Diagnostically Challenging Subtypes of Invasive Lobular Carcinomas: How to Avoid Potential Diagnostic Pitfalls

Invasive lobular carcinoma is the most common special breast carcinoma subtype, with unique morphological (discohesive cells, single-cell files, targetoid pattern) and immunohistochemical (loss of E-cadherin and β-catenin staining) features. Moreover, ILC displays a poor response to neoadjuvant therapy, a different metastatic pattern compared to invasive breast carcinoma of no special type, as well as unique molecular characteristics. In addition to the classic variant of invasive lobular carcinoma, several other well-recognized variants exist, including classic, alveolar, tubulolobular, solid, pleomorphic, signet-ring, and mixed. Furthermore, three novel variants of invasive lobular carcinoma, i.e., with extracellular mucin production, papillary features, and tubular elements, have been described during the last decade. We herewith focus on the unique morphological and immunohistochemical characteristics of these novel varieties of invasive lobular carcinoma, as well as differential diagnostic considerations and potential diagnostic pitfalls, especially when dealing with biopsy specimens.


Introduction
Invasive lobular carcinoma (ILC) is the second most common subtype of breast carcinoma following invasive breast carcinoma of no special type (NST), accounting for 10-15% of breast carcinoma [1]. It has unique clinical, imaging, histological, immunohistochemical, and molecular features.
While usually presenting as a mass-forming lesion, it may remain clinically undetectable.
Mammographic detection may be difficult [2]; ultrasonography is more sensitive but may underestimate tumor size [3]. Magnetic resonance imaging (MRI) may prove more helpful [4,5]. This underestimation of tumor size often results in positive surgical margins, thus requiring additional surgical procedures [6]. Occasionally, ILC may initially manifest as a distant metastatic lesion [7] and thus displays a different metastatic pattern from invasive breast carcinoma of NST [1]. ILC metastasizes less commonly to the lung but more frequently to the peritoneum, gastrointestinal and gynecologic systems [8][9][10][11].
Histologically, ILC is characterized by distinct morphological features, i.e., small cells lacking cohesion, forming single-cell files or arranged in a concentric pattern around existing ducts and lobular units (targetoid pattern), minimal stromal response, and the presence of a variable number of signet-ring cells [6]. Apart from the classic ILC, first described in 1941 by Foote and Stewart [12], a number of histological variants have so far been described, including alveolar [13], tubulolobular [14], solid [15], trabecular [13], signetring [16], pleomorphic [17,18] and mixed [1]. ILC is characterized by deleterious mutations in CDH1 gene mapping in 16q22.1 accompanied by allelic loss of the remaining allele [6] and corresponding to a lack of E-cadherin and aberrant β-catenin immunostaining [19].
ILC usually has a luminal A molecular phenotype and is often estrogen receptor (ER)and progesterone receptor (PR)-positive and HER−2 neu-negative, therefore being a prime candidate for hormonal therapy. It also shows a poor response to neoadjuvant therapy compared to invasive breast carcinoma of NST.
ILC presents specific molecular characteristics compared to other subtypes of invasive breast carcinoma.
Apart from the aforementioned variants, three new ones have recently been described, namely, ILC with extracellular mucin production, ILC with papillary features, and ILC with tubular elements. We herewith review the literature concerning these rare variants that may prove diagnostically challenging, focusing on their special morphological and immunohistochemical features, molecular characteristics, differential diagnostic considerations, and the potential diagnostic pitfalls, especially when dealing with biopsy specimens. This study aims to increase awareness of these rare variants of ILC.
The age ranged from 31 to 87 years (median 63). Most cases had a palpable or a radiographically detected lesion. The tumor size ranged from 7 to 100 mm (median 40 mm). Multifocality was present in several cases, one being a coexistent invasive breast carcinoma of NST [30]. One patient presented with skin ulceration [20] and another one with skin retraction and thickening, suggestive of inflammatory breast carcinoma [32]. Nodal status was positive in 17 cases.
Histologically, ILC with extracellular mucin production consists of two components: a mucinous (with multiple, relatively circumscribed, nodular foci of extracellular mucin and/or patchy extracellular mucin production) with irregular borders and a non-mucinous. The morphological and immunohistochemical characteristics of ILC with extracellular mucin production are shown in Figure 1.
The mucinous component percentage ranges from 5% to 95% of the total tumor area. In both mucinous and non-mucinous components, the architectural pattern can be of the classical, alveolar, and/or solid variants. Tumor cells are of small or intermediate size, pleomorphic or with apocrine morphology. Moreover, in most cases, a variable number of signet-ring cells can be identified in both areas. In many instances, concurrent lobular intraepithelial neoplasia was identified next to the ILC.
ER and PR were positive in 39/39 and 27/38 (one case does not mention PR status) cases, respectively. Her−2 neu was overexpressed in five tumors. The follow-up time ranged from 2 to 171 months (median 50). The clinicopathological data of all reported cases of ILC with extracellular mucin production are summarized in Table 1.    Soong et al. performed molecular analysis interrogating the full coding sequences of 447 genes for mutations and copy number variations and selected introns across for gene rearrangements in 13 cases with adequate material in their series [32]. As expected, alterations of CDH1 at the gene level were present in all but one patient. Notably, concurrent CDH1 mutations and 16q loss were detected in 11 of 13 cases. In the remaining two cases, one harbored 16q loss without detectable CDH1 mutation, and the other harbored CDH1 mutation without loss of a 16q arm. Interestingly, these two cases exhibited signet-ring cell morphology. The most common single-nucleotide variations (SNV) involved CDH1 (10 cases), followed by PIK3CA, POLQ, TP53, ERBB3 (3 cases each), ERBB2, and RUNX1 (two cases each). Other genes with isolated SNV potentially clinically significant included AKT1, FANCD2, GATA3, MAP3K1, MUTYH, PTEN, RB1, and SF3B1 [32].
Important clinicopathological data, including tumor size, lymph node, hormone receptor, Her−2 status, therapy, and follow-up information, are lacking in some of the aforementioned papers. In cases with available information, the age ranged from 61 to 86 years (median 73). All patients presented with a palpable lesion. Imaging findings (mammography and ultrasound) showed a well-circumscribed nodular mass in three cases [35][36][37] and a hypoechoic, highly vascularized mass in one case [34]. The tumor size ranged from 1.2 to 5.5 cm (median 2.8). There was no metastatic lymph node involvement in cases with available information.
Histologically, ILC with papillary features has, in most cases, two distinct components; the first consists of a nodular at least partially encapsulated tumor with a solid papillary growth pattern, fibrovascular cores, and focal hemosiderin deposition. In all but one case, a second component with the morphology of a classic variant ILC was found in close relation with the nodular neoplasm. The single case lacking a classic ILC component showed LCIS around the main tumor [36].
Immunohistochemically, all cases lack E-cadherin expression and are associated with loss of β-catenin [33,34] or aberrant (cytoplasmic) p120 [36,37] expression. GATA3 positivity supported a primary breast tumor [35,37]. Immunostains for various myoepithelial cell markers, such as p63, CK5/6, CK14, calponin, smooth muscle actin, and smooth muscle myosin heavy chain, were negative either within or in the periphery of the nodules. Immunostains for neuroendocrine differentiation markers (Chromogranin A, Synaptophysin, and CD56) were also negative where performed [33,[35][36][37]. ER and PR showed intense staining, while HER−2 neu was uniformly negative. The morphological and immunohistochemical characteristics of ILC with papillary features are presented in Figure 2. The three cases reported by Rakha et al. did not show local recurrence or metastasis within a median follow-up of 13 months [33]. Two additional cases mentioned that patients were alive with no evidence of disease after a follow-up of 8 and 10 months, respectively [36,37]. A summary of clinicopathological characteristics is seen in Table 2.  [34], classified as luminal type B and luminal type A, respectively. Both variants shared common copy number profiles, including the loss of 6q, 11q, 13q, and 16q harboring CDH1 and the gain of chromosome 1q, suggesting a common origin. Interestingly, sequencing analysis revealed a unique CDH1 mutation resulting in a truncated E-cadherin protein in both components, further arguing for a clonal origin. Li et al. performed next-generation sequencing that revealed a frameshift mutation in exon 7 of CDH1 [36].

Invasive Lobular Carcinoma (ILC) with Tubular Elements
This ILC variant displays tubular elements with P-cadherin expression. So far, there is only one manuscript (2020) describing 13 cases [38].
Clinically, the age ranged from 42 to 79 years (median 59). Clinical information concerning the imaging characteristics and tumor size was not mentioned. Five cases had lymph node involvement, two being micrometastases. Some cases were bifocal, multifocal, or bilateral. One case was ovarian metastasis.
Histologically, ILC with tubular elements is characterized by tumor cells arranged in several different architectural patterns seen in ILC (classic, trabecular, solid, and dispersed) admixed with tubular elements of variable shape and size (longitudinal, teardrop shaped, small round tubules with narrow lumina). Tubular elements represent a variable amount of total tumor area. Nuclear atypia is low or intermediate overall. In some cases, concurrent lobular carcinoma in situ can be identified. Figure 3 shows the histopathological and immunohistochemical features of a case of ILC with tubular elements recently diagnosed in our department. Immunohistochemically, E-cadherin expression was strongly reduced in one and completely absent in 12 cases in both components, i.e., ILC and tubular. Despite this E-cadherin loss, most cases retained focal β-catenin expression. P-cadherin was expressed in 12 cases, with stronger staining in tubular elements and weaker or absent staining in the conventional ILC areas. All tumors were ER-positive, and Her−2 neu-negative; PR was positive in eleven cases. The tubular elements had a lower proliferative activity, as measured by Ki67 immunostaining. No information about follow-up time was available. Clinicopathological data of these cases are summarized in Table 3. All tumors underwent molecular analysis. Eleven cases harbored CHD1 mutations, while two lacked detectable CDH1 mutations. Four patients exhibited bifocal, multifocal, and bilateral lesions with tubular elements present in a single focus. Molecular analysis in these cases showed almost always identical CDH1 mutations in all foci, suggesting clonal relatedness of ILC with tubular elements and adjacent ILC with a conventional growth pattern.
Chromosomal analysis revealed gains on 1q, 8q, 11q13, and 16p and losses on chromosomes 8p, 11q, 16q, and 22q [38]. Notably, all cases examined exhibited either a copy number loss and loss of heterozygosity (LOH) or a copy number neutral LOH of 16q22.1 harboring CDH1 gene [38]. In the same study, P-cadherin mRNA expression was assessed by quantitative real-time RT-PCR in two different regions, one with conventional morphology and the second consisting almost exclusively of tubules. The region with tubular morphology showed increased P-cadherin mRNA expression.

Discussion
All three aforementioned ILC variants may be diagnostically challenging, especially in limited biopsy material. This is due to the broad differential diagnosis of these variants, occasional sampling of the mucinous, solid papillary, or tubular component, and finally, lack of awareness.
The differential diagnosis of ILC with extracellular mucin production includes invasive mucinous carcinoma of Capella type B, solid papillary carcinoma (SPC), mixed ILC and mucinous carcinoma, metaplastic matrix-producing carcinoma (MMPC), and polymorphous mammary adenocarcinoma.
Invasive mucinous carcinoma of the Capella B type lacks the classic morphological features of ILC and shows an expression of E-cadherin and β-catenin. SPC may show extracellular mucin production and can be associated with invasive mucinous carcinoma [39]; nevertheless, SPC lacks ILC morphology and shows an expression of E-cadherin and β-catenin. Also, Chromogranin and Synaptophysin are expressed in 50% of cases [40]. In mixed ILC and invasive mucinous carcinoma, immunostaining for E-cadherin will delineate the two different immunophenotypes. MMPC [41] and polymorphous mammary adenocarcinoma [42,43] also resemble ILC with extracellular mucin production since the former has a mucoid/matrix stroma, and the latter may simulate the single-file pattern of ILC [42,44]. Immunohistochemically, they may lack E-cadherin expression [42,45]. Moreover, both tumors lack ER expression, while MMPC also shows positive staining for S-100 with rare exceptions [41,46]. Finally, one should also not forget the possibility (albeit rare) of metastatic signet-ring cell carcinomas to the breast [25]. The morphological and immunohistochemical characteristics of ILC with extracellular mucin production and its mimickers are summarized in Tables 4 and 5.  The differential diagnosis of ILC with papillary features includes solid papillary carcinoma (SPC), encapsulated papillary carcinoma (EPC), the solid or alveolar variant of ILC, an ILC-SPC collision tumor, confluent growth of LCIS, and colonization of a papillary neoplasm by lobular carcinoma in situ. Both SPC and EPC display cellular cohesion and are E-cadherin-positive. The solid and alveolar variants of ILC, the confluent growth of LCIS, and the colonization of a papillary neoplasm by LCIS lack the characteristic supporting fibrovascular cores. In the case of a collision tumor between an ILC and SPC, only the cells of the latter show cohesion. The morphological and immunohistochemical characteristics of ILC with papillary features and its mimickers are summarized in Tables 6 and 7. Tubular carcinoma, the tubulolobular variant of ILC, and mixed-type invasive breast carcinoma of NST and ILC may be considered in the differential diagnosis of ILC with tubular elements. Tubular carcinoma will not display a classic ILC pattern and shows strong E-cadherin, β-catenin, and membranous p120 expression. The mixed-type invasive breast carcinoma of NST and ILC will show positive staining for adhesion molecules in the tubules and an absence of staining in the ILC area [47]. The tubulolobular pattern of ILC shows strong E-cadherin and beta-catenin staining in both tubules and cells lacking cohesion [48][49][50]. Many authors consider this histological pattern a variant of invasive breast carcinoma of NST; however, the latest WHO edition still classifies this type as a variant of ILC [51]. Table 7. Immunohistochemical characteristics of invasive lobular carcinoma with papillary features and its differential diagnoses. The presence of tubular or pseudocribriform structures does not exclude a diagnosis of ILC [25], but an E-cadherin stain should be performed whenever these are present.

ILC with
The morphological and immunohistochemical characteristics of ILC with tubular elements and its mimickers are summarized in Tables 8 and 9.  In the literature, cases of ILC with extracellular mucin production have occasionally been diagnosed as invasive mucinous carcinoma [25,28]. ILC with papillary features has been diagnosed as ductal carcinoma in situ, SPC, or encapsulated papillary carcinoma (EPC) in biopsy specimens [33,34]. In our own experience, we have misdiagnosed ILC with tubular structures as invasive breast carcinoma of NST in biopsy specimens (see Figure 3).
The result of misdiagnosis in these cases can be the misclassification of an ILC as a mucinous carcinoma with a favorable prognosis. In contrast, ILC clinical behavior is more aggressive than mucinous carcinoma, characterized as "initially indolent but slowly progressive" by Rakha et al. [52]. Misdiagnosis of ILC as SPC or EPC may have a worse impact on patient management since these entities are managed as in situ lesions, thus leading to suboptimal staging. The effect of diagnosing ILC with tubular structures as carcinoma of NST or mixed NST-ILC cannot be estimated, with no data concerning the former's biologic behavior being available up to now. It is important for pathologists to keep in mind that extracellular mucin, solid papillary architecture, and tubular structures are not always associated with a ductal phenotype. Careful examination of lesions with the abovementioned characteristics on small biopsy specimens may reveal morphological findings suggestive of ILC (presence of signet-ring cells, lack of cellular cohesion, limited areas with a single-file pattern) that may otherwise be missed. Staining for E-cadherin, β-catenin, and p120 can be helpful in this context.
Concerning molecular findings, the analysis revealed in all three variants biallelic inactivation of CDH1 due to concurrent CDH1 mutation and complete or partial 16q arm loss [32,34,38], which is the genetic hallmark of ILC. Also, copy number alterations consistent with invasive carcinoma of NST, mucinous, SPC, or EPC were not found, thus confirming the morphologic and immunohistochemical findings.
Soong et al. concluded that ILC with extracellular mucin production cases with local recurrence and lymph node involvement bore more mutations with biologic significance compared to those lacking these features, this difference being not statistically significant, at least up to now [32].
In the case of ILC with papillary features presented by Christgen et al., molecular analysis confirmed a common clonal ancestry of the two morphologically distinct tumor components and that the papillary component was the result of subclonal evolution [34].
In what concerns ILC with tubular elements, molecular analysis showed, apart from the lobular phenotype, clonality of ILC with tubular elements with foci having a conventional ILC growth pattern in bifocal, multifocal, and bilateral tumors, and expression of P-cadherin in the mRNA and protein level [38].

Conclusions
In conclusion, we herewith present the clinicopathologic, morphological, immunohistochemical, and molecular characteristics of three rare variants of ILC, emphasizing the potential differential diagnostic issues. Additional cases need to be presented, and additional molecular studies should be performed to better understand both the biological potential and clinical behavior of these rare entities.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
This article is a review and not an original study. All the references are listed.

Conflicts of Interest:
The authors declare no conflict of interest.