In this study, we sought to evaluate the expression of E-cadherin and β-catenin for the immunophenotypical characterization of CISM. We also searched for potential morphological patterns that could help in the diagnosis of different types of CISM lesions. We adopted the morphological classification described by Jacobs et al. that defines CISM as “carcinomas with indeterminate features”. According to this classification CISM lesions are divided in three main groups, namely: (I) presence of necrosis, (II) cytology and / or mixed architecture, and (III) nuclear polymorphism. This classification is highly reproducible and addresses the main morphological groups described in our study.
A total of 25 CISM cases were evaluated in this study. The most common morphological pattern of lesions identified belonged to group (II) with 19 out of 25 cases (76%), followed by group (III) (2/25 cases - 8%), and overlapping patterns between groups (I) and (II) (2/25 cases - 8%) and groups (II) and (III) (2/25 cases - 8%). Our findings are in agreement with those reported by Jacobs et al. who observed, in 28 cases of CISM, 60% of the lesions in group (II) (17/28 cases), 21% in group (I) (5/28 cases) , and 18% in group (III) (5/28 cases).
However, it is noteworthy that the terminology and morphologic criteria used for the diagnosis of CISM are heterogeneous. Fisher et al. termed it as “ductolobular carcinoma in situ” lesions with monomorphic cells with foci of necrosis or cribriform pattern . Acs et al. described 14 cases of CISM referring to the lesions as “with ductal carcinoma in situ and lobular features” and adopted, as a diagnostic criteria, LN in situ lesions with cytological and architectural patterns, with central areas of comedonecrosis or lobules, or large duct units populated by non-cohesive cells with marked nuclear pleomorphism . Maluf et al. analyzed 12 cases of “solid low grade carcinoma in situ of the breast” and included “low-grade solid DCIS, LCIS and DCIS and LCIS associated with invasive carcinomas of any type. Cases showing only unequivocal areas of LCIS or DCIS of nonsolid type were excluded” .
Even among experts in the pathology of breast tumors, the descriptions of these lesions are divergent. Page and Anderson state that in most cases an attempt should be made to classify the lesions as LN or DCIS . However, on rare occasions this might not be possible and the diagnosis of “in situ carcinoma of ductal or lobular type” needs to be made. These authors recommend that if more than one focus of necrosis is found, the lesion should not be classified as LN. Rosen describes two main types of CISM: “concurrent intraductal and lobular carcinoma in situ” for lesions that present a cytology of lobular pattern and distended ducts and central necrosis or calcifications, and “coexistent intraductal and lobular carcinoma in situ in a single duct-lobular unit.” The author uses this description to refer to the more unusual intraductal lesions characterized by the presence of two distinct architectural and cytological patterns .
Recently, in situ lesions with lobular cytological features of classic LCIS but with marked nuclear pleomorphism, comedonecrosis, and with or without apocrine cytology have been described as pleomorphic LCIS . Some reports suggest that these variants are more aggressive than classic LN and a surgical treatment similar to that applied to DCIS is recommended. However , there are no prospective epidemiological studies showing that these variants have different clinical significance and appropriate management of pleomorphic LCIS is currently uncertain .
In our series, we observed a frequent association between immunophenotype and morphology (cytoarchitectural features). Lesions in group (II), with solid architecture and low-grade cytology, were more often associated with expression of E-cadherin. Our data differ from those reported by Acs et al., in which no expression of E-cadherin was observed in the 14 CISM cases analyzed. The most frequent morphological pattern observed in that study was presence of lobular cytology with comedonecrosis (n = 9 ) . Maluf et al. analyzed 12 CISM cases and detected E-cadherin expression in five, while another five cases showed no expression of the protein and two presented a mixed population of cells in this regard. These authors did not observe the prevalence of a specific morphological pattern over others . Similarly to the study by Jacobs et al., lesions in group (II) were the most frequent lesion associated with expression of E-cadherin in our study, however we observed them at a higher frequency. A dual cell population in the same terminal duct-lobular unit was observed in four cases. This is likely due to the coexistence of LN and DCIS in the same terminal duct-lobular unit.
Since first reported, the immunohistochemical reaction of E-cadherin has been proposed as an aiding tool in the differential diagnosis between ductal and lobular lesions, either invasive or in situ. However, it should be noted that up to 15% of lobular lesions may exhibit aberrant expression of E-cadherin and thus, the lack of E-cadherin expression should not be used as the sole criterion for LN diagnosis . Choi et al. observed variability in the immunohistochemical staining of E-cadherin, and detected abnormal staining patterns, both in ductal and lobular lesions, making the differential diagnosis between in situ lobular and invasive lesions very difficult through immunohistochemistry . An alternative to reduce this interference and improve diagnosis is the combined use of immunohistochemical markers of the catenin pathway. Using IHC and molecular biology techniques Da Silva et al. analyzed three cases presenting morphological characteristics and genotyping that agreed with invasive lobular carcinoma, with nonetheless aberrant expression of E-cadherin. Of these three cases, two did not express β-catenin, indicating that the formation of the cadherin-catenin complex, which is required for the normal function of the cell and maintenance of tissue architecture, including cell adhesion, failed [12, 23]. In our study, we observed that expression of E-cadherin agreed with expression of β-catenin in all cases here observed.
Other explanations for the abnormal expression of E-cadherin found in other studies may be related to technical difficulties and pitfalls that may occur during the different stages in the immunohistochemical reaction. In our study, we had some difficulties in the pre-analytical reaction such as material loss and weak staining in some cases. This may reflect the fact that we used specimens coming from the routine diagnosis laboratory of a general hospital; and other cases were sent to us for a second opinion. In many cases, there was no control of the pre-analytical phase or standardization of time of formalin fixation and unbuffered formalin was used. Goldstein et al. showed that the reactivity level varies with the number of blocks and thickness of the sample sections in the pre-analytical process . Different clones of antibodies against E-cadherin and different antigens may also have an effect on the quality of the immunohistochemical staining. A comparison between two types of antibodies revealed discrepancy in the staining of lobular lesions in 6.4% of the cases . Finally, there is a lack of consensus regarding the interpretation of the positivity of immunohistochemical staining of E-cadherin. The established cutoff of a positive signal varies between basal membrane expression and presence of any positivity to 20% of expressing cells [12, 23]. Semi-quantitative evaluations of the intensity of staining and association of different criteria forming scores of staining intensity have also been proposed .
Other immunohistochemical markers have been suggested to aid in the diagnosis of CISM. The p120 catenin is an intracellular protein that promotes the binding between the complex of catenins and cell cytoskeleton. When E-cadherin expression is absent, p120 catenin is dispersed in the cytoplasm, which explains its expression in the cytoplasm in LN, and in the membrane in DCIS [23, 24].