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Flat ductal intraepithelial neoplasia of the breast: a review of diagnostic criteria, differential diagnoses, molecular-genetic findings, and clinical relevance--it is time to appreciate the Azzopardi concept!

Recent widespread use of mammography has resulted in the increased detection of atypical and preinvasive breast lesions such as atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS). In addition, a certain type of breast lesion, which in the past has been called "clinging carcinoma in situ," (1) is currently being encountered with increasing frequency in breast biopsies. Similar to ADH and DCIS, these lesions are commonly associated with abnormal mammographic findings, particularly suspicious microcalcifications. (2) Unlike ADH and DCIS, however, these lesions lack significant intraepithelial proliferation and mainly show cytologic abnormalities rather than architectural alterations. Therefore, these "clinging" or flat lesions can easily be overlooked by histopathologic examination. (1)

The aim of this review article is to discuss (1) historical aspects, (2) histomorphologic features, (3) differential diagnoses, (4) recent molecular-genetic findings, (5) appropriate terminology and classification, and (6) clinical significance and appropriate management of these neoplastic flat lesions of the breast.

HISTORICAL ASPECTS AND EVOLUTION OF THE CONCEPT

The concept of clinging carcinoma in situ was introduced by Azzopardi1 in 1979 in his remarkable book Problems in Breast Pathology to describe a distinct atypical intraepithelial lesion of the breast that can be mistaken for either normal breast tissue or ordinary fibrocystic breast change. Judging from his book, the idea of clinging carcinoma in situ as a distinct neoplastic lesion began after overlooking a case with severe cytologic epithelial atypia. The atypical lesion was confined to 4 ducts only, and because the changes were cytologic rather than architectural, "the atypical focus was missed by two distinguished professors in the University of London and by Azzopardi himself."1 The patient developed infiltrating ductal carcinoma at the same site 8 years later, with axillary lymph node metastases and subsequently died from breast cancer. The histopathologic reexamination of the breast specimen, which was misclassified as fibrocystic changes, however, revealed retrospectively a few dilated ducts showing highly atypical epithelial cells with enlarged, hyperchromatic nuclei, irregular chromatin distribution, prominent nucleoli, loss of polarity of luminal cells, and an abnormal mitosis. The involved ducts were lined by only 2 to 4 layers of highly atypical cells, which were initially overlooked at low magnification but were clearly identifiable by reexamination of the lesion at higher magnification. (1) This particular case with very dramatic consequence for the patient prompted Azzopardi to focus on similar lesions of the breast that lacked significant intraepithelial proliferation but were associated with mild to severe cytologic atypia. The morphologic comparison of these lesions with more conventional types of DCIS, particularly the comparison of cytologic features of these lesions at higher magnification, convinced Azzopardi that these must reflect another type of in situ ductal carcinoma for which he introduced the term "clinging." (1) It was a descriptive term for another type of in situ cancer characterized by only 1, 2, or just a few layers of atypical cells lining the structure of origin--hence clinging in the sense that malignant (atypical) cells are present peripherally while there is no significant intraluminal proliferation. In his book, Azzopardi emphasized that clinging pattern is a difficult one that is not generally recognized and yet deserves much wider recognition.

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It is interesting that 2 years after publication of his book, Azzopardi was not able to convince most members of the World Health Organization (WHO) Working Group, who at that time were in charge of the classification of breast tumors, about the need to add the "clinging" pattern to the upcoming classification. (3) Therefore, clinging carcinoma in situ was not listed among other types of DCIS (intraductal carcinoma) and was not even considered as an atypical breast lesion in the WHO classification published in 1981. (3)

Although not considered as DCIS or atypical by the WHO classification of 1981, several pathologists, particularly those in European countries, started to use the terminology of clinging and indeed classified such lesions as DCIS in their histopathologic reports. A European classification system of DCIS recognized clinging pattern as another variant of DCIS (4) and by using Azzopardi's criteria recommended a separation between well-differentiated (monomorphic variant with low-grade atypia) and poorly differentiated (pleomorphic variant with high-grade atypia) clinging DCIS. (4) The situation in the United States was, however, different. Although a few experts accepted the pleomorphic variant (with high-grade nuclear atypia) as poorly differentiated DCIS and regarded the monomorphic variant (with low-grade atypia) as merely ADH, (5) most pathologists were reluctant to accept the concept of clinging carcinoma in situ.

The main reason for this reluctance was that a diagnosis of carcinoma in situ, regardless of size, type, or grade, would have led to mastectomy. Moreover, many pathologists were uncertain about the true neoplastic nature of atypical epithelial cells lining the ducts, particularly in the well-differentiated or monomorphic variant (with mild nuclear atypia) of clinging DCIS (F. Moinfar, oral communication).

After publication of the first molecular-genetic study6 on the issue of clinging DCIS in 2000, it became clear that both variants of this type of flat lesion represent neoplastic alterations of the breast and share many similarities with other more conventional patterns (types) of DCIS. Meanwhile, several investigators have found association of tubular carcinoma, as well as lobular intraepithelial neoplasia (LIN), with clinging pattern in breast biopsies (7-10) and have published this distinct morphologic change under a variety of terms such as atypical cystic lobules, (11,12) atypical lobules type A, (13) atypical cystic duct, (14,15) columnar cell change with atypia, (16-18) columnar alteration with prominent apical snouts and secretion, (19-21) columnar cell hyperplasia (with or without atypia), (22-25) small ectatic ducts lined by atypical cells with apocrine snouts, (26) ductal intraepithelial neoplasia (DIN), flat type, (2,6,27) flat epithelial atypia, (16,28-30) and enlarged lobular units with columnar alteration. (31) Finally, after more than 20 years, a long period in which the lesion remained widely unrecognized and/or ignored, the most recent WHO Classification of Tumours of the Breast and Female Genital Organs (32) (published in 2003) acknowledged the neoplastic nature of atypical epithelial cells in this variant of intraepithelial breast lesions and accepted the designation of flat epithelial atypia or flat DIN, ductal intraepithelial neoplasia grade 1a (DIN1a), for those lesions associated with mild nuclear atypia. According to the recent WHO classification, flat intraepithelial ductal lesions with severe nuclear atypia, however, are classified as high-grade DCIS or DIN3. (32)

In the following discussion, flat epithelial atypia or DIN1a is designated low-grade flat DIN (see rational for adopting DIN terminology). Similarly, flat DIN with high-grade nuclear atypia (pleomorphic variant of clinging carcinoma in situ) is designated high-grade flat DIN.

MORPHOLOGIC FEATURES/DIAGNOSTIC CRITERIA

The most important morphologic feature is the presence of cytologic atypia of luminal epithelial cells without simultaneous alterations of basally located myoepithelial cells and without significant intraluminal proliferation in the involved ducts or ductules. (1,2) Although one has to examine the lesion at higher magnification to identify cytologic atypia, the appearance of flat DIN at lower magnification is often different from that of the surrounding normal lobules and ducts. (2) Not infrequently, the examination of the involved structures at lower magnification reveals rigid dilatation, particularly within lobules and small ducts (terminal duct-lobular units) (2) (Figure 1, A through D; Figure 2, A and B; Figure 3, A). Even at lower magnification, the involved structures display hyperchromatic nuclei of epithelial cells, which differ from the nuclear staining intensity of the surrounding normal breast tissue. Low-grade flat DIN commonly shows apical snouts, a feature that also can be appreciated at lower magnification (1,2) (Figure 1, A, C, and E; Figure 3, C).

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Once a flat lesion is suspected at lower magnification, one needs to analyze it at higher magnification to evaluate cytologic features (Figure 1, B and F; Figure 2, C and D; Figure 3, D through F; Figure 4, A through D). These features include luminal epithelial cells with at least subtle atypia in the form of nuclear enlargement, higher nuclear-cytoplasmic ratio, and hyperchromatic nuclei (Figure 1, F; Figure 2, C and D; Figure 3, E and F; Figure 4, A through D). Although the low-grade flat DIN shows a monotonous appearance of nuclei with regular chromatin distribution, has a regular nuclear membrane, and usually lacks prominent nucleoli, high-grade flat DIN commonly shows nuclear pleomorphism (pleomorphism of anaplasia), irregular chromatin clumping, nuclear membrane irregularity, and prominent and often multiple nucleoli (1,2) (Figure 1, F; Figure 2, C and D; Figure 3, E and F; Figure 4, A through D). It is of note that the atypical epithelial cells show round, ovoid, or even elongated nuclei. The atypical cells are of flattened, cuboidal, or columnar cell shape (Figure 3, B). Although in low-grade flat lesions mitotic figures are hardly present, high-grade flat lesions usually show marked mitotic activity, eventually associated with atypical mitotic figures (Figure 4, C).

Flat DIN can be present in either pure flat form or may show transition into focal micropapillary or cribriform growth pattern. In such cases with transition, the cytologic features in flat areas are identical to those areas with more conventional micropapillary/cribriform growth patterns (2) (Figure 2, D).

Regarding the luminal contents, flat DIN is commonly associated with abnormal luminal secretion. It is important to keep in mind that any deviations from the normal, usually uniformly eosinophilic staining and thin-looking secretion need to be examined carefully. (1) Variations in staining intensity and texture of the secretory material in a given lumen, particularly the presence of granular and fragmented products with or without microcalcifications, should raise some degree of suspicion about the presence of flat DIN (2) (Figure 2, A and B; Figure 4, B; Figure 5, A). In that setting, a close look at higher magnification not infrequently reveals apoptotic figures and very small amounts of cell debris within the abnormal secretory material of flat DIN. (1,2)

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Tables 1 and 2 summarize the characteristic morphologic features of low-grade and high-grade flat DIN.

MAIN DIFFERENTIAL DIAGNOSES AND MAJOR PITFALLS

Low-grade flat DIN needs to be distinguished from adenosis, particularly blunt duct adenosis (BDA). In contrast to flat DIN, BDA shows simultaneous alteration (hypertrophy) of epithelial and myoepithelial cells with increased intralobular (specialized) connective tissue (Figure 6, A and B; Figure 7, A). As apposed to rigid dilatation of flat DIN, the dilated tubules of BDA show curved structures (blunt lateral outlines, blunt endings) (Figure 6, A and B; Figure 7, A and B). (1,2) Whereas in flat DIN high-power magnification reveals mild to moderate nuclear atypia, no cytologic atypia should be found in simple BDA. In contrast to BDA, which is not associated with any intraluminal proliferations, the involved ducts in low-grade flat DIN often display a slight proliferation of luminal cells (2-4 cell layers) showing a monotonous epithelial appearance. It is in the presence of tall columnar epithelium (columnar cell change) that the changes in the myoepithelial layer are of greatest significance. (1,2) It is important to keep in mind that in adenosis (BDA, columnar cell change), the outer cell layer is easily identified and frequently hypertrophic. Indeed, there is an organoid hypertrophy of all cellular elements in BDA (Figure 6, A and B; Figure 7, A through D and F). In contrast to BDA, the alterations in flat DIN concern only one cell type, namely luminal epithelial cells. (1,2) The myoepithelial cell layer in flat DIN is often barely discernible and usually not hypertrophic (Figure 1, C and F; Figure 2, C; Figure 3, E and F; Figure 4, A through D; Figure 5, A through C; Figure 7, C and E). Although the neoplastic cells of flat DIN are negative for high-molecular-weight cytokeratin such as cytokeratin (CK) 5/6, luminal cells in BDA often show a heterogeneous positive reaction for CK 5/6 (see also immunohistochemistry) (2) (Figure 6, C and D).

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Lactational-type (like) changes within the lobules and acinar structures may also be confused with flat DIN. The involved acini in lactational-type change show marked cytoplasmic vacuolization and luminal cells with hobnail changes that are similar to those of Arias-Stella phenomenon. These changes, however, are usually absent in flat DIN. (2)

Low- and high-grade flat DIN may be associated with hypersecretory activity and show thick, eosinophilic, and colloidlike luminal material. In this condition, the neoplastic epithelial cells can easily be overlooked and misinterpreted as fibrocystic change, particularly at low magnification. It is the presence of unusual thick and colloid-like secretory material often associated with rigid dilatation of the involved ducts that should prompt examination at higher magnification to evaluate the cytologic features of the lining epithelial cells2 (Figure 2, A and B).

IMMUNOHISTOCHEMISTRY

Immunohistochemistry of low-grade flat DIN does not differ from that of other types of low-grade DIN (DCIS) with cribriform, micropapillary, or solid growth patterns. (2,20,21,27,28,32) Like other types of low-grade DIN (DCIS), the neoplastic cells of flat DIN are negative for high-molecular-weight cytokeratins such as CK34BE12, CK 5/6, or CK 14. (2,17,32) The negative immunoreaction for CK 5/6 (high-molecular-weight cytokeratin) in flat DIN is in contrast to its heterogeneous positive reactivity in adenosis (BDA) (2,32) (Figure 6, C and D). All variants of low-grade intraepithelial neoplastic lesions are positive for estrogen receptor and progesterone receptor but are negative for HER2/neu. (2,17,32) MIB-1 (KI-67) activity is always low (ranging from less than 5% to 8%). (17,33) The neoplastic cells of high-grade flat DIN are usually negative for estrogen receptor and progesterone receptor but commonly overexpress HER2/ neu (2) (Figure 5, D and E). A positive nuclear immunoreaction for MIB1 (Ki-67) in more than 20% of the cells is frequently present in high-grade flat DIN. (2) Most of both low- and high-grade flat DIN are positive for E-cadherin. (2,17)

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MOLECULAR-GENETIC FINDINGS

Loss of Heterozygosity

The first study of genetic alterations in flat DIN was published in 2000 and clearly showed that genetic alterations in this type of breast lesion are quite common. (6) Most importantly, this study demonstrated that flat DIN showed the same genetic alterations (loss of heterozygosity [LOH]) identified in adjacent in situ and infiltrating ductal carcinoma. (6) Using polymerase chain reaction, this study examined DNA extracts from microdissected areas of 22 cases with extensive flat DIN (clinging carcinoma in situ), including 13 cases associated with well-differentiated infiltrating ductal carcinoma (mostly pure or mixed tubular carcinoma) as well as 5 cases associated with more conventional types of DIN (DCIS) with cribriform and/or micropapillary growth pattern. Among 22 cases, 13 cases (59%) were of low-grade (monomorphic variant of clinging carcinoma in situ) flat type, and 9 cases (41%) were of the high-grade (pleomorphic variant) flat lesion. The flat lesions in 17 of 22 cases (77%) showed LOH at a minimum of 1 locus. There were 9 of 13 low-grade (monomorphic variant) and 8 of 9 high-grade (pleomorphic variant) flat lesions that definitely showed LOH. Among the 5 cases associated with conventional types of DIN (DCIS), 4 showed an identical LOH pattern in the DCIS and the flat lesions on at least 1 locus. Furthermore, the examined tubular carcinomas and the low-grade flat lesions in this study shared a common LOH pattern on at least 1 locus in 9 cases (70%). The most common genetic alterations were at chromosomes 11q21-23.2, 16q23.1-24.2, and 3p14.2 with LOH in 50%, 45%, and 41%, respectively, of informative cases. Loss of heterozygosity at several loci were more common in high-grade (polymorphic variant) compared with low-grade (monomorphic variant) flat DIN. (6)

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A recent study performed by Dabbs et al (34) has further confirmed the neoplastic nature of flat lesions with mild atypia (designated as columnar cell change with atypia or atypical columnar cell hyperplasia) and has found comparable LOH rate in the flat lesion to that of the Armed Forces Institute of Pathology study. (6) This second LOH study evaluated allelic imbalances on chromosomes 1p, 3p, 5q, 9p, 9q, 10q, 17p, 17q, 19q, and 22q by using 20 polymorphic DNA markers. Loss of heterozygosity was frequently identified at loci 17q12, 9q22.1,17p13.1, 9p23, and 5q23.3 particularly in cases with atypical columnar cell hyperplasia. (34) Of note, cases with columnar cell change without atypia (BDA, 2 cases) in this study were not associated with allelic imbalances. Based on the genetic alterations, the authors of the second LOH study (34) concluded that a select group of atypical columnar cell lesions are precursors to invasive carcinoma.

Clonality Analysis

Only one study has examined the issue of clonality in the flat lesion of the breast.6 In this study, the flat lesion and the adjacent invasive ductal carcinoma in 2 cases were selected for clonality analysis by assessing the DNA methylation pattern at a short tandem repeat (CAG) on the human androgen receptor (HUMARA) gene (analysis of X chromosome inactivation). The flat lesion in both cases was of the low-grade or monomorphic type. Both were associated with pure tubular carcinoma. One case also contained LIN with mild nuclear atypia (low-grade LIN). Importantly, the cells from both cases clearly showed an identical monoclonal pattern (nonrandom X chromosome inactivation) in the flat lesion and the associated infiltrating carcinoma. (6) In addition to nonrandom X chromosome inactivation, both of these cases revealed identical LOH at a minimum of 1 locus. Interestingly, the neoplastic cells of LIN showed the same monoclonal pattern as the adjacent flat lesion and tubular carcinoma (clonally related neoplastic breast lesions). (6)

Comparative Genomic Hybridization

One study performed by Simpson et al (17) examined a variety of columnar cell lesions from 18 patients by means of classic comparative genomic hybridization analysis. Although several cases included intraductal proliferative areas of usual ductal hyperplasia, ADH, and low-grade DCIS, flat epithelial atypia (columnar cell change with cytologic atypia and columnar cell hyperplasia with cytologic atypia) were found in 10 cases. Regarding flat epithelial atypia, although 4 of 5 columnar cell hyperplasia with atypia showed changes with recurrent losses on 16q (2 of 5) and 18p (2 of 5), 3 of 5 columnar cell changes with atypia revealed chromosomal aberrations with recurrent losses on 16q and 17p (both 2 of 5 cases). (17) Columnar cell lesions in this study showed low numbers of chromosomal abnormalities and recurrent 16q loss, features that are similar to those identified in low-grade intraepithelial neoplasia (DCIS) and invasive carcinoma. Based on the comparative genomic hybridization (and immunohistochemical) data from this study, the authors concluded that columnar cell lesions are a nonobligate, intermediary step in the development of some forms of low-grade in situ and invasive carcinoma. (17)

A very recent study performed by Stacher and Moinfar (unpublished data, 2009) investigated 12 cases with low-grade flat DIN by means of array comparative genomic hybridization and found a characteristic 1q gain and 16q loss in almost 50% of the cases. It is of note that the combination of 1q gains and 16q losses is a common cytogenetic finding in low-grade DCIS and low-grade infiltrating ductal carcinoma. (35,36) More interestingly, identical chromosomal abnormalities (1q gain and 16 q loss) were commonly found in LIN that accompanied cases of low-grade flat DIN in this recent study (Stacher and F. Moinfar, unpublished data, 2009).

RATIONAL FOR ADOPTING THE DIN TERMINOLOGY

There are several and confusing names for this type of breast lesion in the literature. These include atypical cystic lobules, atypical lobules type A, clinging carcinoma, clinging carcinoma in situ, columnar alteration of lobules, columnar cell change with atypia, atypical columnar change, columnar alteration with prominent apical snouts and secretion, columnar cell hyperplasia (with or without atypia), small ectatic ducts lined by atypical cells with apocrine snouts, cancerization of small ecstatic ducts of the breast by ductal carcinoma in situ cells with apocrine snouts, and enlarged lobular units with columnar alteration. * The most recent WHO classification of breast tumors, published in 2003, favors the designation of flat epithelial atypia and also uses DIN1a as a synonym for this type of breast lesion. (32)

In my opinion, the most appropriate term for this recently more appreciated breast lesion is "flat DIN." (2) Like other types of DIN (DCIS), based on the degree of cytologic atypia (mild vs severe), flat DIN should be separated into a low- or high-grade lesion. The main reasons for advocating the DIN terminology are

1. It points out the intraepithelial (noninvasive) nature of the lesion. The very alarming term of cancer or carcinoma in situ incorporated the designation of clinging carcinoma in situ or DCIS is avoided.

2. It emphasizes the flat nature of the lesion; hence there is no significant intraluminal proliferation of cribriform, micropapillary, or solid growth pattern. The native luminal epithelial cells are simply replaced by one or a very few cell layers of atypical cells.

3. The atypical epithelial cells are neoplastic, as shown by recent molecular-genetic findings.

4. The intraepithelial neoplastic cells are morphologically and immunohistochemically of ductal type, even if they arise in ductules and lobules (terminal duct-lobular units).

5. The descriptive term of flat DIN is appropriate because it incorporates all important properties of the lesion, namely the flat character, the ductal type of the cells, and intraepithelial (noninvasive) neoplastic nature of the involved structures.

6. It follows the meaningful concept of intraepithelial neoplasia as a unifying concept in the breast as used in many other organs.

7. Based on the degree of nuclear atypia (mild versus severe cytologic atypia), it can simply be classified into clinically relevant low- and high-grade flat DIN.

8. It avoids the artificial separation between columnar cell change with atypia and columnar cell hyperplasia without and with atypia. By definition, the degree of cytologic atypia in low-grade flat DIN is minimal and often very subtle. Indeed, a close evaluation of some illustrations (publications) as examples of columnar cell hyperplasia without atypia reveals flat epithelial atypia, if one pays more attention to the diagnostic criteria as introduced and emphasized by Azzopardi. (1) This would also explain why columnar cell hyperplasia without atypia has been reported to be common within or at the periphery of tubular carcinoma and/or LIN. (10,22,23,34)

WHAT IS THE SIGNIFICANCE OF FLAT DIN?

Clinical Significance and Issues Concerning Management of the Patients

Studies with follow-up information on biopsies with only flat DIN as the most advanced atypia, whether in core biopsies or excisional biopsies, are limited. Eusebi et al (37) identified 21 cases of clinging carcinoma, in a retrospective review of more than 4000 breast biopsies initially diagnosed as benign (fibrocystic change) between 1965 and 1971. This study, however, did not distinguish between clinging carcinoma of the monomorphic variant and more proliferative micropapillary DCIS (DIN) or a polymorphic type (high-grade DCIS). Two of the 21 patients died of breast carcinoma. One patient developed an advanced ipsilateral breast cancer several years after the initial biopsy and died 12 years after the biopsy, and the second patient presented with infiltrating ductal carcinoma 6 years after the initial biopsy and died 4.9 years later. In addition, 2 more patients were found to have persistent clinging carcinoma, one in the ipsilateral breast 9 years later and the other in the contralateral breast 11 months later. The results from the study by Eusebi et al (37) indicated an absolute risk of 9.5% with a mean follow-up of 16.7 years, but this study was, unfortunately, inclusive of lesions beyond flat DIN (pure clinging carcinoma in situ).

Bijker et al, (38) analyzing the data of the European Organization for Research and Treatment of Cancer (trial 10853, excision with or without radiotherapy), reported no recurrences among 59 women with well-differentiated DCIS with clinging pattern after a relatively short median follow-up of 5.4 years. Of the 63 patients diagnosed as having clinging DCIS (well-differentiated or monomorphic variant) on core needle biopsy (CNB) retrospectively, 7 women developed an infiltrating carcinoma in the ipsilateral breast indicating an absolute risk of 11.1%. The absolute risk was higher in the ipsilateral breast compared with 3.2% in the contralateral breast, with a mean follow-up of 6.2 years available for 55 of 63 patients. (37)

Although the available data are quite limited and do not permit assessment of precise level of risk of developing subsequent carcinoma, they appear to indicate an increased risk of developing carcinoma compared with the absolute risk in women with ADH as shown by the study performed by Tavassoli and Norris (39) in which 9.8% of women with ADH developed invasive carcinoma with a mean follow-up of 12.4 years. It is, however, of note that the development of subsequent infiltrating carcinoma after a diagnosis of ADH significantly varies in the literature from 3.7% to 22%. (40-42)

Currently, there are limited data concerning the significance of low-grade flat DIN (flat epithelial atypia) detected on CNB and its appropriate management. Most of the data regarding flat epithelial atypia in core biopsies are limited to abstracts that looked at the findings in subsequent excisions.43-46 Based on these limited data, a subsequent excision revealed a more advanced neoplastic lesion (DCIS or invasive carcinoma) in almost one-third of cases. However, it should be emphasized that, given the variation in terminology used to designate this lesion and the inclusion in many of these studies of more advanced neoplastic lesions (ADH) showing rigid arcades, epithelial tufts, and focal cribriform or micropapillary growth pattern in addition to flat epithelial atypia, it is difficult to determine how many of the reported cases are truly pure flat epithelial atypia and how many constitute ADH. Because most published follow-up studies on flat epithelial atypia include ADH among the studied cases, the results are of limited value and need to be interpreted more cautiously.

Regarding the presence of pure flat lesion (flat epithelial atypia, flat DIN) in breast core biopsies and the subsequent excisional biopsy, there are 2 recent studies that deserve more consideration: One study, performed by Lakshimi et al, (47) reported 3 of 14 (21%) pure flat epithelial atypia upgraded to DCIS and/or invasive carcinoma on excisional biopsy and concluded that flat epithelial atypia shows a risk of upgrade to carcinoma (in situ or invasive) similar to that of ADH and, hence, should be managed by a follow-up excisional biopsy. The second recent study, done by Martel et al, (48) reviewed 1751 core biopsies retrospectively and found 63 cases with pure flat epithelial atypia (low-grade flat DIN). Of the 63 patients, 24 had a subsequent biopsy up to 10 years after the initial CNB; an invasive carcinoma was identified in 9 patients (14.3%), 7 (11.1%) in the ipsilateral and 2 (3.2%) in the contralateral breast. It is of note that in the study done by Martel et al (48) 5 women underwent an excisional biopsy of the ipsilateral breast within less than 3 months of the initial needle biopsy; none had either an invasive or advanced intraepithelial neoplasia (DCIS). The authors of this study concluded that low-grade flat DIN (flat epithelial atypia or flat DIN1) is a marker of slightly increased risk for subsequent development of infiltrating breast carcinoma. Martel et al (48) concluded that when pure low-grade flat DIN (flat epithelial atypia) is found on needle biopsy as the most advanced lesion after mammographic correlation, an excisional biopsy is not mandatory but close follow-up is advised with repeat mammograms for early detection of any clinically occult carcinoma.

Obviously, one major limitation of the previously mentioned studies is the small number of examined cases with pure flat DIN (flat epithelial atypia) with correlation to the subsequent excisional biopsy of the breast. With that regard, what we definitely need are studies with large numbers of patients that allow reliable statistical analysis before one can draw a clinically relevant conclusion whether excisional biopsy after identification of pure low-grade flat DIN on needle biopsy is mandatory or not. Furthermore, the use of standardized criteria for the diagnosis and uniform terminology is essential in establishing the significance and determining guidelines for management of this lesion, particularly on CNB. For the time being, however, it is reasonable to inform the women diagnosed with this type of atypical (neoplastic) breast lesion on CNB about the limitation of our current knowledge for its biologic significance and the lack of uniform management whether it should definitely be excised or be managed by close clinical-mammographical follow-up. In cases with a more conservative approach, a close follow-up has been recommended with repeat mammogram every 6 months for 2 to 3 years to detect early a more advanced DIN (DCIS) or invasive carcinoma. (48)

Practical Consideration for Histopathologists

There are several practical considerations and issues that need to be emphasized as follows:

1. Cases with pure low-grade flat DIN (flat epithelial atypia) identified on CNB should be examined more thoroughly: The current use of the mammotome and the larger 11-gauge (or 8-gauge) needle provide a larger and more adequate sampling of the mammographically detected abnormalities. If a pure low-grade flat DIN (flat epithelial atypia) is encountered on CNB and it is the only explanation for the mammographically detected abnormalities (suspicious microcalcifications), it is advised to perform additional (3-4) levels, beyond the usual 3 initial levels. This is to exclude a more advanced neoplastic lesion deeper in the block. (48) If intraepithelial proliferations such as rigid bridges (arcades) or micropapillary structures focally become apparent in deeper levels, the lesion should be diagnosed as ADH, and an excisional biopsy needs to be performed. (2,48,49)

2. Excisional breast biopsies with areas of pure low-grade DIN flat type should be embedded and worked up in their totality and meticulously searched for more advanced intraepithelial lesions or invasive carcinoma. (2,9)

3. Due to the common coexistence of low-grade flat DIN and LIN, one needs to carefully examine the tissues (CNB, excisional biopsy) to identify or exclude LIN. One should be aware of the possibility of small areas of tubular carcinoma (or well-differentiated infiltrating ductal carcinoma) in the setting of such flat lesion. Therefore, a careful search to exclude an infiltrating carcinoma is prudent. The infiltrating carcinoma in association with low-grade flat DIN is usually of low grade and is commonly of pure tubular, mixed tubular (well-differentiated invasive ductal carcinoma), or even invasive lobular type (invasive lobular carcinoma).

4. Rare cases of pure flat lesion showing severe cytologic (nuclear) atypia should not be designated flat epithelial atypia or low-grade flat DIN. Those cases represent high-grade DIN (DCIS, grade 3) and need to be managed like other types of high-grade intraepithelial lesions. (1,2,32)

5. Similar to LIN, low-grade flat DIN occurs not infrequently multifocal, multicentric, or even bilateral. (2,48) Like LIN, the assessment of resection margins of excisional biopsies containing such low-grade flat lesions is meaningless and definitely not required. (2) The situation is different in cases with high-grade flat DIN (DCIS, grade 3, polymorphic variant of clinging carcinoma in situ), which often, like other types of high-grade intraepithelial lesions, represent a more localized neoplastic proliferation. Therefore, like other types of high-grade DIN (DCIS, grade 3), the assessment of the resection margins is mandatory for high-grade flat DIN.

6. Incorporation of the columnar cell in the designation of this lesion is misleading, and the value of a complex subcategorization of columnar cell lesions as suggested by some investigators (16,17) is doubtful. The normal epithelium of the breast varies from cuboidal to low columnar and tall columnar in shape with or without apical snouts in its many normal physiologic variations. Furthermore, flat epithelial atypia also varies from flat cells with scant cytoplasm to cuboidal/low columnar or tall columnar atypical cells. In flat DIN or flat epithelial atypia, the involved structures are replaced by 1 or very few (up to 4) cell layers with mild (and often very subtle) cytologic atypia. It is, therefore, not clear what is accomplished by inclusion of columnar in the terminology or classification of these lesions in a complex way (including lesions with significant intraepithelial proliferations with cribriform or micropapillary growth pattern) that has very little in common with the original concept of clinging (flat) lesion as introduced by Azzopardi several years ago. Once again, the designation of columnar is misleading as it may imply that only lesions with columnar cells should be placed in this category. Furthermore, if one follows Azzopardi's criteria and accepts his clinging (flat) concept, one would realize that the separation of these lesions into columnar cell change with atypia, columnar cell hyperplasia without atypia, and columnar cell hyperplasia with atypia is misleading. Finally, as discussed in this review, pathologists should be familiar with the main differential diagnosis of flat DIN (clinging carcinoma in situ), namely BDA, which often simulates flat DIN.

SUMMARY WITH CONCLUSIVE REMARKS

Flat DIN represents one of the earliest, morphologically recognizable, neoplastic alterations of the breast. It constitutes another type of DIN that may or may not progress to invasive breast carcinoma. It is characterized by mildly to severely atypical (neoplastic) cells simply replacing the single layer of native epithelial cells without appreciable proliferation; architectural alterations of tufting, intraluminal bridging, and micropapillary structures are typically absent. This type of early breast lesion can easily be missed because the alteration is mainly cytologic rather than architectural. Flat DIN has to be divided into low and high grades based on the degree of cytologic (nuclear) atypicality. Low-grade flat DIN can commonly be found within and/or at the periphery of tubular carcinoma and displays very similar cytologic, immunohistochemical, and molecular-genetical abnormalities as those identified in more advanced low-grade DIN (DCIS) and tubular (or well-differentiated ductal) carcinoma. The high-grade flat DIN (pleomorphic variant of clinging carcinoma in situ) in pure form rarely occurs in the breast. The cytomorphology, immunohistochemistry, and genetic abnormalities in this rare variant are also very close to those seen in high-grade DCIS.

The risk for subsequent invasive carcinoma for the pure low-grade flat DIN seems to be low but needs to be assessed in well-designed, large studies with long-term follow-up. Furthermore, we need studies with large numbers of patients that allow reliable statistical analysis before one can draw a clinically relevant conclusion whether excisional biopsy after identification of pure low-grade flat DIN on needle biopsy is mandatory or not. It is important to note that the use of standardized criteria for the diagnosis and uniform terminology is essential in establishing the significance and determining guidelines for management of this lesion, particularly on CNB.

Azzopardi had recognized many years ago the very significant problem with the traditional terminology of lobular carcinoma in situ and showed in his work sympathy for an alternative, and more appropriate, designation as stated in the following:

One cannot leave this subject without discussing which term is most appropriate to designate the condition currently called carcinoma lobular in situ. The writer is in agreement with the increasing number of workers who are dissatisfied with the designation of "carcinoma" for this condition ... The dissatisfaction with the term "carcinoma" in this context remains and is to a great extent justified ... the term "carcinoma" is too emotive and alarming to patient and to surgeon, It seems that the name "lobular neoplasia" adopted by Haagensen (1971), satisfies our need for a different name. It does not dismiss CLIS as a marker while, at the same time, it does not elevate it to the frightening stature of a "carcinoma". It is short, accurate, and reasonably distinctive. (1(p232))

Finally, I would like to conclude that similarly the term DCIS is no longer the appropriate one; it is too emotive and alarming to patients and clinicians, and the name (concept) of DIN satisfies our need for a different term (concept).

In this context, the best designation for clinging ductal carcinoma in situ would be flat DIN. Certainly, it is short, accurate, and reasonably distinctive!

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Farid Moinfar, MD

Accepted for publication January 20, 2009.

* References 1, 2, 8-17, 19, 22, 25, 26, 31, 32.

From the Unit of Breast and Gynecologic Pathology, Department of Pathology, Medical University of Graz, Graz, Austria.

The author has no relevant financial interest in the products or companies described in this article.

Reprints: Farid Moinfar, MD, Unit of Breast and Gynecologic Pathology, Department of Pathology, Medical University of Graz, Auenbruggerplatz 25 Graz, Styria 8036, Austria (e-mail: farid.moinfar@ meduni-graz.at).
Table 1. Morphologic Features of Low-Grade Flat Ductal
Intraepithelial Neoplasia (a)

Changes identifiable at low magnification (architectural
alterations)

* Either no significant architectural changes or rigid dilatation
of small ducts and ductules

* Sometimes ducts (ductules) with focal and minimal intraepithelial
proliferation showing tufts or rigid micropapillary or cribriform
growth pattern (early roman bridges)

* Abnormal luminal contents showing fragmented, nonhomogeneous and
often densely staining secretory material (variations in staining
intensity and texture of the luminal material)

* Not infrequently luminal microcalcifications associated with
(abnormal) secretory material

* Sometimes involvement of the acini within the lobules closely
mimicking an adenosis growth pattern. The involved acini, however,
show a much darker nuclei of epithelial cells compared with those
of normal breast tissue or adenosis

* Often numerous apical snouts

Changes identifiable at higher magnification (cytologic
alterations)

* The involved structures (ductules and ducts) are lined by a very
few (mostly 2-4) cell layers of epithelial cells with monotonous
appearance. There is no significant intraepithelial proliferation
with formation of rigid bridges or solid structures

* Mild nuclear atypia of epithelial cells including enlarged,
hyperchromatic nuclei with increased nuclear-cytoplasmic ratio

* Round, ovoid, or even elongated nuclei showing regular chromatin
distribution and regular nuclear membrane

* Round/ovoid or tall columnar cytoplasm can be present. Epithelial
cells with round, hyperchromatic nuclei with scant cytoplasm are
common

* Small nucleoli

* Mild degree of loss of the normal orientation (loss of nuclear
polarity): the nuclei, when ovoid, tend to point in slightly
different directions

* Not infrequently, changes in cytoplasmic staining or cytoplasmic
pallor of atypical cells: The round or ovoid, hyperchromatic nuclei
stand out in stark relief against a nearly colorless cytoplasmic
pallor

* Mitoses are very rare

* The above-mentioned changes affect only one cell type
(homogeneous cell population of luminal epithelial cells): The
basally located myoepithelial cells are often attenuated and barely
visible

* As apposed to adenosis, there is no simultaneous cell alterations
in epithelial and myoepithelial cells. There is no organoid
hypertrophy of the involved ducts (ductules)

* As apposed to adenosis, luminal epithelial cells with apical
snouts are not associated with easily identifiable (and frequently
hypertrophic) myoepithelial cells

* Sometimes, a few apoptotic bodies and cell debris within the
abnormal secretory, fragmented luminal material

(a) Synonyms according to the World Health Organization, 2003: flat
epithelial atypia or DINla. (32) For morphologic features, see also
Azzopardi (1) and Moinfar. (2)

Table 2. Morphologic Features of High-Grade Flat Ductal
Intraepithelial Neoplasia (a)

Changes identifiable at low magnification (architectural
alterations)

* Either no significant architectural changes or rigid dilatation
of ducts and ductules

* Abnormal luminal contents showing fragmented, nonhomogeneous and
often densely staining secretory material (variations in staining
intensity and texture of the luminal material)

* Not infrequently luminal microcalcifications associated with
(abnormal) secretory material

* Although in pure form there is no significant intraepithelial
proliferation with formation of rigid bridges or micropapillary or
solid structures, not infrequently more conventional and easily
recognizable solid or cribriform growth patterns even with
comedo-type central necrosis can be identified at the periphery of
high-grade flat lesions (combined growth pattern)

Changes identifiable at higher magnification (cytologic
alterations)

* The involved structures (ductules and ducts) are lined by a very
few (mostly 1-3) cell layers of highly atypical and pleomorphic
epithelial cells (pleomorphism of anaplasia). Severe nuclear atypia
of epithelial cells including very large, hyperchromatic nuclei
with high nuclear-cytoplasmic ratio

* Mostly, round or ovoid nuclei showing irregular chromatin
distribution and irregular nuclear membrane

* Prominent and often multiple nucleoli

* Increased mitotic activity, eventually including atypical mitotic
figures

* The above-mentioned changes affect only one cell type
(homogeneous cell population of luminal epithelial cells): The
basally located myoepithelial cells are often attenuated and barely
visible

* There is no simultaneous cell alterations in epithelial and
myoepithelial cells. There is no organoid hypertrophy of the
involved ducts (ductules)

* A close look at the luminal contents often reveals a few
apoptotic bodies and cell debris in the lumina of the involved
ducts

(a) Synonyms according to the World Health Organization, 2003:
ductal carcinoma in situ (DCIS), G3, pleomorphic variant of
clinging carcinoma in situ or DIN3. (32) For morphologic features,
see also Azzopardi (1) and Moinfar. (2)
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Publication:Archives of Pathology & Laboratory Medicine
Article Type:Disease/Disorder overview
Date:Jun 1, 2009
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