Differential diagnosis of solid carcinoma in situ

Differential Diagnosis of Solid Carcinoma In Situ Horacio M. Maluf, MD ● Although most cases of carcinoma in-situ are easily classified as either ductal or lobular on the basis of their pattern of involvement and the nature of their cell-to-cell relation, a few pose diagnostic problems. Attention to the presence of associated lesions and the results of immunohistochemical staining for E-cadherin and, to a lesser extent, cytokeratin can help to categorize problematic cases. This article reviews the histological criteria employed to separate ductal carcinoma in situ from lobular carcinoma in situ, the role of immunohistochemistry in the diagnosis of equivocal lesions, and the evidence suggesting the existence of combined and truly hybrid forms. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Lobular carcinoma in situ, ductal carcinoma in situ, E-cadherin, high molecular weight keratin

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N 1941, DRS FRANK W. FOOTE, JR. and Fred W. Stewart, two of the most distinguished mammary pathologists of the 20th century (Fig 1), published the first thorough description of lobular carcinoma in situ (LCIS).1 Based on the features presented in this publication, the distinction between LCIS and ductal carcinoma in situ (DCIS) seemed relatively straightforward. Low-grade carcinomas in situ growing in a loosely cohesive sheet were classified as lobular and those forming glands or exhibiting nuclear pleomorphism or necrosis as ductal. Subsequent experience has revealed two limitations to this approach. First, the assessment of certain classical morphological features such as pleomorphism and, to a lesser extent, cell size is subjective, and this subjectivity makes it difficult to apply these criteria reproducibly. Second, it has become clear that each form of carcinoma in situ can displays findings more characteristic of the other. For example, LCIS can display pleomorphism or necrosis; certain ductal carcinomas in situ lack gland formation, pleomorphism, and necrosis; and occasional cases of carcinoma in situ exhibit a mixture of lobular and ductal characteristics. As a result, the classification of solid carcinomas in situ has become complex. The approach to this task can be best understood by separately considering the following features.

ducts. They are the frequent residence of in situ carcinomas with cribriform, micropapillary, and solid patterns of growth, conventionally classified as DCIS. The mammary lobule, with its closely arranged acini, terminal ductule and specialized stroma, is the seat of classical LCIS, and, despite the acinar expansion provoked by the LCIS, one can still recognize this basic anatomic unit at low magnification. Although these patterns hold true in many cases, exceptions to this topographical distribution occur frequently for both forms of in situ carcinoma, and preexisting changes can obscure the underlying anatomy. DCIS can involve mammary lobules while preserving their shape, a phenomenon known as “cancerization” of the lobules, and LCIS frequently extends into ducts. Ductal involvement by LCIS can take the form of either solid growth, in which the cells fill the duct completely, or pagetoid spread, in which the neoplastic cells grow in clusters and single cells underneath the ductal epithelium. This former pattern is a source of great diagnostic difficulty since it closely resembles the appearance of solid DICS. Pagetoid spread is infrequent in DCIS, but can be occasionally observed. Finally, postmenopausal atrophy can reduce the lobules to small buds of terminal ducts and the intralobular stroma to dense collagen, making it difficult to recognize terminal duct-lobular

PATTERN OF INVOLVEMENT Pathologists have come to classify certain patterns of involvement by in situ carcinoma as either ductal or lobular. Although this nomenclature does not reflect an anatomic origin,2 the patterns designated as DICS and LCIS are sufficiently distinctive to allow separation in most cases. Thus, large round structures, lacking the organized, cluster-like arrangement of acini and identifiable connecting terminal ductules, are interpreted as

From the Lauren V. Ackerman Division of Surgical Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO. Address reprint requests to Horacio M. Maluf, MD, Division of Surgical Pathology, Department of Pathology and Immunology, Box 8118, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110-1093; e-mail: [email protected]. © 2004 Elsevier Inc. All rights reserved. 0740-2570/04/2101-0005$30.00/0 doi:10.1053/j.semdp.2003.10.009

Seminars in Diagnostic Pathology, VOL 21, NO 1 (FEBRUARY), 2004: pp 25-31

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Fig 1. A moment of relaxation and consultation in the pathology laboratory of the Memorial Hospital in New York City (circa 1946). In the center is Dr. Fred W. Stewart, then Chairman of the department, and to the left is his colleague and co-worker, Dr. Frank W. Foote, Jr. To the right is the eminent dermatopathologist, Dr. Sophie Spitz. (Paul Parker Photo, Courtesy of the Memorial Sloan-Kettering Cancer Center Archives.)

nature of the proliferation. The presence of cells with the classical cytological attributes of LCIS or intracytoplasmic lumens, a pagetoid pattern of spread (Fig 4), or coexisting invasive lobular carcinoma allows one to classify such an ambiguous solid carcinoma in situ as lobular carcinoma in situ. In the same way, the presence of conventional forms of DCIS (eg, micropapillary or cribriform) will favor the diagnosis of ductal carcinoma in situ. One must remember, however, that LCIS can coexist with any form of DCIS, including papillary carcinoma. In uncommon cases, examination of the cell-to-cell relation will not provide diagnostic information because the cells do not display either a noncohesive, “disheveled” appearance or closely apposed, sharp membranes (Fig 5). Evaluation of cytological and immunohistochemical characteristics are required to establish a diagnosis in this situation.5,6

units. Thus, the anatomic site of involvement is neither sufficient nor necessary to classify in situ carcinoma; therefore, one must give precedence to the following architectural, cytological, and immunohistochemical features. CELL-TO-CELL RELATION The character of the cell-to-cell relation represents the only type of architectural evidence that aids in the classification of types of solid carcinoma in situ; thus, this property takes on great importance in classifying a tumor as either ductal or lobular. Low-grade lesions displaying a non-cohesive “disheveled,” to use Dr. Page’s description,3 arrangement and poorly defined cell borders almost always represent LCIS (Fig 2), and one can safely make that diagnosis based on this appearance. Tumors composed of closely apposed cells with sharp membranes usually represent DCIS (Fig 3), but this pattern is not diagnostic. Occasional examples of LCIS will show this appearance, which Dr. Rosen refers to as the “mosaic pattern” of LCIS4; the presence of other features more characteristic of lobular carcinoma establishes the lobular nature of this variety of LCIS. Thus, when confronted with a solid form of carcinoma in situ that does not display the characteristic “disheveled,” noncohesive cell-to-cell relationship, one should search for associated evidence to define the

Fig 2. Lobular carcinoma in a duct. The cells are noncohesive, some display intracytoplasmic lumens.

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NUCLEAR FEATURES The nuclei of lobular carcinoma are classically defined as small, round to ovoid, monomorphic, and generally devoid of nucleoli. Lesions with these characteristics are known as type A LCIS. Type B LCIS, on the other hand, exhibits some nuclear pleomorphism and nucleoli are commonly identified. Essentially the same nuclear features can be found in all forms of DCIS, so nuclear features do not provide help distinguishing the 2 lesions. In the past, the presence of high-grade nuclei or marked pleomorphism was considered a feature exclusively seen in DCIS. This belief is no longer tenable given the growing acceptance of pleomorphic lobular carcinoma as a lesion in the spectrum of lobular cancer.7,8 Separation of pleomorphic LCIS from DCIS can be achieved by focusing on ancillary features of pleomorphic LCIS such as the noncohesive appearance of the tumor cells, the presence of signet ring cells, the

Fig 3. DCIS in lobules. The cells are widely and evenly spaced. Cell borders appear sharply defined, and there is abundant clear cytoplasm.

CYTOPLASMIC CHARACTERISTICS Although cytoplasmic features will not separate LCIS from some solid forms of DCIS, certain findings are sufficiently more common in one form of in situ neoplasia or the other that they strongly suggest a certain diagnosis. The presence of intracytoplasmic lumens containing stainable mucin is more frequently seen in LCIS than DCIS. The extreme manifestation of this phenomenon is the existence of signet ring cells in lobular carcinoma in situ. In our experience, these lesion are usually associated with conventional or pleomorphic lobular carcinomas in both their in situ and invasive phases. Less value is placed on cytoplasmic lumens with targetoid inclusions since they are not rare in DCIS. Clear cytoplasm, with its accompanying sharply defined cellular membranes, is a feature that favors a diagnosis of DCIS, the exception again being the “mosaic pattern” of LCIS.

Fig 4. Lobular carcinoma in situ with well defined cell borders. The lesion predominantly involves the lobules, occasional cells contain cytoplasmic lumens, and there is a pagetoid pattern of spread in the terminal duct.

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different molecules have been the targets of these studies. One, the transmembranous glycoprotein, E-cadherin, which participates in calcium dependent intercellular adhesion and embryonic development, exhibits a linear membranous pattern of staining in the vast majority of cases of DCIS, and almost all case of lobular carcinoma are negative. Molecular studies have demonstrated mutations of the E-cadherin gene.11,12 The rare cases of lobular carcinoma that show reactivity for E-cadherin do so in a paranuclear cytoplasmic pattern, probably reflecting abnormal processing of E-cadherin by the Golgi apparatus. The results have been so consistent in the different studies,13-16 that, although no clinical correlations have been carried, it seems reasonable to use the pattern of E-cadherin staining to classify lesions having equivocal histological finding. This approach should be necessary in only a small fraction of cases. Other studies have focused on cytokeratin staining to

Fig 5. Indeterminate lesion. Neither sharp membranes nor lack of cohesion are evident in this lesion. Lobular carcinoma was present in other areas and the cells in this duct were E-cadherin negative.

higher N/C ratio (Fig 6), the presence of pagetoid spread, and the association with more conventional forms of LCIS. NECROSIS Like high nuclear grade, necrosis has been considered a feature exclusive to DCIS. It has become clear, however, that cases with otherwise typical features of lobular carcinoma can show central necrosis and calcification.5,6,9,10 The necrosis in LCIS is not as extensive as that seen in high-grade DCIS of the comedo type; it generally involves less than 50% of the luminal cross section of a duct or lobule. Necrosis occurs in both pleomorphic and conventional forms of LCIS (Fig 7). Complete necrosis of the carcinoma cells within a given duct, as seen in high-grade DCIS, is not seen in LCIS. IMMUNOHISTOCHEMISTRY In recent years, numerous articles addressing the role of immunohistochemistry in distinguishing different forms of solid in situ carcinoma have appeared. Two

Fig 6. Pleomorphic lobular carcinoma. The cells are large and pleomorphic. The high N/C ratio, the noncohesive arrangement, and the signet ring cells distinguish this lesion from high-grade DCIS.

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ful in discriminating certain equivocal cases, they offer no advantage over staining for E-cadherin alone, and the latter should be the stain of choice in difficult cases. A final note of caution is that studies exploring the relationship between the immunophenotype and the clinical outcome have not been conducted; therefore, conventional microscopy remains the pillar of diagnosis. HYBRID (INDETERMINATE) AND COMBINED CASES Even with careful histological analyses and immunohistochemical studies, a few cases will defy classification. Some will be examples of DCIS and LCIS coexisting in the same area (Fig 8) or even within the same duct (Fig 9),5,19,20 and both diagnoses should be rendered if the cytological features and/or E-cadherin stains support that interpretation. More difficult are cases showing a mixture of both histological pheno-

Fig 7. Lobular carcinoma with necrosis. Lobular carcinoma in a duct showing lack of cohesion and central necrosis.

discriminate between solid DCIS and LCIS. Using antibodies to cytokeratin 8 (CAM5.2; Becton-Dickinson, Mountain View, CA), Lehr et al17 found that ductal carcinomas display a diffuse cytoplasmic staining with accentuation of the at the cell borders (“molding”). Lobular carcinomas, on the other hand, demonstrate a ring like perinuclear distribution (“bag of marbles”). These findings seem to represent the immunohistochemical correlate of the classical, histological, noncohesive appearance of LCIS. Bratthauer et al18 found a similar perinuclear pattern of staining in lobular carcinoma utilizing antibody 34␤E12 against high molecular weight (HMW) cytokeratins (1, 5/6, 10, 14), and essentially no reactivity in classic DCIS. The authors advise strict adherence to protocol in order to obtain consistent results. The typical profile for LCIS in this latter study is negative for E-cadherin and positive for HMW keratin, whereas cases of DCIS displayed the opposite pattern. Some case designated as hybrid reacted with E-cadherin and HMW keratin or, conversely, failed to stain with either antibody. Although the staining patterns seen with cytokeratins seem help-

Fig 8. DCIS (top) and LCIS (bottom) in contiguous lobules. Subtle differences in cells size and cell membranes are seen.

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types (Fig 10) and E-cadherin positive and negative cells. Equally problematic are hybrid cases like those described by Bratthauer, in which the cells stain for both E-cadherin and HMW keratin or fail to stain for either protein. A possible explanation for some of these forms of in situ neoplasia is that they represent transitional forms between DCIS and LCIS. The existence of a common precursor for both these lesions has found support in both molecular and morphological studies.18,21,22 Interestingly, patients with LCIS showing focal E-cadherin reactivity developed ipsilateral carcinomas with ductal features more frequently and did so after shorter intervals than patients with E-cadherin negative LCIS, thus behaving in manner akin to DCIS.23

Fig 10. Admixture of DCIS and LCIS. LCIS cells appear as pale, noncohesive cells. The cells of DCIS appear darker and form microacini.

SUMMARY The classification of solid forms of carcinoma in situ can be challenging. The character of the cell-to-cell relation provides important information and often suffices to identify a proliferation as lobular in type. When the architectural features appear ambiguous or nondistinctive, the nature of coexisting carcinomas often helps to identify the nature of the solid carcinoma in situ. In the absence of coexisting lesions, cytological and immunohistochemical characteristics of the malignant cells will usually point to a diagnosis. REFERENCES

Fig 9. DCIS and LCIS within the same duct. The central cell population represents DCIS and is E-cadherin positive. The peripheral rim is LCIS and did not react with antibodies to E-cadherin. Notice the larger, paler cytoplasm and the occasional microacini in the DCIS.

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