Immunohistology of the Breast

Cell Adhesion: Ductal Versus Lobular Carcinoma

Based on cell cohesiveness, the two broad categories of breast carcinoma (invasive or in situ) are ductal and lobular types. Ductal carcinoma in-situ increases the risk of invasive malignancy at the local site whereas lobular carcinoma in-situ is considered a marker of generalized increased risk of invasive malignancy, although some recent data suggests precursor properties for lobular carcinoma in-situ. Invasive ductal carcinomas (IDCs) are often unifocal lesions compared to invasive lobular carcinoma, which are often multifocal and/or more extensive than what is estimated on clinical and mammographic examination. Distant metastases from ductal carcinoma preferentially involves lung and brain, whereas metastases from lobular carcinoma more often involve the peritoneum, bone, bone marrow, and visceral organs of gastrointestinal and gynecologic tracts. Despite the above differences, at present, with the combined multi-modality therapy, there appears to be no difference in disease free or overall survival between ductal and lobular carcinomas. ^{, ,} However, there are enough significant differences in patient preoperative evaluation and subsequent treatment, that an accurate diagnosis is warranted at the time of core biopsy. At some breast cancer centers, a preoperative (before lumpectomy or mastectomy) magnetic resonance imaging (MRI) of the breast is performed to evaluate the extent of disease with a core biopsy diagnosis of invasive lobular carcinoma. ^{, , ,} A core biopsy diagnosis of ductal versus lobular carcinoma is also important if the patient will be treated by neo-adjuvant chemotherapy. Although therapy response is better predicted with tumor receptor status and grade rather than morphologic tumor type, studies do show response to neoadjuvant chemotherapy (NACT) only in a subset of ductal cancers with no or minimal effect on lobular cancers. Therefore, pathologists have to strive hard to give the best diagnosis possible for current management and also for the future as specific therapies become available. One study has shown relative effectiveness of aromatase inhibitor, letrozole over tamoxifen for patients with lobular carcinoma.

Strong E-Cadherin (ECAD) membranous staining has been long used to define ductal carcinomas. Ductal carcinomas (in situ or invasive) retain membranous ECAD because they do not show homozygous mutation/silencing of the ECAD gene. Mutation of the ECAD gene either leads to a mutant protein that loses its adhesive properties or there is not enough protein to function as adhesive molecule.

The ECAD gene, CDH1 , is a large gene located on 16q22.1. The ECAD protein has an intracytoplasmic portion, an intramembranous portion, and an extracellular domain. Cell-to-cell adhesion through ECAD is also critically dependent on the subplasmalemmal cytoplasmic catenin complexes (α, β, γ and p120 isoforms) that link ECAD to the actin cytoskeleton of the cell. Abnormalities of the catenins or ECAD gene expression can result in a variety of immunohistochemical ECAD pathologies. Lobular carcinomas studied at the genetic level have often shown ECAD mutation that accounts for the loss of cohesiveness of the tumor cells. ^, The majority of these mutations have been found in combination with a loss of heterozygosity (LOH) of the wild type ECAD locus (16q22.1), a hallmark of classical tumor suppressor genes. Immunohistochemically, this correlates with either a complete absence of the ECAD protein or abnormal localization (apical or perinuclear). This abnormal localization may be dependent on the type of mutation. Truncation mutations produce an ECAD product that is inept at binding to neighboring cells, resulting in a histologic pattern of widely dyshesive cells that are completely negative for ECAD by IHC (e.g., classic infiltrating lobular carcinoma [ILC]) ( Fig. 19.18 ). Loss of membrane staining may be associated with a granular cytoplasmic immunostaining ( Fig. 19.19A and B ) that represents cytoplasmic solubilization of a portion of the truncated protein. Proximal truncation mutations may result in the inability of ECAD to bind to the catenin complex, resulting in a short ECAD represented by focal or dot-like membrane immunostaining ( Fig. 19.19C ). Patients with focal staining of LCIS cells with ECAD may have an ipsilateral risk of carcinoma akin to DCIS. Mutations in the catenin complex can also lead to dysfunctional ECAD and loss of membrane staining. ^, While deletions of the CDH1 gene as a result of LOH are seen in ductal carcinomas, they are not early events and are not usually associated with the point mutations seen in lobular neoplasia.

In most cases, ECAD staining is unequivocal (positive or negative) and can be solely used in distinguishing ductal from lobular carcinomas. In a minority (approximately 15% cases) of cases, the stain may be difficult to interpret. This aberrant staining for ECAD is often seen as partial membranous and beaded. Another stain that could be used in such situations is p120. This stain represents p120 catenin, which binds with ECAD on the internal aspect of the cell membrane to form the cadherin-catenin complex ( Fig. 19.20A ). The complex is essential for the formation of intercellular tight junctions, and is composed of an external domain of calcium-dependent ECAD, and an internal domain of ECAD to which is bound the α, β, and p120 catenins. The α and β catenin are complexed with the carboxy-terminal cytoplasmic tail of ECAD, whereas the p120 catenin is anchored to ECAD in a juxta-membranous site. P120 is actively involved in the status of cell motility, ECAD trafficking, ECAD turnover, promotion of cell junction formation, and regulation of the actin cytoskeleton. The binding of p120 to ECAD stabilizes the complex and increases the half-life of membrane ECAD by slowing the normal turnover of ECAD that normally occurs by cellular endocytosis. P120 that is bound to ECAD exists in equilibrium with a small cytoplasmic pool of p120. When ECAD is absent, the cytoplasmic pool of p120 increases. Therefore, in normal ducts and ductal carcinomas, p120 shows a membranous pattern of staining ( Fig. 19.20B and C ). In contrast, lobular carcinomas (with absent or non-functional ECAD) show strong cytoplasmic p120 immunoreactivity ( Fig. 19.20D and E ). This positive cytoplasmic staining for lobular carcinoma is much easier to interpret than ECAD negative staining. A combination of ECAD and p120 drastically reduces the number of ambiguous diagnoses and better delineates (or diagnosed with increased confidence) the category of mixed ductal and lobular carcinoma ( Fig. 19.20F ). These mixed carcinomas comprise no more than 10% of all breast carcinomas and probably arise due to “late” ECAD inactivation within a ductal carcinoma. In contrast, loss of ECAD protein occurs very early in lobular carcinogenesis. Lack of ECAD staining and strong p120 cytoplasmic staining is observed in all morphologically characterized lobular carcinoma in situ and atypical lobular hyperplasias ( Fig. 19.21 ). Additionally, lack of ECAD within minimal epithelial proliferation in the breast terminal duct lobular unit defines atypical lobular hyperplasia and distinguishes it from mild ductal hyperplasia. This distinction is important because patients with ALH are typically referred to a risk clinic. Although some studies have advocated surgical excision with core needle biopsy diagnosis of atypical lobular hyperplasia, ^, most breast care providers recommend referral to high risk clinic rather than surgical excision. The IHC stains support the notion that the term “lobular hyperplasia” has no significance in breast pathology.

Immunohistochemical and molecular methods not only aid with the diagnostic issues, but to some extent they also demand that one looks at the morphology that has been learned in a new light. All invasive breast carcinomas that infiltrate in a “single-file” pattern with a low nuclear grade are not lobular carcinomas, as IDC also have this pattern.

The morphologic assessment of a ductal or lobular phenotype is not without controversy and has limitations. The classic ILC is composed of small cells with bland cytology and some “plasmacytoid” features. The growth pattern is completely dyshesive. Breast tissue can grossly appear normal (as well as the mammogram) yet show widespread dyshesive carcinoma of the classic type. These tumors are uniformly ECAD negative and associated with specific patterns of systemic metastases. IDC can also show patterns seen in ILC, for example, “single-filing” of tumor cells, targetoid patterns, and regional dyshesiveness. Such patterns may be confusing but are readily resolved with ECAD immunostaining. There are subgroups of morphologically “indeterminate” lobular/ductal phenotypes. ECAD separates these groups distinctly in most cases and demonstrates the existence of mixed lobular-ductal phenotypes in a minority of cases. ^{, ,} ECAD stains MECs, a pitfall for misinterpretation of LCIS as DCIS (see Fig. 19.18A ).

The morphologic reproducibility of distinguishing IDC from ILC and LCIS from DCIS is less than optimal. There can be substantial variation in the interpretation of ILC versus IDC and LCIS versus DCIS. For this reason alone, ECAD and similarly p120 IHC could be justified to aid in correctly classifying these lesions.

Lobular Carcinoma Variants and Former Lobular Variants

Pleomorphic Lobular Carcinoma

Described by Bassler in 1980 and further detailed by Weidner, Eusebi, and Reis-Filho, the genetic, immunohistologic, and clinical features have been sufficiently detailed to recognize invasive pleomorphic lobular carcinoma (PLC) and pleomorphic lobular carcinoma in situ (PLCIS) as a distinct clinicopathologic entity. Based on cell cohesiveness, PLC and PLCIS are basically a subtype of lobular carcinoma. The histologically recognizable PLC and PLCIS are almost always ECAD negative (or show aberrant staining) and demonstrates strong cytoplasmic immunoreactivity for p120. Histologically, these show grade 3 nuclei with a dyshesive pattern of growth in both in situ and infiltrating varieties ( Fig. 19.22 ). The in-situ component may be discovered on mammograms as calcifications. The core biopsies demonstrate in-situ dyshesive grade 3 nuclei with some cases showing comedonecrosis and calcification. A comprehensive analysis of 26 PLCs revealed a closer association between PLC and classic lobular carcinoma than between PLC and ductal carcinoma. The authors analyzed 26 cases of PLC, 16 cases of classic lobular carcinoma and 34 cases of IDC by IHC, array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and chromogenic in situ hybridization (CISH). Comparative analysis of aCGH data suggested the molecular features of PLC (ER/PR+, ECAD-negative, 1q(+), 11q(−), 16p(+) and 16q(−)) were more closely related to those of classic ILC than IDC. However, PLCs also showed some molecular alterations that are more typical of high-grade IDC than ILC (p53 and HER2 positivity in some cases, 8q(+), 17q24-q25(+), 13q(−) and amplification of 8q24, 12q14, 17q12 and 20q13). Some of these IDC-like alterations may be responsible for the aggressive biology of PLC. However, it is to be noted that pleomorphic histology may not always be associated with increased mitotic activity which is important for breast cancer prognosis. In fact, in a multivariable model, pleomorphic morphology was not prognostic but rather mitosis score, receptor subtype and pathologic stage were independently and significantly associated with disease free survival.

Sneige et al. studied 24 cases of PLCIS by IHC and found them to be universally positive for ER (100%). They also showed frequent p53 reactivity (25%) and moderate to high proliferative activity; and HER2 positivity was seen in 1/23 cases (4%). Of these 24 cases, 14 were associated with PLC, which showed similar IHC profile. Our experience with PLC and PLCIS is also very similar. Although HER2 over-expression/amplification may be seen in PLCs, the HER2+ rate is not very high as previously reported, and PLCs are ER+ in the majority of cases, albeit expression levels may vary from case to case. Since, there is high likelihood for developing invasive carcinoma in the vicinity of PLCIS, these lesions should be managed similar to DCIS.

Tubulolobular Carcinoma

Described originally by Fisher in 1977 as a lobular growth pattern with tiny tubules and single-filing characteristic of lobular carcinoma, the prognosis was described as intermediate between that of pure tubular carcinoma and ILC. ^, This lesion had been categorized as a variant of ILC because of the small cells and characteristic ILC pattern of single-filing, and targetoid infiltration.

Wheeler as well as Esposito documented uniform membranous ECAD immunostaining in the tubules and lobular-appearing components ( Fig. 19.23 ), ^, and discovered that pure LCIS and mixed LCIS/DCIS predominate in these lesions. The combination of a small, rounded tubule profiles with infiltrating lobular-like patterns that is ECAD+ is a ductal immunoprofile.

Invasive Micropapillary Carcinoma-Use of Epithelial Membrane Antigen

Tight clusters of neoplastic cells surrounded by clear spaces characterize the invasive micropapillary carcinoma. ^, The cell clusters are devoid of fibrovascular cores (unlike papillary carcinoma) and often display tubular structures in the center. The stroma is typically described as “spongy” with little or no desmoplasia of the surrounding tissue. ^, Some ductal carcinomas of “no special type” (NST) also show clear spaces around neoplastic cells, which are likely due to retraction of the intervening fibrotic stroma and should not be confused with micropapillary morphology. Fortunately, the distinction between true micropapillary carcinoma and NST carcinoma with retraction artifact can be easily made by epithelial membrane antigen (EMA or MUC1) stain. ^{, ,} Ductal carcinoma of NST shows an apical or cytoplasmic staining with EMA. In contrast, invasive micropapillary carcinomas show accentuation of the basal surface (stroma facing) of the neoplastic cells ( Fig. 19.24 ). This “reverse polarity” of the neoplastic cells is a characteristic feature of invasive micropapillary carcinoma. The distinction between a ductal NST and micropapillary carcinoma may not be clinically very significant, because stage for stage, there is no significant difference between the two entities. However, micropapillary morphology (even when small) is highly predictive of lymph node metastases, and these tumors also more commonly tend to involve the skin and chest wall. The incidence of axillary lymph node metastasis has been reported to be as high as 95%. However, we believe the actual incidence of axillary lymph node involvement in routine clinical testing is close to 60%. Acs et al. showed that even partial reverse cell polarity, defined as prominent linear EMA reactivity on at least part of the periphery of tumor cell clusters, has the same implication as micropapillary differentiation and these tumors may represent part of a spectrum of invasive micropapillary carcinoma. A confident diagnosis of a true micropapillary carcinoma on a core biopsy helps the surgeon to plan appropriate management, viz. special attention and clinical exam of the axilla, to perform fine needle aspirate if axillary nodes are slight enlarged (but not definitely suspicious), and to request intraoperative frozen section even if the lymph node is grossly negative.

“Basal-Like” Carcinoma-Use of Basal Cytokeratins

Routine hormone receptor (HR) protein and HER2 oncoprotein analysis on invasive breast carcinomas in the last decade has delineated clinically significant subgroups. One such group is that of “triple negative” tumors, that is, tumors that are negative for all three biomarkers ER, progesterone receptor (PR), and HER2. These tumors have been known to be clinically aggressive, and therapeutic options are limited since these are not amenable to HR-based therapy or HER2 targeted therapy. The so called “basal-like” breast carcinomas constitute at least 80% of the “triple negative” tumors. The “basal-like” subtype was initially recognized by gene expression profiling studies. ^, Basal-like carcinomas are histologically characterized by high Nottingham grade, geographic necrosis, good circumscription, and mild to moderate host lymphocytic response. These tumors generally show reduced (not absent) ECAD membranous expression, but show strong membranous p120 immunoreactivity (unpublished data); therefore, we consider them as subtypes of ductal carcinomas. Basal-like carcinomas are characteristically “triple negative” and show expression of basal-type cytokeratin (CK5/6, CK14, CK17), EGFR, vimentin, and p53. ^, Often, a panel of basal-type cytokeratins and EGFR in triple negative tumors is used to identify basal-like carcinomas ( Fig. 19.25 ). The antibodies used to identify the basal-like variant are an example of “genomic application” of IHC. This details fundamental immunohistologic profiles, used as surrogate markers, that reflect a genomic profile. Antibody to CK5 (clone XM26) is more sensitive (but equally specific) than CK5/6 (clone D5/16B4) for identifying basal-like breast carcinomas. The immunohistochemical studies have also confirmed the existence of in-situ carcinoma of basal phenotype. ^, Gene-expression studies have consistently identified basal-like carcinomas to have poor prognosis. ^{, ,} These tumors occur in both pre and post-menopausal patients; however, identifying basal-like carcinoma in a young pre-menopausal patient may suggest the presence of hereditary breast and ovarian carcinoma syndrome. At present, triple negative basal-like breast carcinomas are not treated any differently compared to non-basal triple negative breast carcinomas. Immunotherapy and poly ADP-ribose polymerase (PARP) inhibitors are frequently used to treat triple negative basal-like breast cancers. At present, PARP inhibitor are only effective in patients who have germline mutations in BRCA1/2 genes or defective homologous recombination repair pathway. The basal cytokeratins in triple negative breast cancers can be used for diagnostic purposes to define true basal-like breast cancers as a surrogate to molecular profiling. Basal cytokeratins can also be used in clinically problematic weak ER+ cases to determine if these tumors represent luminal or basal-like breast cancers. Other markers such as nestin and inositol polyphosphate-4-phosphatase (INPP4b) have been utilized for this distinction. In a small study of 58 cases, positive nestin expression and loss of INPP4b in weak ER+ tumors (Allred score 3 to 5) showed a worse medial survival of 45.8 months versus 65 months for other weak ER+ tumors ( P = .012).

Metaplastic Carcinoma-Use of Keratins, Melanoma, and Vascular Markers

Metaplastic carcinoma comprises a group of heterogeneous neoplasms that exhibit pure epithelial or a mixed epithelial and mesenchymal phenotypes. ^, Diagnosis is not problematic when there is a recognizable component of metaplastic carcinoma, that is, an obvious adenocarcinoma, adenosquamous or squamous cell carcinoma, osseous or chondroid differentiation. The most problematic cases are the ones, which predominantly show spindle cell morphology without an obvious epithelial or DCIS component ( Fig. 19.26 ). This is usually the issue on a core biopsy rather than on an excision specimen. IHC stains can be helpful in this situation. A panel composed of multiple keratin stains (CAM5.2, AE1/3, 34βΕ12, CK5, and CK7) and EMA is more useful than a single keratin. Another sensitive and specific marker for spindle cell metaplastic carcinoma is p63 and should always be included in the panel. ^, Vimentin expression in the tumor does not exclude a spindle cell carcinoma. ^{, ,} Vimentin expression has been found in 50% of hormone-independent cell lines, and since metaplastic carcinomas are usually negative for receptors, vimentin expression is actually expected. If all the keratins, EMA, and p63 fail to show any immunoreactivity on a core biopsy, complete excision of the lesion should be recommended. In many cases, an epithelial component is present only focally. Although every effort should be made to prove an atypical/malignant-appearing spindle cell lesion to be a metaplastic carcinoma, the differential diagnosis also includes melanoma, angiosarcoma, and stroma of phyllodes tumor. At least two melanoma markers should be performed. S100 is a very sensitive melanoma marker, but has been reported to stain between 20% and 50% of metaplastic breast carcinomas and therefore is not the best stain for this differential diagnosis. ^, Strong keratin reactivity or multiple keratin positivity would also exclude a melanoma. However, CAM5.2 positivity alone is not enough to exclude a melanoma unless it is strong and diffuse. ^, Another significant malignant lesion with which metaplastic carcinoma can be confused is an angiosarcoma. These tumors may occur after radiation treatment or de novo . It is obvious to think about angiosarcoma in a malignant spindle or epithelioid lesion of the breast if there has been a prior history of radiation treatment. However, in absence of such a clinical history, the lesion should be extensively examined by available IHC stains. More than one vascular marker should be used due to the heterogeneous expression of vascular markers. Of the 3 commonly used vascular markers (CD31, CD34, and Factor VIII), CD31 is generally considered as the most specific vascular endothelial marker, but occasional weak staining of carcinomas has been described. We have also seen staining of carcinoma cells and histiocytes with CD31. It is a diagnostic pitfall, especially in small samples. A diagnosis of a de novo primary angiosarcoma of the breast should be made only if there is unequivocal IHC staining for vascular markers, negative staining for p63 and HMWKs, and appropriate histology of the lesion. Other vascular markers that can be positive in angiosarcoma include D2-40, ERG, and FLI-1. In absence of staining for epithelial markers, the possibility of phyllodes stroma should also be considered. The stroma of phyllodes tumor is often (not always) positive for CD34, but is negative for other vascular markers. P63 is often negative in phyllodes tumor. However, p63 and p40 reactivity has been reported in 57% and 29% of malignant phyllodes tumor ( n = 14), respectively. In the same publication, focal cytokeratin reactivity was also reported in 21% of malignant phyllodes tumor. In our experience this is uncommon, but we suggest taking overall morphology and results of all IHC markers into account for making a definitive diagnosis. In summary, a malignant spindle cell lesion is a metaplastic carcinoma unless proven otherwise. A panel comprising of multiple keratins (especially basal cytokeratins), EMA, p63, melanoma, and vascular markers is required in the workup of a malignant spindle cell lesion.

Other Spindle Cell Neoplasms (Myoepithelial and Mesenchymal Tumors)

Tumors of the breast in which MEC differentiation predominantly include adenomyoepithelioma, myoepithelioma, and myoepithelial cell carcinoma (MECC). Although the majority of myoepithelial tumors are benign, occasional tumors may exhibit aggressive behavior in the form of carcinoma or myoepithelial carcinoma. ^, The typical immunostaining pattern of the myoepithelial components of these tumors is strong cytoplasmic staining for 34βE12, CK5, and nuclear p63. Tumor cells are typically positive with S-100 protein (90%) and may be positive with muscle markers such as calponin (86%), muscle-specific actin, desmin (14%), and α-smooth muscle actin (36%). ^, Occasional cells exhibit immunostaining with glial fibrillary acidic protein (GFAP). The presence of smooth muscle markers and immunostaining for GFAP is more in keeping with pure myoepithelial differentiation as opposed to metaplastic carcinomas (discussed above), which are largely negative for these markers. ^, Expression of SMA is very nonspecific and is not a definitive marker for muscle differentiation. Metaplastic carcinomas of the breast (carcinosarcoma, spindle cell carcinoma, sarcomatoid carcinoma) have an immunoprofile very similar to myoepithelial differentiation, as they regularly coexpress weak cytoplasmic CAM5.2 for low molecular weight keratins, strong cytoplasmic immunostaining for HMWK 34βΕ12, CK5/6 or CK5, vimentin, and nuclear immunostaining for p63 (90%). However, GFAP and SMMHC are largely negative. Immunostaining with the muscle markers is most indicative of a pure myoepithelial neoplasm as opposed to a metaplastic carcinoma. The immunoprofile of metaplastic carcinoma is shared to a great degree with myoepithelial neoplasms, with some investigators suggesting that the MEC is the progenitor cell for metaplastic carcinomas. ^{, , ,} Leibl also demonstrated that the experimental myoepithelial markers CD29 and 14-3-3 sigma stain metaplastic carcinomas, supplying further evidence of the myoepithelial nature to these tumors. The literature suggests that using the terms myoepithelial carcinoma versus metaplastic carcinoma is a matter of semantics and may not have any clinical significance.

Myoepithelial tumors need to be separated from the rare primary spindle cell sarcoma of the breast, which may include fibrosarcoma (vimentin positive) leiomyosarcoma and rhabdomyosarcoma (positive with muscle markers), synovial sarcoma (positive with CK7 and CK19), malignant nerve sheath tumors (patchy S-100+ and vimentin+), and the so-called malignant fibrous histiocytomas (vimentin+). Although each of these tumors may have characteristic light microscopic features, immunostaining patterns may be useful in the diagnostic distinction ( Table 19.4 ). Primary liposarcomas (S100+) of breast are rare tumors that may arise in a preexisting phyllodes tumor (CD34+ stroma).

When the spindle cell lesion in the breast demonstrates bland cytomorphologic features, then the differential diagnosis includes myofibroblastoma, fibromatosis, fibromatosis-like metaplastic carcinoma, inflammatory myofibroblastic tumor, nodular fasciitis, scar tissue, leiomyoma, and cellular pseudoangiomatous stromal hyperplasia (PASH). Most of these lesions have characteristic morphology, but IHC stains are frequently used to confirm cellular differentiation. The myofibroblastoma of the breast ( Fig. 19.27 ) is distinguished immunohistochemically from myoepithelial tumors by lack of immunostaining for keratins, S-100 protein, and other myoepithelial markers. Myofibroblastomas demonstrate CD34+ cells in vast majority of the cases. Myofibroblastomas are often positive for HRs and show variable reactivity for actin and desmin. Myofibroblastomas have been previously referred to as solitary fibrous tumor of the breast, but immunohistochemical and genetic studies fail to find the link. Solitary fibrous tumors are often STAT6 positive, and mammary myofibroblastomas have been found to be negative. The genetic studies on myofibroblastomas have shown partial monosomy of 13q and 16q with deletion of the region 13q14 in greater than 50% cases. These findings are similar to 13q and 16q rearrangements in spindle cell lipoma. Moreover, it is not uncommon to find adipose tissue within myofibroblastomas supporting the concept that myofibroblastomas are likely related to spindle cell lipomas. PASH, which could be part of fibroadenoma or phyllodes tumor, is also positive for CD34. In contrast, fibromatosis involving the breast is negative for CD34, but demonstrate abnormal (nuclear) localization of beta-catenin. ^, It is to be noted that nuclear β-catenin expression has been documented in phyllodes tumor and metaplastic carcinomas, but expression is diffuse and strong in fibromatosis compared to other diagnoses. Scar tissue can be extremely difficult to distinguish from fibromatosis but is thought to be negative for nuclear β-catenin expression. Fibromatosis-like metaplastic carcinoma is distinguished from fibromatosis by expression of keratin stains and p63 reactivity in the former. In addition to the characteristic morphology, inflammatory myofibroblastic tumor shows reactivity for actin and ALK protein. Leiomyomas are extremely rare in the breast but are expected to be positive for smooth muscle markers, actin, desmin, and caldesmon. Patchy actin and desmin reactivity can be seen in many other myofibroblastic lesions, and therefore diagnosis of leiomyoma requires appropriate morphology and uniform strong expression for muscle markers.

Sentinel Lymph Node Immunohistochemistry

For the surgical pathologist, the appropriate triage and examination of the SLN is of utmost importance, but even here some controversy exists. When SLN mapping procedure began to be the standard of care a few years ago, the SLNs were histologically examined by multiple levels and cytokeratin stains on at least two levels. Since then, more experience has been gained with the procedure and the reporting of SLNs. It was soon realized that majority of micrometastases (metastases between 0.2 and 2 mm) can be identified by H&E alone and IHC for cytokeratin stains generally highlight isolated tumor cells (tumor cell aggregates ≤0.2 mm). Although the exact clinical significance of isolated tumor cells, and even micrometastases, remains uncertain, studies have shown that they both are associated with non- SLN positivity in approximately 10% of cases, especially when the tumor size is larger than 1 cm (pT stage 1C or more). However, completion axillary dissection with one to two positive sentinel nodes in lumpectomy patients, does not translate into improved disease free or overall survival as shown in Z0011 trial. If the primary breast carcinoma is of ductal type, it would be difficult (not impossible) to identify isolated tumor cells by H&E stain, and most pathologists would agree that they would be able to identify micrometastases ( Fig. 19.31A ). Therefore, cytokeratin stains on SLN do not add any significant information beyond H&E stain in a primary ductal cancer. However, there are significant differences, when the primary breast tumor shows a lobular morphology. Due to single cell infiltration, small (micro) metastases of lobular carcinoma (of the classical type) in a lymph node are extremely difficult to identify ( Fig. 19.31B and C ). Occasionally, cytokeratin stains would identify macro-metastases, not readily apparent on H&E stain. Cserni et al. have reported that sentinel node positivity detected by IHC in lobular carcinomas was associated with further nodal metastases in 12 of 50 (24%) cases. Therefore, it is not unreasonable (although not required) to perform cytokeratin stains on SLNs in cases of lobular carcinoma. As far as lymph node examination after neoadjuvant therapy is concerned, routine cytokeratin staining of the lymph node is not required. However, IHC staining can be performed if findings are equivocal or suspicious on H&E staining.

When performing cytokeratin immunostaining of SLNs, one should use a cocktail such as AE1/AE3 cocktail; CAM5.2 is less desirable because of the manner in which it stains dendritic cells in the lymph node. MM cells occur in small clusters less than 2 mm in diameter within the lymph node or subcapsular sinus, and they need to be distinguished from the dendritic appearance of the interstitial reticulum cells of the lymph node, which are also keratin positive. Studies with larger numbers of patients are needed to discern if the site of lymph node micrometastasis (peripheral sinus vs. parenchyma of lymph node) is clinically significant.

Aggregates of breast epithelial cells in the subcapsular sinus of axillary lymph nodes have been described by Carter and associates as occurring as a result of “mechanical transport” after a breast biopsy. Some impugn the core biopsy itself or the breast massage that follows isotope/dye injection as sources of mechanical displacement of cells into the SLN. Solitary keratin-positive cells may be transported to the SLN, and the histologic feature often associated with true benign transport is the association of CK-positive cells with altered red blood cells and hemosiderin and macrophages ( Fig. 19.32 ). Diaz et al. described benign epithelial tissue in skin dermal lymphatics and SLN(s) from patients with pure DCIS. This lends morphologic documentation to the concept of “benign mechanical transport.” The distinction between benign transport and “true” metastasis is easy if the cells in lymph node appear “benign,” but there is no objective way to distinguish benign transport from true metastasis when the cells appear cytologically malignant.

Intraoperative Molecular Testing of Sentinel Lymph Node

In the past few years, a few studies have shown the usefulness of intraoperative molecular tests in determining metastatic disease. ^, These are reverse transcriptase polymerase chain reaction assays (RT-PCR) which use a completely closed system and are fully automated from RNA extraction to final interpretation. One such assay was called the GeneSearch Breast Lymph Node (BLN) Assay (Veridex LLC, Warren, NJ), which was approved by United States Food and Drug Administration (FDA) for axillary lymph node testing. The GeneSearch BLN assay was composed of sample preparation kit, all reagents required for performing RT-PCR and protocol software to be used with the Cepheid SmartCycler System (Sunnyvale, CA). According to the company, the test was optimized for detecting metastatic disease larger than 0.2 mm. The test analyzed the expression of CK19 and MGB genes. The studies showed high sensitivity, specificity, positive, and negative predictive values for the test. Overall, this molecular assay was very much comparable to the frozen section examination, permanent sections, and even IHC. However, just like any other test, one should be aware of the false positive and false negative test results as well as the usefulness and pitfalls of a particular test. Since molecular tests are not morphologic assays, one has to be extremely careful with any sources of contamination. A cutting bench metastasis (“floater”) can be easily recognized on an H&E-stained slide as such, but will give a false positive result by RT-PCR, and there will be no definite way to identify this as an error. The SLNs identified in the axillary tail may contain minute amount of breast tissue in the surrounding adipose tissue which may also give a false positive result. Therefore lymph nodes should be completely trimmed of the adipose tissue before sectioned for the molecular analysis. Moreover, fat interferes with the assay itself, and this could be an issue when the SLN is diffusely replaced by adipose tissue. Occasionally a benign epithelial inclusion (>0.2 mm) within the lymph node could also be a source of a false positive result ( Fig. 19.33 ). Given the significance of the treatment decision based on a positive SLN result (CALND, which cannot be undone) and several sources of false positive result with molecular tests, we believe that currently there is insufficient data to replace the morphologic methods with molecular assay. At present, we suggest that a positive molecular result should be confirmed by morphology either by frozen or permanent sections before a final decision is made. In contrast a negative result is highly valuable given the very high negative predictive value of the molecular tests. Notably, the commercial assay mentioned earlier (GeneSearch BLN assay) was taken off the market due to a variety of reasons including workflow problems and lack of interest in identifying micrometastasis and isolated tumor cells not seen with routine testing. However, one test that is still in clinical use is One Step Nuclei Acid (OSNA) amplification assay. It is used in 12% of the UK hospitals. It is a quantitative RT-PCR for CK19. The original publication correlated the mRNA copy number to size of metastasis (negative, micro, or macrometastasis). In a meta-analysis, OSNA was found to be highly sensitive (87%) and specific (98%) for detecting a macrometastasis. However, up to 21% of cases classified as macrometastasis by OSNA would have tumor reclassified as micrometastasis on corresponding microscopic examination. To address this limitation, a new cutoff has been proposed. However, due to the reasons mentioned earlier, it is unclear if this test will be widely adopted.