal hyperplasia components were arranged

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peripherally. Thus the presence of different patterns of ductal carcinoma in situ within individual lesions (46 out of 100) and the coexistence of atypical ductal hyperplasia and ductal carcinoma in situ (17 out of 100) strongly supported the presence of heterogeneity within a lesion. Similarly, if adequately sampled, the spectrum of morphological alterations of fibrocystic change, proliferative breast disease, and atypical hyperplasia are commonly seen in association with carcinoma in situ or invasive breast lesions. Multiple sampling of a breast lesion by FNAB may overcome the problem of heterogeneity of individual lesions to some extent; however, this remains a limiting factor in interpretation of FNAB, and consequently in further management of patients. Aside from morphology, attempts have been made to use ancillary studies to distinguish between atypical hyperplasia and carcinoma in situ. The author’s group has already used cell image analysis to assess the DNA ploidy pattern of our breast fine needle aspirates to differentiate between atypia and neoplasia. Although the frequency of aneuploidy was higher in carcinoma (59%), the presence of aneuploidy in 28% of the cases of atypical ductal hyperplasia limited the used of this technology. Similar experiences are reported by Crissman and coworkers [27], who found that aneuploidy is more common in ductal carcinoma in situ, although 36% of atypical ductal hyperplasia were aneuploid. Aneuploidy was further correlated with poor nuclear grading. Teplitz and colleagues [28] reported concordance in ploidy values of atypical ductal hyperplasia and concurrent carcinoma. Norris and coworkers [29] also used cell image analysis for DNA ploidy study as well as nuclear measurements in breast lesions. These authors concluded that ‘‘Thus, the difficult distinction of atypical hyperplasia from well differentiated intraductal carcinoma by light microscopy was not aided by DNA analysis or by nuclear measurements.’’ In a similar study conducted by King and colleagues [30] using 12 nuclear parameters, the study authors were able to correctly classify six of seven cases of moderate and atypical ductal hyperplasia as abnormal. Moderate hyperplasia, however, fell into the spectrum of usual intraductal hyperplasia without atypia. This image analysis study also had a low sensitivity for malignancy, ascribed by the study authors to a large percentage of in situ carcinoma. In a follow-up study, the same authors found that image cytometry could not distinguish atypical hyperplasia from intraductal hyperplasia without atypia or ductal carcinoma in situ, although it could separate atypical ductal hyperplasia from invasive carcinoma and nonproliferative lesions [31]. Thus, neither intra-active morphometry nor DNA ploidy study is likely to assist in differentiating atypical ductal hyperplasia from ductal carcinoma in situ on cytologic specimens. It was also intriguing to imagine that the study of oncogene expression may aid in the differentiation between atypical hyperplasia and carcinoma in situ; however, there is no support for the use of oncogene study as an adjunct to differentiate hyperplasia from neoplasia [32].

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Among various antibodies, monoclonal antibody B72.3 has received most attention as an adjunct in cytology. Monoclonal antibody B72.3 demonstrates selective reactivity for carcinoma cell over normal human adult tissue [33]. This is the basis of an immunocytochemical approach employing the antibody to stain breast fine needle aspirates [34]. Monoclonal antibody B72.3 obtained by standard hybridoma technology is directed against a membrane protein of human breast cancer [35]. Using this immunoperoxidase technique on 50 breast fine needle aspirates, Lundy and coworkers [36] reported a 10% increase in their diagnostic accuracy. The author’s group used monoclonal antibody B72.3 in 52 of our breast fine needle aspirates. The results of this study demonstrated a diagnostic accuracy of 96%, sensitivity of 94% and specificity of 100% [37]. Despite these promising reports, the use of monoclonal antibody B72 as an aid in differentiation between atypical hyperplasia and carcinoma has remained limited. This is mainly due to scattered reports of false-positive immunostaining seen in fibrocystic changes and apocrine cells [38]. The author’s group believes that, morphologically, the usual pattern of fibrocystic change and apocrine cells are recognizable. The validity of the use of MAB B72.3 should only be exclusively limited in differentiation between atypical hyperplasia and carcinoma. Muscle specific actin (MSA) immunostaining is potentially another marker that can be used to differentiate between atypical hyperplasia and carcinoma in situ. Actin is a contractile protein that is present in smooth and striated muscle, in pericytes, and in myoepithalial cells of salivary glands, breast, and sweat glands [39]. Papotti and colleagues [40] used MSA immunostianing to differentiate between benign and malignant papillary lesions of the breast. In a similar study, Raju and coworkers [41] demonstrated the lack of MSA immunostaining for myoepithelial layer is strong evidence in favor of papillary carcinoma. The author’s group has demonstrated the value of MSA in differentiation between atypical hyperplasia and carcinoma in situ in surgical cases [42]. In these studies, MSA was found to be a specific marker for detecting myoepithelial cells. Aside from the persistence of clustering and cellular cohesion and absence of significant numbers of isolated single cells, we believe that the presence of myoepithelial cells within the clusters of atypical cells in a breast aspirate is a significant finding, and can separate hyperplasia from neoplasia [12,13,22]. Based on these observations, The author’s group has assessed the feasibility of the use of MSA as a marker for myoepithelial cells in aspirated smears and cell block preparations obtained from breast fine needle aspirates [43,44]. The observed staining of myoepithelial cells in benign and highrisk proliferative breast disease can be used as a strong differentiating feature in interpretation of atypical breast fine needle aspirates. This will potentially maximize the diagnostic accuracy of fine needle aspiration biopsies and help to reduce the number of inconclusive cytologic diagnoses.

724

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Our study further confirms that the morphologic distinction between myoepithelial cells and spindle cells of nonmyoepithelial origin is not always possible. Lack of immunostaining for MSA is strong evidence against the presence of myoepithelial cells. Muscle-specific, actin-positive myoepithelial cells may also be seen in the background of malignant breast lesions. This may occur during passage of the needle through the benign component of a malignant breast lesion. To summarize, based on the available information in selected cases, the differentiation between atypical hyperplasia and carcinoma may be possible; however, in clinical practice, recognition of atypical hyperplasia requires tissue confirmation. Ductal carcinoma in situ Ductal carcinoma in situ presents with variable cytologic features, which reflects its morphologic diversity. Divided into comedocarcinoma and noncomedocarcinoma, there are distinct cytological differences between the two entities. Comedocarcinoma It has been recognized for many years that in addition to the high-grade nuclear morphology, comedo ductal carcinoma in situ (DCIS) has a higher proliferative rate by cell kinetic study and is more frequently associated with microinvasion [45–49].Comedo DCIS is known to have a higher local recurrence rate than other types of DCIS, frequently expresses HER-2/neu oncogenes, and is often aneuploid [50–52]. In addition to nuclear grade, comedo-type necrosis contributes to the identification of DCIS that is more likely to produce local failure after excision. Thus, it appears that comedocarcinoma represents a unique type of DCIS that is most closely related to invasive carcinoma [52]. In the experience of the author and colleagues, FNAB of comedo DCIS is usually cellular, and displays loosely cohesive clusters of malignant cells with individual cell necrosis and mitosis. Nuclear membrane abnormality, clumping of chromatin, and conspicuous nucleoli are often present. Nuclear pleomorphism and irregularly shaped nucleoli are characteristic features of comedo DCIS. Microcalcified particles may or may not be present. Similarly, Moriya and colleagues [53] found that comedo DCIS revealed necrosis, hypercellularity, marked nuclear atypia, and loss of cellular cohesion. In contrast, noncomedocarcinomas were characterized by minimal nuclear atypia, lack of single cells, predominance of benign elements, or scant cellular yield. Malamud and coworkers [54] showed that comedo DCIS is more likely to be diagnosed as positive on FNAB than is noncomedo DCIS. These authors suggest that nuclear features are the most helpful differentiating features between the two types of carcinoma in situ. Large, irregularly shaped nucleoli are the most characteristic feature for comedo lesions (Fig. 4).

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Fig. 4. High nuclear grade ductal carcinoma in situ (Masood Cytology Index of 22). (A) Direct smear cytology exhibiting clusters of pleomorphic epithelial cells with evidence of necrosis (Pap stain
400). (B) Higher view of the same case showing extreme pleomorphism (Pap stain
600). (C) Another view of the same case displaying the presence of macronucleoli (Pap stain
600). (D) The corresponding histopathology (H & E stain
600).

The presence of comedo DCIS in fine needle aspirates often indicates a comedo DCIS with or without associated invasion. Lilleng and Hagman [55] compared invasive comedocarcinoma with minimally invasive and insitu lesions. In this study, the authors found that in-situ and minimally invasive comedocarcinoma could be separated morphologically from comedo DCIS. Minimally invasive and in-situ lesions demonstrated a background of necrotic cellular debris and tumor cells. In contrast, the invasive comedocarcinoma revealed numerous single scattered tumor cells intermingled with the cell clusters, and in most cases, a background with inconspicuous or no necrotic material. In the experience of the author’s group, stromal infiltration defined by strict criteria may predict the status of tumor invasion in breast fine needle aspirates [56] (Masood et al, submitted for publication, 2005). The interpretation of fine needle aspirates for diagnosis of the high risk and premalignant breast lesions pose a diagnostic dilemma for cytopathologists. Although it has been suggested that DCIS may be separated from atypical ductal hyperplasia [22], other evidence indicates that distinguishing between DCIS and infiltrating carcinoma is virtually impossible [57]. This latter pitfall is not associated with significant clinical implication for malignant palpable breast masses, because most are infiltrating carcinoma. Palpable DCIS often represents extensive disease, for which mastectomy is considered the appropriate therapy. This difficulty, however, is most pertinent for nonpalpable breast lesions, because a considerable percentage of these are small and are in situ, and may not require mastectomy [58].

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Therefore, attempts should be made to identify comedocarcinoma, the type of DCIS that requires more aggressive therapy. Noncomedo ductal carcinoma in situ Aspirates from noncomedo type DCIS vary in cellularity and are characterized by a monomorphic cell population of small- to medium-sized epithelial cells arranged singly or as loosely cohesive clusters. The cell clusters may have a solid, cribriform, or papillary pattern with no accompanying myoepithelial cells present. This is in contrast with atypical hyperplasia, in which the myoepithelial cells are seen intermingled within the groups of atypical cells and appear as part of the cellular aggregate. Microcalcified particles, foamy histiocytes, and a few isolated myoepithelial cells may be seen in the background (Fig. 5). The cytological features of DCIS are not exclusive and are often seen in invasive lesions. Nevertheless, associated with specific mammographic features and defined clinical presentations, it may be possible to maximize the ability of FNAB and to suggest the diagnosis of an invasive carcinoma. This includes the recognition of cytological features of special subtypes of ductal carcinoma such as mucinous, medullary, sarcomatoid, and smallcell carcinoma, which most often present as an invasive lesion, although in-situ mucinous carcinoma and small-cell carcinoma can rarely occur.

Fig. 5. Low nuclear grade ductal carcinoma in situ (Masood Cytology Index of 19). (A) Direct smear demonstrating a monomorphic population of epithelial cells forming cribriform type patterns (Pap stain
400). (B) Another view of the same case displaying the lack of significant pleomorphism (Pap stain
600). (C) A single cluster of cells with one cell type arranged in a cribriform pattern (Pap stain
600). (D) The corresponding histopathology (H & E stain
600).

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Neoplastic cells in a breast aspirate plus the presence of skin retraction, fixed nipple, ulceration, and inflammatory carcinoma or evidence of metastasis also indicate advanced invasive breast carcinoma. Similarly, intracystic papillary carcinomas often present as a cystic lesion by mammography or sonography. The aspirates are often bloody, rich in cellularity, and contain three-dimensional cell clusters with small or large papillae and scattered single epithelial cells. Because the recommended management of DCIS may be similar to that of invasive ductal carcinoma, the separation between these two entities may not be critical at some institutions; however, the decision to perform lumpectomy versus modified radical mastectomy is often based on the size of the lesion, the extent of in-situ carcinoma, or the status of the lumpectomy margins. In addition, the separation becomes essential for those patients selected for preoperative chemotherapy or radiotherapy. In these circumstances, assessment of tumor invasion by confirmatory core needle biopsy/surgical biopsy is the recommended procedure of the author’s group. Lobular carcinoma in situ (lobular neoplasia) Lobular carcinoma in situ (LCIS) is often an incidental finding on the pathologic study of tissue removed from palpable breast lesions or by a needle localization of a mammographic abnormality [59,60]. In published series, LCIS has been found in 0.8% to 3.6% of breast biopsy specimens. A similar incidence has been reported for LCIS discovered by mammographic localization of nonpalpable breast lesions (1%–2%) [61,62]. It is important to distinguish between LCIS and infiltrating lobular carcinoma (ILC), because the management of these two entities is quite different. LCIS is now considered only a marker for the subsequent development of breast cancer, with bilateral mastectomies or close observation as management. In contrast, the treatment of ILC is similar to that of other invasive cancers. The author and colleagues are in agreement with the observation of Kline, Salhany and Page, and Sneige [21,63,64] that the cytomorphological distinction between atypical lobular (ALH), LCIS, and ILC may be difficult. Aspirates of LCIS and ALH may show loosely cohesive groups of small uniform cells with eccentric regular nuclei and occasional intracytoplasmic lumina. The nuclei are hyperchromatic, with fine chromatin clumping and occasional inconspicuous nucleoli. Occasionally, small cell groups forming ‘‘cell balls’’ conforming to acini of lobular neoplasia can be seen. Aspirates of ILC show overlapping features, but are more cellular and contain more atypical single cells. Characteristic cytomorphologic findings in ILC include low-to-moderate cell yield. The smears show a relatively uniform population of small to medium-sized cells. The cells have scanty, ill-defined cytoplasm with increased nuclear cytoplasmic ratio, and tend to occur in small groups ‘‘Indian file,’’ in cords, or singly. The cytoplasm may contain sharply punched-out vacuoles. Occasionally, signet ring forms may be seen. The

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author and colleagues believe that the cellular aspirates that contain significant numbers of small uniform cells characteristic of ILC should be diagnosed accordingly, and that treatment should proceed to definite therapy; however, patients whose aspirates are scanty in cellularity should undergo an excisional biopsy to establish the diagnosis (Fig. 6) [22]. Follow-up management It is generally agreed and commonly practiced that the diagnosis of highrisk proliferative breast disease and premalignant breast lesion obtained from FNAB or other minimally invasive procedure requires a follow-up excisional biopsy. The significance of recognition of the above-mentioned borderline breast lesions is the ability to identify those high-risk breast cancer patients who may benefit from variety of risk reduction modalities. Because the author’s group has defined the cytology of borderline breast lesions, several studies have validated the Masood Cytology Index as the only semiquantitative measure to assess the effect of chemopreventive agents. It has also been used to study the relationship between our defined morphology and the corresponding alterations associated with the pattern of expression of biomarkers and genetic profile in borderline breast lesions [65–71].

Fig. 6. An example of a lobular neoplasia/lobular carcinoma in situ (Masood Cytology Index of 18) versus an infiltrating lobular carcinoma (Masood Cytology Index of 21). (A) Direct smear of an atypical lobular hyperplasia/lobular carcinoma in situ characterized by clustering of small-sized epithelial cells with uniform nuclei (Pap stain
400). (B) Higher view of the same case features distended acini (Pap stain
600). (C) A cluster of mildly atypical epithelial cells forming Indian-file appearance (Pap stain
400). (D) The corresponding histopathology of an infiltrating lobular carcinoma (H & E stain
600).

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References [1] Emsler VL. The epidemiology of benign breast disease. Epidemiol Rev 1981;3:184–202. [2] Lov SM, Gelman RS, Silen W. Fibrocystic ‘‘disease’’ of the breastda nondisease? N Engl J Med 1982;307:1010–4. [3] Page DL, Vander Z, Weig R, et al. Relation between component parts of fibrocystic disease complex and breast cancer. J Natl Cancer Inst 61:1055–63. [4] Kodlin D, Winger EE, Morgenstern NL, et al. Chronic mastopathy and breast cancer, a follow-up study. Cancer 1977;39:2603–7. [5] Hutchinson WB, Thomas DB, Hamlin WB, et al. Risk of breast cancer in women with benign breast disease. J Natl Cancer Inst 1980;65:13–20. [6] Bach MM, Barclay THC, Cutler SJ, et al. Association of atypical characteristics of benign breast lesions with subsequent risk of breast cancer. Cancer 1972;29:338–43. [7] Page DL. Cancer risk assessment in benign breast biopsies. Hum Pathol 1986;17:871–8. [8] Dupont WD, Parl FF, Hartman WH, et al. Breast cancer risk associated with proliferative breast disease with atypical hyperplasia. Cancer 1993;71:1258–65. [9] Azzopardi JG. Benign and malignant proliferative epithelial lesions of the breast: a review. Eur J Cancer Clin Oncol 1983;19(2):1717–20. [10] Wellings SR. Development of human breast cancer. Adv Cancer Res 1980;31:287–314. [11] Ashikari R, Huros AG, Snyder RE, et al. A clinicopathologic study of atypical lesions of the breast. Cancer 1974;33:310–7. [12] Masood S, Frykberg ER, McLellan GL, et al. Prospective evaluation of radiologically detected fine needle aspiration biopsy of nonpalpable breast lesions. Cancer 1990;66:1480–7. [13] Masood S, Frykberg ER, McLellan GL, et al. Cytologic differentiation between proliferative and nonpro-liferative breast disease in mammographically guided fine-needle aspirates. Diagn Cytopathol 1991;7:581–90. [14] Linsk JA, Krunzer G, Zajicek J. Cytologic diagnosis of mammary tumors from aspiration biopsy smears. II. Studies on 210 fibroadenomas and 210 cases of benign dysplasia. Acta Cytol 1977;16:8. [15] Bottles K, Chan JS, Holly EA, et al. Cytologic criteria for fibroadenoma. A stepwise logistic regression analysis. Am J Clin Pathol 1988;89:707–13. [16] Schwartz GF, Feig SA, Patchefsky AS. Significance and staging of nonpalpable carcinomas of the breast. Surg Gynecol Obstet 1988;166:6–10. [17] Wanebo HJ, Feldman PS, Wilhelm MC, et al. Fine needle aspiration cytology in lieu of open biopsy in management of primary breast cancer. Ann Surg 1984;199:569–78. [18] Gent HJ, Sprenger E, Dowlatshahi K. Stereotaxic needle localization and cytological diagnosis of occult breast lesions. Ann Surg 1986;204:580–4. [19] Dziura BR, Bonfiglio TA. Needle cytology of the breast. Acta Cytol 1979;23:332–40. [20] Masood S, Hardy NM, Assaf-Munasifi N. Cytologic grading system in diagnosis of highrisk and malignant breast lesions. Merits and Pitfalls. Acta Cytol 1994;38:798. [21] Sneige N, Staerkel GA. Fine needle aspiration cytology of ductal hyperplasia with and without atypia and ductal carcinoma in situ. Hum Pathol 1994;25:485–92. [22] Silverman J, Masood S, Ducatman BS, et al. Can FNA biopsy separate atypical hyperplasia, carcinoma in situ, and invasive carcinoma of the breast? Cytomorphologic criteria and limitations in diagnosis. Diagn Cytopathol 1993;9(6):713–28. [23] Abendroth CS, Wang HH, Ducatman BS. Comparative features of carcinoma in situ and atypical ductal hyperplasia of the breast on fine needle aspiration biopsy. Am J Clin Pathol 1991;96:654–9. [24] Dawson AE, Mulford DK, Shiels LA. The cytomorphology of proliferative breast disease. Am J Clin Pathol 1995;103(4):438–42. [25] Stanley MW, Henry-Stanley MJ, Zera R. Atypia in breast fine-needle aspiration smears correlates poorly with the presence of a prognostically significant proliferative lesion of ductal epithelium. Hum Pathol 1993;24(6):630–5.

730

MASOOD

[26] Lennington WJ, Jensen RA, Dalton LW, et al. Ductal carcinoma in situ of the breast: heterogeneity of individual lesions. Cancer 1994;73:118–24. [27] Crissman JD, Visscher DW, Kubus J. Image cytophotometric DNA analysis of atypical hyperplasia and intraductal carcinomas of the breast. Arch Pathol Lab Med 1990;114:1249–53. [28] Teplitz RL, Butler BB, Tesluk H, et al. Quantitative DNA patterns in human preneoplastic breast lesions. Anal Quant Cytol Histol 1990;12:98–102. [29] Norris HJ, Bahr GF, Mikel UV. Quantitative DNA patterns in human preneoplastic breast lesions. Anal Quant Cytol Histol 1990;12:98–102. [30] King ER, Chen KL, Duarte L, et al. Image cytometric classification of premalignant breast disease in fine-needle aspirates. Cancer 1988;62:114–24. [31] King ER, Chen KL, Hem JD, et al. Characterization of image cytometry of ductal epithelial proliferative disease of the breast. Mod Pathol 1991;4:291–6. [32] Masood S. HER-2/neu oncogene expression in atypical ductal hyperplasia, carcinoma in situ and invasive breast cancer. Mod Pathol 1991;4(1):12A. [33] Johnson WW, Szpak CA, Thor A, et al. Applications of immunocytochemistry to clinical cytology. Cancer Invest 1987;5(6):593–611. [34] Nuti M, Teramoto YA, Mariani-Constantini R, et al. A monoclonal antibody (B72.3) defines patterns of distribution of a novel tumor-associated antigen in human mammary carcinoma cell populations. Int J Cancer 1982;29:539–45. [35] Colchen D, Hovan Hand P, Nuti M, et al. A spectrum of monoclonal antibodies reactive with mammary tumor cells. Proc Natl Acad Sci USA 1981;78:3199–203. [36] Lundy J, Lozowski M, Mishviki Y. Monoclonal antibody B73.2 as a diagnostic adjunct in fine needle aspirates of breast masses. Ann Surg 1986;203:399–402. [37] Prey MU, Bedrossian CW, Masood S. The value of monoclonal antibody B72.3 for the diagnosis of breast carcinoma: experience with the first commercially available source. Hum Pathol 1991;22(6):598–602. [38] Lundy J, Kline TS, Lozowski M, et al. Immunoperoxidase studies by monoclonal antibody B72.3 applied to breast aspirates: Diagnostic considerations. Diagn Cytopathol 1988;4:95–8. [39] Mukai K, Schollmeyer JV, Rosai J. Immunohistochemical localization of actin. Am J Surg Pathol 1981;5:71–97. [40] Papotti M, Eusebi V, Gagliotta P, et al. Immunohisto-chemical analysis of benign and malignant papillary lesions of the breast. Am J Surg Pathol 1983;7:451–61. [41] Raju VB, Lee MW, Zarbo RJ, et al. Papillary neoplasia of the breast: immunohistochemically defined myoepithelial cells in the diagnosis of benign and malignant papillary breast neoplasms. Mod Pathol 1989;2(6):569–76. [42] Masood S, Sim SJ, Lu L. Immunohistochemical differentiation of atypical hyperplasia versus carcinoma in situ of the breast. Cancer Detect Prev 1992;16(4):225–35. [43] Masood S, Lu L, Assaf-Munasifi N, et al. Application of immunostaining for muscle specific action in detection of myoepithelial cells in breast fine needle aspirates. Diagn Cytopathol 1995;13:71–4. [44] Masood S, Assaf-Munasifi N, Hardy NM. The value of muscle specific actin immunostaining in differentiation between atypical hyperplasia and carcinoma in breast fine needle aspirates. Acta Cytol 1994;38:860–1. [45] Rosai J. Ackerman’s surgical pathology. St. Louis (MO): Mosby; 1989. p. 1219–26. [46] Carter D, Smith RRL. Carcinoma in situ of the breast. Cancer 1977;40:1189–93. [47] Meyer JS. Cell kinetics of histologic variants of in situ breast carcinoma. Breast Cancer Res Treat 1986;7:171–80. [48] Patchefsky AS, Schwartz GF, Finkelstein SD, et al. Heterogeneity of intraductal carcinoma of the breast. Cancer 1989;63:731–41. [49] Lagios MD, Westdahl PR, Margopin FR, et al. Duct carcinoma in situ. Relationship of extent of noninvasive disease to the frequency of occult invasion, multiplicity, lymph node metastasis and short-term treatment failures. Cancer 1982;50:1309–14.

CYTOMORPHOLOGY OF FIBROCYSTIC CHANGE

731

[50] Crissman JD, Visscher DW, Kubus NS. Image cytophotometric DNA analysis of atypical hyperplasia and intraductal carcinoma of the breast. Arch Pathol Lab Med 1990;114: 1249–53. [51] Bellamy COC, McDonald C, Salter PM, et al. Noninvasive ductal carcinoma of the breast. The relevance of histologic categorization. Hum Pathol 1993;24:16–23. [52] Frykberg ER, Masood S, Copeland EM, et al. Ductal carcinoma in situ of the breast. Surg Gynecol Obstet 1993;177(4):425–40. [53] Moriya T, Silverberg SG. Intraductal carcinoma (ductal carcinoma in situ) of the breast. A comparison of pure noninvasive tumors with those including different proportions of infiltrating carcinoma. Cancer 1994;74(11):2972–8. [54] Malamud YR, Ducatman BS, Wang HH. Comparative features of comedo and noncomedo ductal carcinoma in situ of the breast on fine needle aspiration biopsy. Diagn Cytopathol 1992;8:571–6. [55] Lilleng R, Hagman B. The comedo subtype of intraductal carcinoma. Cytologic characteristics. Acta Cytol 1992;36(3):345–52. [56] Klijanienko J, Katsahian S, Vielh P, Masood S. Stromal infiltration as a predictor of tumor invasion in breast fine needle aspiration biopsy. Diagn Cytopath 2004;30:182–6. [57] Wang HH, Ducatman BS, Eich D. Comparative features of ductal carcinoma in situ and infiltrating ductal carcinoma of the breast on fine needle aspiration biopsy. Am J Clin Pathol 1989;92:736–40. [58] Bradley SJ, Weaver DW, Bouwman DL. Alternatives in the surgical management of in situ breast cancer. A meta-analysis of outcome. Ann Surg 1990;56:428–32. [59] Rosen PP, Lieberman PH, Braun DW, et al. Lobular carcinoma in situ of the breast. Am J Surg Pathol 1978;2:225–50. [60] Anderson JA. Lobular carcinoma in situ: a long term follow up in 52 cases. Acta Pathol Microbiol Scand 1974;82:519. [61] Meyers J, Kopans PE, Stomper PC, et al. Occult breast abnormalities; percutaneous preoperative needle localization. Radiology 1984;150:333–7. [62] Rosenberg AL, Schwartz GF, Feig SA, et al. Clinically occult breast lesions: localization and significance. Radiology 1987;162:167–70. [63] Kline TS, Kline IK, Howell LP. Guides to clinical aspiration biopsy. Philadelphia: Lippincott Williams and Wilkins; 1999. [64] Salhany K, Page DL. Fine needle aspiration of mammary lobular carcinoma in situ and atypical lobular hyperplasia. Am J Clin Pathol 1989;92:22–6. [65] Fabian CJ, Zalles C, Kamel S, et al. Biomarkers and cytologic abnormalities in women at high and low risk for breast cancer. J Cell Biochem 1993;176 163–160. [66] Masood S, Rasty G. Potential value of cytology in the detection of breast cancer precursors by fine needle aspiration biopsy: ‘‘The future pap smear for breast cancer.’’ Acta Cytol 1999; 43(5):890. [67] Khan SA, Masood S, Miller L, et al. Random fine needle aspiration of the breast of women at increased risk and standard risk controls. Breast J 1998;4(6):4420. [68] Fabian CJ, Kimler BF, Zalles CM, et al. Short term breast cancer prediction by random periareolar fine needle aspiration cytology and the gail risk model. J Natl Cancer Inst 2000;92: 1217–27. [69] Fabian C, Kilmer B, Brady D, et al. Phase II chemoprevention trial of DFMO using the random FNA model. Breast Cancer Res Treat 2000;64(1):48. [70] Fabian C, Kimler B, Brady D, et al. A phase II breast cancer chemoprevention trial of oraldifluoromethylornithine. Clin Cancer Res 2002;8:3105–17. [71] Bean GR, Scott V, Yee L, et al. Retinoic acid reciptor-beta2 promoter methylation in random periareolar fine needle aspiration. Cancer Epidemiol Biomarkers Prev 2005;14(4): 790–8.