Differential Expression of E-Cadherin in Lobular and Ductal Neoplasms of the Breast and Its Biologic and Diagnostic Implications

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1 Anatomic Pathology / E-CADHERIN EXPRESSION IN BREAST CARCINOMA Differential Expression of E-Cadherin in Lobular and Ductal Neoplasms of the Breast and Its Biologic and Diagnostic Implications Geza Acs, MD, PhD, 1 Thomas J. Lawton, MD, 3 Timothy R. Rebbeck, PhD, 2 Virginia A. LiVolsi, MD, 1 and Paul J. Zhang, MD 1 Key Words: Breast neoplasm; Lobular carcinoma; Ductal carcinoma; E-cadherin Abstract We studied the pattern of E-cadherin expression in 183 invasive carcinomas (100 ductal, 42 lobular, 41 with mixed ductal and lobular features) and 198 in situ carcinomas (131 ductal, 53 lobular, 14 in situ with ductal and lobular features) by immunohistochemistry. We found a highly significant correlation of E-cadherin membrane expression with the histologic phenotype of the tumors. While moderate to strong membrane expression of E-cadherin was seen in all invasive and in situ ductal carcinomas, 41 of 42 invasive and 50 of 53 in situ lobular carcinomas showed complete loss of expression. All in situ carcinomas diagnosed histologically as showing mixed ductal and lobular features demonstrated complete loss of staining. Invasive carcinomas with ductal and lobular features showed 3 staining patterns: (1) complete or almost complete lack of membrane staining similar to that seen in lobular carcinomas, (2) uniform membrane expression throughout the tumor similar to ductal carcinomas, and (3) focal loss of E-cadherin staining, which correlated well with the histologic impression of focal lobular features. In tumors with histologically equivocal features, immunohistochemical detection of E-cadherin expression can be a useful diagnostic tool for the differentiation of ductal and lobular carcinomas of the breast. The classification of mammary carcinoma traditionally is based on the histologic phenotype of the tumor. Breast cancers are classified as ductal or lobular carcinomas, according to certain distinct morphologic features noted by microscopic examination. Although generally accepted histologic criteria serve to distinguish lobular from ductal carcinoma of the breast, 1-3 this differential diagnosis may present a challenge in poorly differentiated carcinomas showing equivocal histologic features with a diffuse infiltrating pattern and in the pleomorphic variant of invasive lobular carcinoma. 1,4,5 A similar diagnostic challenge also occurs in in situ carcinomas of the breast. Although some in situ carcinomas composed of distinct lobular and ductal components have been categorized as truly mixed lesions, 6 other tumors defy clear classification owing to the presence of equivocal histologic features. Lobular carcinoma in situ (LCIS) may be mimicked by low-grade ductal carcinoma in situ (DCIS) with a solid growth pattern involving terminal ducts and lobules. 7,8 A related problem is presented by lesions with typical lobular cytology that have large areas of central comedo-type necrosis. 7,9 Finally, an in situ carcinoma involving lobules that is composed of large, pleomorphic, discohesive tumor cells and signet-ring cells, with or without necrosis (so-called pleomorphic LCIS), also may be difficult to classify. 7,9-11 A diagnosis of in situ carcinoma with ductal and lobular features (DLCIS) has been suggested for these cases. 7,8,12 Although the clear distinction of ductal and lobular types in invasive mammary carcinoma is more or less an academic exercise, the distinction of in situ lesions would be clinically important. LCIS generally is managed conservatively, since it is currently regarded as a high-risk factor for the development of invasive carcinoma, rather than an obligate precancerous lesion. DCIS, on the other hand, is considered to be an American Society of Clinical Pathologists Am J Clin Pathol 2001;115:

2 Acs et al / E-CADHERIN EXPRESSION IN BREAST CARCINOMA obligate precancerous lesion and is managed surgically with the goal of complete removal of the tumor. 7,13-15 Although little is known about the biology and clinical significance of DLCIS, these lesions tend to be managed clinically as DCIS. E-cadherin is a member of a family of transmembrane glycoproteins responsible for the Ca 2+ -dependent cell-cell adhesion mechanism and has been demonstrated to be involved in organogenesis and morphogenesis In epithelial cells, E-cadherin is considered one of the key molecules for the formation of the intercellular junctional complex and for the establishment of cell polarization. 19 Expression of reduced numbers of functionally active E-cadherin molecules has been observed in aggressive tumors of the esophagus, ovary, and stomach, and decreased expression of E-cadherin is thought to be associated with invasiveness of the tumor cells. 20 Previous studies have shown reduced expression of E- cadherin in approximately 50% of mammary carcinomas in association with high histologic grade, nodal metastases, and loss of estrogen and progesterone receptors However, not all studies confirmed these findings. 30 In invasive lobular carcinoma (ILC) and LCIS, a complete loss of expression has been observed. 23,24,31 It was shown that E-cadherin gene mutations and loss of the wild-type allele by loss of heterozygosity is the predominant mechanism by which E- cadherin protein expression frequently is lost in lobular carcinoma, indicating that E-cadherin acts as a classic tumor suppressor gene E-cadherin germline mutations in gastric and lobular breast carcinoma were reported recently, 35,36 suggesting the importance of E-cadherin mutations in tumorigenesis. Thus, there is emerging evidence that E-cadherin is associated specifically with the lobular phenotype of breast carcinoma, and E-cadherin inactivation might have a crucial role in the dispersed and discohesive growth pattern in LCIS and ILC. 31,32 However, the practicality of using E-cadherin expression to differentiate between ductal and lobular carcinomas in a large series, including cases with equivocal features, has not been evaluated. In the present study, we examined the pattern of E- cadherin expression in a large series of ductal and lobular carcinomas of the breast. We analyzed the correlation between E-cadherin expression and various clinicohistopathologic features of the tumors, including those with equivocal ductal and lobular features, to further evaluate its role in the morphogenesis of breast carcinomas and its role in the classification of carcinomas with equivocal features. Materials and Methods We selected 214 breast biopsy specimens containing at least in situ carcinoma from the surgical pathology files of the Department of Pathology of the University of Pennsylvania Medical Center, Philadelphia. H&E-stained slides were reviewed independently by 2 pathologists (G.A. and P.J.Z.) to establish a diagnosis with histologic type and tumor grade based on established criteria 1-3 without knowledge of the results of the immunohistochemical assays. Discrepancies in diagnosis regarding tumor type and grade were resolved by simultaneous viewing and consultation with a third pathologist (V.A.L.). All invasive carcinomas were graded using the modified combined histologic grading system as described by Elston and Ellis. 37 Ductal carcinomas in situ were graded as described by Scott et al 38 using primarily nuclear grade. After the initial histologic examination, there were 183 invasive carcinomas, which were divided into invasive ductal carcinoma (IDC; n = 100), invasive lobular carcinoma (ILC; n = 42), and invasive carcinoma with ductal and lobular features (IDLC; n = 41), and 198 in situ carcinomas, which were further classified as DCIS (n = 131), LCIS (n = 53), and DLCIS (n = 14) Table 1. Invasive carcinomas showing mixed ductal and lobular features were examined further by analyzing the following features within the tumor: presence of nests or trabeculae of tumor cells, formation of tubules or true lumina within nests of tumor cells, presence of intracellular lumina, and presence of discohesion. Data on tumor size, axillary lymph node status, estrogen and progesterone receptor status, and erbb-2 overexpression were retrieved from the pathology reports. Immunohistochemical assays were performed on formalin-fixed paraffin-embedded sections. Five-micrometerthick sections were cut and deparaffinized in xylene and rehydrated in graded alcohols. Slides were steamed in a 0.01-mol/L concentration of sodium citrate buffer (ph 6.0) for 20 minutes. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 20 minutes. After blocking with 1.5% normal horse serum (Vector Laboratories, Burlingame, CA) in Automation Buffer (ph 7.0, Biomeda, Foster City, CA), slides were incubated with the monoclonal antibody against E-cadherin (1:200 dilution, HECD-1 clone, Zymed Laboratories, South San Francisco, CA) overnight at 4 C. Slides then were washed 3 times with Automation Buffer and incubated for 30 minutes at 37 C with biotinylated horse antimouse immunoglobulin G (heavy and light chains) secondary antibody (1:200 dilution, Vector Laboratories). After incubation with horseradish peroxidase conjugated streptavidin (Streptavidin HRP Detection System, Research Genetics, Huntsville, AL) for 40 minutes at 37 C, slides were developed with stable diaminobenzidine (Research Genetics) for 5 minutes and counterstained with hematoxylin. Immunohistochemical stains were evaluated independently by 2 pathologists (G.A. and P.J.Z.). Slight differences 86 Am J Clin Pathol 2001;115:85-98 American Society of Clinical Pathologists

3 Anatomic Pathology / ORIGINAL ARTICLE Table 1 Classification of Invasive and In Situ Breast Carcinomas Based on Histologic Features No. of Cases IDC Alone 15 With DCIS 81 With both DCIS and LCIS 4 Total 100 DCIS Alone 14 With LCIS 8 With IDC 81 With IDC and LCIS 4 With ILC 1 With IDLC 16 With IDLC and LCIS 7 Total 131 ILC Alone 11 With LCIS 22 With DCIS 1 With DLCIS 8 Total 42 LCIS Alone 6 With ILC 22 With IDC 1 With IDC and DCIS 4 With IDLC 7 With IDLC and DCIS 5 With DCIS 8 Total 53 IDLC Alone 10 With DCIS 16 With LCIS 7 With both DCIS and LCIS 5 With DLCIS 3 Total 41 DLCIS Alone 3 With ILC 8 With IDLC 3 Total 14 DCIS, ductal carcinoma in situ; DLCIS, in situ carcinoma with mixed ductal and lobular features; IDC, invasive ductal carcinoma; IDLC, invasive carcinoma with mixed ductal and lobular features; ILC, invasive lobular carcinoma; LCIS, lobular carcinoma in situ. in interpretation were resolved by simultaneous viewing and reevaluation by a third pathologist (V.A.L.). Only clear membranous staining was considered positive. The intensity of the membrane reactivity was scored semiquantitatively on a 4-tiered scale as follows: 3, strong complete membrane staining, comparable to benign ductal and lobular epithelial cells, clearly visible with the 10 objective; 2, moderate clear membrane staining visible with the 20 objective; 1, weak but still complete membrane staining visible with the 40 objective; 0, absent or incomplete membrane staining. Then a score was generated by multiplying the intensity of staining with the percentage of cells exhibiting positive staining, resulting in a possible score of 0 to 300. The presence of E-cadherin staining in epithelial cells of normal ducts and acini served as an internal positive control in every case. We studied the correlation between E-cadherin expression and the following variables: tumor type, size, combined tumor grade and the various components of the combined grading system, lymph node status, estrogen and progesterone receptor status, and erbb-2 overexpression. For IDLC, the correlation between E-cadherin staining and the following variables was analyzed: presence or absence of tumor cell nests and trabeculae, tubule and lumen formation, intracytoplasmic lumina, discohesion, and different in situ components. For statistical analysis, the Mann-Whitney rank sum test and Kruskal-Wallis 1-way analysis of variance by ranks were used, followed by the Dunn multiple comparison test, when appropriate. The correlation between the levels of E-cadherin staining in invasive and in situ components of ductal carcinomas was estimated using the Spearman rank correlation test. A 2-sided P value less than.05 was considered statistically significant. Results Moderate to strong membrane expression of E-cadherin was identified in benign mammary epithelium and all invasive (n = 100) and in situ (n = 131) tumors histologically classified as ductal carcinomas Table 2 Table 3 Figure 1 Image 1. E-cadherin expression was present throughout the tumor in 96 (96.0%) of 100 IDC and 128 (97.7%) of 131 DCIS cases, whereas focal complete loss of membrane staining involving a proportion of tumor cells was seen in 4 IDCs (4.0%; score 50 and 70 in 1 case each; score 80 in 2 cases) and 3 DCISs (2.3%; score 50 in 2 cases; score 80 in 1 case). The pattern and intensity of staining in the invasive and in situ components of the same tumors were very similar (r = ; P <.001, Spearman test). There was no correlation between E-cadherin staining and the following variables: tumor size, tumor grade, tubule formation, nuclear pleomorphism, mitotic activity, axillary lymph node status, estrogen and progesterone receptor status, and erbb-2 overexpression (P >.05, Kruskal-Wallis or Mann-Whitney test; Table 2). Similarly, no correlation was found between E- cadherin expression and histologic type and grade of DCIS (P >.05, Kruskal-Wallis test; Table 3). In contrast with ductal carcinomas, 41 (98%) of 42 ILCs showed complete loss of membrane expression of E- cadherin (Figure 1) and Image 2A, although weak to moderate cytoplasmic staining was present in 14 cases. No difference in staining was detected between classic (n = 31), pleomorphic (n = 9), and solid (n = 2) variants of ILC Image 2B. One tumor, although histologically classified as American Society of Clinical Pathologists Am J Clin Pathol 2001;115:

4 Acs et al / E-CADHERIN EXPRESSION IN BREAST CARCINOMA Table 2 Membrane Expression of E-Cadherin in Invasive Ductal Carcinoma (IDC) E-Cadherin Staining Score IDC No. of Cases Median Range Mean ± SEM P * Total ± 6.5 Size >.05 T ± 8.5 T ± 12.3 T ± 22.5 Grade >.05 I ± 12.4 II ± 10.1 III ± 11.6 Tubule formation > ± ± ± 8.4 Nuclear pleomorphism > ± ± ± 8.9 Mitotic activity > ± ± ± 16.2 Nodal status >.05 Positive ± 9.8 Negative ± 9.7 ER status >.05 Positive ± 9.8 Negative ± 9.3 PR status >.05 Positive ± 9.0 Negative ± 10.5 ErbB-2 status >.05 Positive ± 9.6 Negative ± 9.8 ER, estrogen receptor; PR, progesterone receptor. * Comparison with expression of E-cadherin. Mann-Whitney or Kruskal-Wallis test. As part of the combined histologic grading system. 37 Image 1 Strong membrane expression of E-cadherin in cells of in situ and invasive ductal carcinoma (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 100). pleomorphic ILC, showed moderate membrane staining in 70% of the tumor cells (score 140) Image 3. This tumor was associated with an intermediate-grade solid DCIS. Similarly, 50 (94%) of 53 LCIS cases showed complete loss of membrane expression of E-cadherin (Figure 1) with weak focal membrane staining (scores 20-50) identified in the remaining 3 cases. Perinuclear dot-like staining also was seen in 2 cases of LCIS Image 4A. In ducts showing intramural spread of LCIS, the E-cadherin negative tumor cells were interspersed between the myoepithelial cells and an inner layer of remnant E-cadherin positive luminal cells Image 4B. In lobules partially occupied by LCIS cells, the tumor cell population was outlined by its negativity for E- cadherin, contrasting with the positive staining of the residual terminal ductule epithelium. Separate foci of IDC and ILC were identified in the same breast in 1 case. The tumors exhibited a marked difference in E-cadherin expression. While the IDC showed strong uniform membrane staining (score 300), the ILC demonstrated a 88 Am J Clin Pathol 2001;115:85-98 American Society of Clinical Pathologists

5 Anatomic Pathology / ORIGINAL ARTICLE A B Image 2 A, Complete loss of E-cadherin expression in cells of invasive lobular carcinoma, classic type. Note the targetoid pattern of infiltration by the tumor cells around a small duct, which shows strong membrane staining for E-cadherin (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 100). B, Complete loss of E-cadherin expression in cells of pleomorphic invasive lobular carcinoma (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 200). complete loss of membrane expression of E-cadherin Image 5. In general, when both DCIS and LCIS were present in the same biopsy specimen, with or without an invasive component, they showed a marked difference in E-cadherin staining Image 6A. Even when DCIS and LCIS were present within the same terminal duct lobular unit (true mixed in situ carcinoma, n = 3), the distinct components showed a clear difference in E-cadherin staining Image 6B and Image 6C. Comparison of E-cadherin staining in IDC and ILC revealed a highly statistically significant difference between the 2 groups (P <.001, Kruskal-Wallis test with Dunn multiple comparison test) (Figure 1). Similarly, a highly significant difference was observed between DCIS and LCIS (P <.001, Kruskal-Wallis test with Dunn multiple comparison test) (Figure 1). Fourteen in situ carcinomas were identified that showed equivocal, mixed ductal and lobular features histologically (DLCIS), because the lesion was composed of small, uniform cells, similar to the cells of typical LCIS but that filled and distended larger ducts as well as lobular units and demonstrated large central areas of comedo-type necrosis (n = 9) Image 7A, or of discohesive, large tumor cells with abundant cytoplasm, intracytoplasmic lumina, and pleomorphic nuclei, filling large ducts and adjacent lobular units with or without central necrosis (n = 5) Image 7B. 7,12 Eleven (79%) of 14 DLCIS cases were associated with an invasive carcinoma: 5 with pleomorphic ILC, 3 with classic ILC, Table 3 Membrane Expression of E-Cadherin in Ductal Carcinoma In Situ (DCIS) E-Cadherin Staining Score DCIS No. of Cases Median Range Mean ± SEM P * Total ± 5.1 Grade >.05 I ± 12.0 II ± 8.7 III ± 6.6 Type >.05 Comedo ± 7.3 Cribriform ± 8.2 Solid ± 11.0 Micropapillary ± 19.2 * Compared with expression of E-cadherin. Kruskal-Wallis test. American Society of Clinical Pathologists Am J Clin Pathol 2001;115:

6 Acs et al / E-CADHERIN EXPRESSION IN BREAST CARCINOMA E-Cadherin Staining Score IDC ILC IDLC DCIS LCIS DLCIS Figure 1 Distribution of the level of E-cadherin membrane expression in invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), invasive carcinoma with ductal and lobular features (IDLC), ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and in situ carcinomas with ductal and lobular features (DLCIS). Bars indicate the median value of E- cadherin staining scores. * P <.01. P <.001. NS (not significant), P >.05 (Kruskal-Wallis test with Dunn multiple comparison test). and 3 with IDLC. All 14 DLCIS cases showed a complete lack of membrane expression of E-cadherin (Figure 1) Image 7C Image 7D. A statistically significant difference was observed between DCIS and DLCIS (P <.001, Kruskal-Wallis test with Dunn multiple comparison test). No NS difference in E-cadherin staining was found between LCIS and DLCIS cases. Forty-one invasive carcinomas were classified as having mixed ductal and lobular features histologically (IDLC), owing to the presence of a mixture in variable degrees of ductal cytology and growth pattern (large, pleomorphic cells with cohesive cellular arrangement with or without lumen formation) and typical lobular pattern (dispersed infiltrating fashion) in the same lesion, or to occasional tubules or small nest formation in a lesion that otherwise showed a typical lobular type of infiltration and cytology. In the 41 IDLC cases, LCIS or DCIS was identified in 7 cases (17%) and 16 cases (39%), respectively; concurrent LCIS and DCIS were seen in 5 cases (12%), and DLCIS was present in 3 cases (7%). As a group, the level of E-cadherin expression in IDLC was between those in IDC and ILC, with significant differences from both IDC and ILC (P <.001, Kruskal-Wallis test with Dunn multiple comparison test) (Figure 1). However, 3 patterns of E-cadherin expression were recognized in this group of tumors Figure 2 : (1) In the first group ( lobularlike IDLC, n = 10), there was complete (n = 9, score 0) or almost complete (n = 1, score 20) loss of E-cadherin staining, similar to that seen in typical ILCs. In situ lesions were present in all but 1 case and were mainly LCIS (n = 5) and DLCIS (n = 2), with 1 concurrent LCIS and DCIS and 1 DCIS. (2) In the second group ( ductal-like IDLC, n = 24), there was uniform membrane expression of E-cadherin throughout the tumor similar to that seen in typical ductal carcinomas. Seventeen (71%) of 24 cases contained in situ A B Image 3 A, Invasive carcinoma showing a dispersed, single-cell infiltrating pattern. The tumor cells show moderate nuclear pleomorphism. This tumor was classified histologically as pleomorphic invasive lobular carcinoma (H&E, original magnification 200). B, The tumor cells show moderate membrane expression of E-cadherin (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 200). 90 Am J Clin Pathol 2001;115:85-98 American Society of Clinical Pathologists

7 Anatomic Pathology / ORIGINAL ARTICLE A B Image 4 A, Complete lack of membrane expression of E-cadherin in lobular carcinoma in situ. Note the perinuclear dot-like reactivity in some tumor cells (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 400). B, Ductal involvement by cells of in situ lobular carcinoma. The E-cadherin negative tumor cells are interspersed between the myoepithelial cells and an inner layer of remnant E-cadherin positive luminal cells (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 200). E-Cadherin Staining Score ILC IDLC I IDLC III IDLC II IDC Figure 2 Distribution of the level of E-cadherin membrane expression in invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), and invasive carcinoma with ductal and lobular features (IDLC). Three patterns of staining were recognized in IDLC: lobular-like (IDLC I) showed complete or almost complete lack of E-cadherin staining similar to that seen in ILC; ductal-like (IDLC II) demonstrated uniform membrane expression of E-cadherin throughout the tumor similar to that seen in typical ductal carcinomas; intermediate (IDLC III) showed focal loss of E-cadherin staining. Statistical analysis revealed no significant difference between IDLC I and typical ILC or between IDLC II and typical IDC. A significant difference was found between IDLC I and IDLC II (P <.001), IDLC I and typical IDC (P <.001), and IDLC II and typical ILC (P <.001). No statistically significant difference was found between IDLC III and IDLC I or IDLC II or between typical IDC and ILC (P >.05, Kruskal-Wallis test with Dunn multiple comparison test). Bars indicate the median value of E-cadherin staining scores. lesions that, different from those of the first group, were mainly DCIS (n = 13), with 3 concurrent DCIS and LCIS and 1 LCIS. (3) In the third group ( intermediate IDLC, n = 7), there was focal complete loss of E-cadherin staining (score ) Image 8. The associated in situ lesion was DCIS in 2 and LCIS, DLCIS, and concurrent DCIS and LCIS in 1 case each. Statistical comparison of the E-cadherin staining scores of these groups with each other and with the scores of invasive ductal and lobular carcinomas was performed (Figure 2). There was no significant difference between the lobularlike group and typical ILC or between the ductal-like group and typical IDC. Significant differences were found between the lobular-like and the ductal-like groups (P <.001), the lobular-like group and typical IDC (P <.001), and the ductal-like group and typical ILC (P <.001). However, we found no statistically significant difference between the intermediate group and either groups 1 and 2 in IDLC or typical IDC and ILC (P >.05, Kruskal-Wallis test with Dunn multiple comparison test). The complete loss of immunostaining correlated well with the histologic impression of lobular features and lack of tubule or lumen formation (P <.005, Mann-Whitney test) and presence of intracytoplasmic lumina (P <.001, Mann- Whitney test) exhibited by IDLC Table 4. E-cadherin staining also was reduced significantly in IDLC accompanied by LCIS compared with IDLC without an LCIS component (P <.01, Mann-Whitney test) and IDLC with only a DCIS component (P <.05, Mann-Whitney test). American Society of Clinical Pathologists Am J Clin Pathol 2001;115:

8 Acs et al / E-CADHERIN EXPRESSION IN BREAST CARCINOMA A B C D Image 5 Separate foci of invasive ductal (A) and lobular (B) carcinomas in the same breast specimen (H&E, original magnification 100 [A] and 200 [B]). The tumors exhibit a marked difference in E-cadherin expression: While the invasive ductal carcinoma shows strong membrane staining (C), the invasive lobular carcinoma demonstrates a complete loss of membrane expression of E-cadherin (D) (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 100 [C] and 200 [D]). No statistically significant correlation was found between E-cadherin expression and the presence of discohesive growth in IDLC. Discussion We analyzed the expression of E-cadherin in 198 in situ and 183 invasive mammary carcinomas. We found a highly significant correlation of E-cadherin membrane expression with the histologic phenotype of tumors. All 131 DCIS and all 100 IDC cases showed moderate to strong membrane expression of E-cadherin, as seen in the nonneoplastic mammary epithelium. In contrast, membrane expression of E-cadherin was identified in only 1 (2%) of 42 ILCs (moderate reactivity) and 3 (6%) of 53 cases of LCIS (weak and focal reactivity). Based on our data, the sensitivity and specificity of the complete lack of membrane expression of E-cadherin (score 0) to predict the lobular phenotype in invasive and in situ carcinomas were 97.6% and 96.0%, and 94.3% and 97.7%, respectively. The sensitivity and specificity of any positive membrane staining (score >0) to predict the ductal phenotype in invasive and in situ carcinomas were 96.0% and 97.6%, and 97.7% and 94.3%, respectively. If a score of 92 Am J Clin Pathol 2001;115:85-98 American Society of Clinical Pathologists

9 Anatomic Pathology / ORIGINAL ARTICLE A B C Image 6 A, Separate foci of in situ ductal and lobular carcinomas in the same breast specimen show a marked difference in E-cadherin staining. While the in situ ductal carcinoma shows strong membrane staining, the in situ lobular carcinoma demonstrates a complete loss of membrane expression of E-cadherin. Note the complete loss of E-cadherin membrane staining in the associated invasive lobular carcinoma as well (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 200). B, In situ mammary carcinoma, truly mixed ductal and lobular type. The ductal carcinoma in situ (DCIS) component shows strong membrane expression of E-cadherin in sharp contrast with the adjacent lobular component, which demonstrates a complete lack of staining (immunoperoxidase stain for E- cadherin with hematoxylin counterstain, original magnification 200). C, Truly mixed in situ ductal and lobular carcinoma. The cribriform areas of low-grade DCIS show strong membrane expression of E-cadherin, while the adjacent lobular carcinoma in situ component demonstrates complete loss of membrane staining (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 400). more than 50 is considered positive for E-cadherin membrane staining, the sensitivity and specificity of the lack of membrane expression of E-cadherin (score 50 or less) to predict the lobular phenotype in invasive and in situ carcinomas were 97.6% and 99.0%, and 100.0% and 98.5%, respectively. In this case, the sensitivity and specificity of positive membrane staining (score >50) to predict the ductal phenotype in invasive and in situ carcinomas were 99.0% and 97.6%, and 98.5% and 100.0%, respectively. Interestingly, the only E-cadherin positive ILC case was diagnosed histologically as pleomorphic lobular carcinoma and was associated with an intermediate-grade solid DCIS component. Given the well-known difficulty of differentiating the pleomorphic variant of lobular carcinoma from ductal carcinoma with a dispersed infiltrating pattern, 3 this tumor likely represents an example of the latter. Our data are similar to the findings of Moll et al 24 and Gamallo et al, 23 who also reported a complete loss of E- cadherin expression in the majority of ILCs, while all cases of ductal carcinoma retained at least some expression of E- cadherin. Interestingly, we found perinuclear dot-like staining in 2 cases of LCIS. Such perinuclear staining is thought to result from mutant E-cadherin, which is incorrectly processed within the Golgi apparatus, or from accelerated protein turnover. 22 Currently, the classification of breast carcinomas is based purely on histologic examination. Although wellaccepted histologic features have served to classify the vast American Society of Clinical Pathologists Am J Clin Pathol 2001;115:

10 Acs et al / E-CADHERIN EXPRESSION IN BREAST CARCINOMA A B C D Image 7 A, In situ mammary carcinoma with ductal and lobular features (DLCIS) involving ducts and lobular units. While the growth pattern is ductal, the tumor is composed of small uniform cells characteristic of lobular carcinoma in situ (LCIS) and demonstrates large central areas of comedo-type necrosis (H&E, original magnification 50). B, DLCIS. The lesion is composed of discohesive, large tumor cells with abundant cytoplasm, intracytoplasmic lumina, and pleomorphic nuclei that fill large ducts and adjacent lobular units (H&E, original magnification 100). C, The tumor cells show complete loss of membrane expression of E-cadherin, characteristic of LCIS. Note the strong membrane staining in the residual normal epithelium (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 100). D, The tumor cells show complete loss of membrane expression of E-cadherin, characteristic of LCIS (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 100). majority of tumors, in a small number of cases, a clear-cut histologic classification into ductal or lobular types is difficult owing to the presence of both ductal and lobular growth patterns within the same tumor or other equivocal histologic features. In routine practice, many of these tumors are diagnosed as mammary carcinoma with ductal and lobular features or are classified as ductal or lobular type arbitrarily. We found 14 in situ and 41 invasive carcinomas with mixed features in our series. While the ductal vs lobular distinction in invasive carcinomas has little clinical significance, the classification in the case of in situ carcinomas bears significant clinical importance in patient management. Because of the strong correlation of E-cadherin expression in otherwise typical lobular and ductal carcinomas, as demonstrated in previous reports 23,24 and the present study, it seems reasonable to use immunohistochemical staining for E-cadherin to 94 Am J Clin Pathol 2001;115:85-98 American Society of Clinical Pathologists

11 Anatomic Pathology / ORIGINAL ARTICLE Table 4 Membrane Expression of E-Cadherin in Invasive Carcinoma With Ductal and Lobular Features (IDLC) E-Cadherin Staining Score IDLC No. of Cases Median Range Mean ± SEM P * Total ± 18.0 Tubules <.005 Present ± 18.0 Absent ± 32.7 Nests and trabeculae Present ± 18.5 Absent 1 90 Intracellular lumina <.001 Present ± 26.8 Absent ± 14.7 Discohesion >.05 Present ± 63.8 Absent ± 18.7 LCIS <.01 Present ± 32.7 Absent ± 17.6 DCIS >.05 Present ± 21.8 Absent ± 31.8 Presence of LCIS only ± 42.1 <.05 Presence of DCIS only ± 21.3 DCIS, ductal carcinoma in situ; LCIS, lobular carcinoma in situ. * Compared with expression of E-cadherin. Kruskal-Wallis test. Mann-Whitney test. Statistical analysis was not performed. enhance our ability to classify breast carcinomas in difficult cases with histologically equivocal features. In the 41 IDLCs, 10 cases (24%) and 24 cases (58%) showed E-cadherin expression similar to typical lobular and ductal carcinomas, respectively, and, therefore, these cases could be further classified accordingly based on immunohistochemical detection of E-cadherin. Only 7 (3.8%) of 182 invasive breast carcinomas remained histologically and immunophenotypically mixed ductal and lobular carcinoma. Interestingly, in in situ lesions, all 14 DLCIS cases showed a lobular type of E-cadherin expression with complete loss of staining in all cases. Furthermore, the invasive components associated with DLCIS were of the lobular type in 8 (73%) of 11 cases and mixed in the remaining 3 cases (27%). These findings suggest that DLCIS and typical LCIS share similar biologic characteristics and are closely related. It seems most likely that the DLCIS cases identified in our series represent LCIS with primarily ductal involvement and comedo-like necrosis 9 or pleomorphic LCIS 10,11 with ductal involvement. Currently, any in situ carcinoma diagnosed to have mixed lobular and ductal features likely would be treated as DCIS. 12 Although this approach might be appropriate for tumors with mixed distinct ductal and lobular components (truly mixed ductal and lobular in situ carcinomas), it might not be so in lesions with equivocal ductal and lobular features (DLCIS), which failed to show E-cadherin expression (a feature of ductal differentiation). Our findings suggest that it is necessary and feasible to distinguish between truly mixed in situ carcinomas and those with equivocal features owing to the potential difference in their biologic behavior. Furthermore, excluding truly mixed lesions, DLCISs are likely lobular neoplasms, which could be confirmed by E-cadherin immunostaining. Larger series of such cases with E-cadherin staining and clinical follow-up are needed to elucidate further the biologic behavior and clinical significance of DLCIS. 7,12 The fact that 11 of the 14 cases in our series were associated with an invasive component suggests a more aggressive biologic behavior compared with typical LCIS. We found that IDLC associated with LCIS showed significantly lower E-cadherin expression compared with cases without an LCIS component, while the presence or absence of DCIS in itself showed no correlation with E- cadherin staining. Overall, in IDLC there was a strong correlation between loss of E-cadherin membrane expression and both lack of tubule formation and the presence of intracellular lumina. It was suggested that the discohesive growth pattern in lobular carcinomas may be the result of loss of E- cadherin function. 23,24 Although we found a clear tendency for reduced E-cadherin staining in IDLCs showing discohesion, it did not reach statistical significance, likely owing to the small number of cases with discohesive growth (Table 4). Another explanation may be that loss of E-cadherin cellular adhesion is associated more with the formation of LCIS than with the maintenance of the discohesive, typical lobular invasive growth pattern. 31 IDCs also may exhibit a dispersed American Society of Clinical Pathologists Am J Clin Pathol 2001;115:

12 Acs et al / E-CADHERIN EXPRESSION IN BREAST CARCINOMA A B Image 8 Invasive carcinomas with ductal and lobular features showing focal loss of E-cadherin staining (IDLC III). While the majority of the tumor was composed of large tumor cells forming cohesive nests and trabeculae with lumen formation characteristic of ductal carcinoma (A), a portion of the tumor exhibited discohesive, dispersed infiltrative growth and intracytoplasmic lumina in the tumor cells, reminiscent of lobular carcinoma (B). While the former areas of the tumor showed moderate membrane expression of E-cadherin (A), a complete loss of staining was found in the latter areas (B) (immunoperoxidase stain for E-cadherin with hematoxylin counterstain, original magnification 200). growth pattern, including infiltration around benign ducts in a targetoid fashion. Recently, cell motility factors, such as scatter factor and acidic fibroblast growth factor, that induce invasiveness of diverse epithelial cell lines in vitro without changing E-cadherin expression have been described Such factors might promote invasiveness of ductal carcinomas with high E-cadherin expression. Alternatively, perturbation of the cadherin cell adhesion system without loss of cadherin may occur as a result of loss of catenin function, eg, owing to phosphorylation of its tyrosine residues. 20 Evidence suggests that in fact a wide variety of tyrosine kinases leads to the perturbation of the cadherin system, possibly through phosphorylation of cadherin-associated proteins. 20 In our series, we found no correlation between E- cadherin membrane expression and the following variables: tumor size, grade, tubule formation, nuclear pleomorphism, mitotic activity, estrogen and progesterone receptor status, and erbb-2 overexpression in IDCs. While this finding is similar to that of Lipponen et al, 30 others have demonstrated a correlation between reduced expression of E-cadherin and tumor grade, lymph node status, 25,29 negative estrogen receptor status, 30 lack of tubule formation, 23 and mitotic activity. 23 Although most studies analyzed the same clinicopathologic features, no consistent correlation of E-cadherin staining emerged with any of these features. In contrast with the findings of Gupta et al, 28 we did not find a significant correlation between E-cadherin expression and grade or type of in situ ductal carcinomas. Although Berx et al 32 also observed a variation in E- cadherin staining intensity in ductal carcinomas, they suggested that this finding is most likely due to technical artifact without any biologic significance. In our series, a minority of IDLC showed real heterogeneity in E-cadherin expression. In such cases, areas of the tumor showing complete lack of membrane staining correlated well with the histologic impression of lobular features exhibited by the tumor (Image 8). There are several possible explanations for these findings. These tumors, especially when associated with both DCIS and LCIS, may represent truly mixed ductal and lobular carcinomas. Alternatively, since loss of heterozygosity and other alterations of chromosome 16q are frequent aberrations in ductal carcinomas, 42,43 it also is possible that loss of E-cadherin expression owing to loss of chromosome 16q or mutation of the E-cadherin gene may be a secondary random event in the evolution of ductal carcinomas and, therefore, difficult to detect 32 ; so far, no E-cadherin mutations have been demonstrated in ductal carcinomas. On the other hand, reversible and focal down-regulation of E- cadherin expression was demonstrated in in vivo experiments. 44 Reversible down-regulation of E-cadherin may occur by hypermethylation of the promoter region of E- cadherin. 45 The reversibility of the down-regulation of E- cadherin expression in ductal carcinomas also is demonstrated by the finding that the invasion suppressor activity of E-cadherin can be restored by treating the tumor cells with the antiestrogen tamoxifen Am J Clin Pathol 2001;115:85-98 American Society of Clinical Pathologists

13 Anatomic Pathology / ORIGINAL ARTICLE It was shown that in the development of lobular breast carcinoma, mutation of the E-cadherin gene occurs at a very early stage and is already present in LCIS without invasion and in LCIS adjacent to an invasive component, as well as in ILC The common lack of E-cadherin as a distinctive feature of LCIS and ILC supports the hypothesis that both are related closely and constitute one distinct tumor entity 24 and suggests that, in contrast with current theories, LCIS may in fact represent a true precancerous lesion. Complete loss of E-cadherin expression has never been observed in DCIS, and no inactivating E-cadherin gene mutations have been detected in ductal carcinomas, 32,47 indicating different genetic pathways for the development of LCIS and DCIS 28,33 and providing the clearest evidence of molecular difference between the two main histologic types of breast carcinomas. 32 Molecular analysis of the E-cadherin gene in carcinomas with mixed ductal and lobular features could help further in the classification of these tumors with equivocal features. We confirmed that there is a strong correlation between the histologic phenotype of mammary carcinomas and membrane expression of E-cadherin by the tumor cells. These findings support the significant role of E-cadherin in the morphogenesis of breast carcinomas. In tumors with histologically equivocal features, immunohistochemical detection of E-cadherin expression can be used as a useful diagnostic tool in the differentiation of ductal and lobular carcinomas of the breast. Tumors that show complete lack of E-cadherin membrane staining most likely represent lobular carcinomas, while tumors demonstrating diffuse membrane expression of E-cadherin most likely represent ductal carcinomas. From the 1 Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center and the 2 Center for Clinical Epidemiology and Biostatistics and Cancer Center, University of Pennsylvania, Philadelphia; and the 3 Department of Pathology, University of Washington Medical Center, Seattle. 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