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 (LCIS) is considered a marker of generalized increased risk of invasive malignancy, although some recent data suggest precursor properties for lobular carcinoma in situ. Invasive ductal carcinomas (IDCs) are often unifocal lesions compared with 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 the lung and brain, whereas metastases from lobular carcinoma more often involve the peritoneum, bone, bone marrow, and visceral organs of gastrointestinal and gynecologic tracts. In spite of the listed differences, at present, with the combined multimodality therapy, there appears to be no difference in disease-free and 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 neoadjuvant chemotherapy. Although therapy response is better predicted with tumor receptor status 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 the 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 an 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 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 (see 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. Although 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.

A classic invasive and in situ lobular carcinoma demonstrating complete lack of E-cadherin (ECAD) staining (A). Note the staining of myoepithelial cells with ECAD (A). The growth pattern of in situ carcinoma is indeterminate for cell type (B), but positive membranous staining for ECAD indicates lobular involvement by ductal carcinoma in situ (C).

Aberrant staining with E-cadherin (ECAD). (A) Cytoplasmic ECAD staining in invasive lobular carcinoma. (B) Kobular carcinoma in situ. (C) An example of “dot-like” ECAD staining in invasive carcinoma.

In the majority of cases, ECAD staining is unequivocal (positive or negative), and can be solely used in distinguishing ductal from lobular carcinomas. In a minority (~15% cases) of cases, the stain may be difficult to interpret. 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 catenins 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 (see Fig. 19.20B and C ). In contrast, lobular carcinomas (with absent or nonfunctional ECAD) show strong cytoplasmic p120 immunoreactivity (see 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 (see 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 ). In addition, 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.

Diagrammatic representation of E-cadherin (ECAD) relationship to p120 (A). The dynamic biology of ECAD-p120 can be illustrated using a dual ECAD (brown) and p120 (red) stain (B and C). In this example of invasive ductal carcinoma, strong membranous reactivity (reddish-brown) is identified for both ECAD and p120 (B). Similar reddish-brown membranous staining is identified in acinar cells within this lobule (C). An example of invasive and in situ lobular carcinoma (D) demonstrating strong cytoplasmic immunoreactivity for p120 using a single color stain (E). (F) A dual ECAD-p120 stain demonstrating membranous ECAD and p120 (reddish-brown) immunoreactivity in the ductal component with lack of ECAD (absence of brown staining) but strong cytoplasmic p120 (red) staining in this example of mixed ductal and lobular carcinoma.

An example of lobular neoplasia (A). The cells of lobular neoplasia demonstrate strong cytoplasmic staining compared with membranous staining of normal duct cell with p120 (B). Another example of lobular carcinoma in situ with pagetoid extension into ducts (C). A dual E-cadherin (ECAD)-p120 stain showing a thin layer of residual luminal cells staining with ECAD (brown) and the duct largely replaced by LCIS cells demonstrating strong cytoplasmic reactivity (red) for p120 (D). Another example of lobular neoplasia stained with dual ECAD-p120 stain demonstrating intense red cytoplasmic staining with p120 (E).

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 IDCs 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 dyscohesiveness. 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 Bässler 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 demonstrate 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.

Pleomorphic LCIS (PLCIS) in lobular arrangement showing nuclear grade 3 and prominent nucleoli (A and B). PLCIS is ECAD negative (C). Low magnification of PLCIS, with comedonecrosis and calcification simulating DCIS (D). Note the dyshesion and plasmacytoid cellular features characteristic of PLCIS (E). ECAD is negative in PLCIS and positive in myoepithelial cells (F).

Sneige and colleagues 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; in addition, 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 overexpression/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, although expression levels may vary from case to case. Because 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 small, rounded tubule profiles with infiltrating lobular-like patterns that are ECAD+ is a ductal immunoprofile.

Pattern of infiltration of tubulolobular carcinoma is similar to lobular carcinoma (A). Tiny tubules populate the tumor that otherwise simulates lobular carcinoma (B). ECAD is positive in tiny tubules and single cells (C).

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 and colleagues 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, namely 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.

An invasive micropapillary carcinoma of the breast (A) demonstrating “reverse polarity” of the neoplastic cells by EMA (B). Note the intense staining by EMA at the stroma-facing side of the cells.

“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, PR, and HER2. These tumors have been known to be clinically aggressive, and therapeutic options are limited because 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), and 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), epidermal growth factor receptor (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 premenopausal and postmenopausal patients; however, identifying basal-like carcinoma in the young pre­menopausal patient may suggest the presence of hereditary breast and ovarian carcinoma syndrome. Although there are no specific chemotherapeutic drugs currently available to treat these patients, data (especially on PARP inhibitors) are emerging, and it is important to recognize these tumors as therapies become more refined.

A “basal-like” carcinoma (A) demonstrating immunoreactivity for CK5/6 (B) and epidermal growth factor receptor (C).

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, or osseous or chondroid differentiation. The most problematic cases are the ones that 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, 34βE12, 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 because 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 a 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 three 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 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 the phyllodes tumor. However, Cimino-Mathews reported p63 and p40 reactivity 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 multiple keratins, EMA, p63, melanoma, and vascular markers is required in the workup of a malignant spindle cell lesion.

This predominantly spindle cell neoplasm (A) showed only a focal area of epithelioid malignant cells (B). The tumor was completely negative for AE1/3 and CAM5.2 but demonstrated staining for basal cytokeratins (CK5/6, CK14, CK17) and p63 (C and D, respectively), supporting the diagnosis of spindle cell metaplastic carcinoma.

Other Spindle Cell Neoplasms (Myoepithelial and Mesenchymal Tumors)

Tumors of the breast in which there is 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 S100 protein (90%), and may be positive with muscle markers such as calponin (86%), muscle-specific actin, desmin (14%), and α-SMA (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 previously), 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 34βE12, 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 term 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 S100+ and vimentin positive), and the so-called malignant fibrous histiocytomas (vimentin positive). 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 the breast are rare tumors that may arise in a preexisting phyllodes tumor (CD34+ stroma).

When a 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 pseudoangiomatoses 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, S100 protein, and other myoepithelial markers. Myofibroblastomas demonstrate CD34+ cells in at least 50% 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 demonstrates abnormal (nuclear) localization of β-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 with 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.

Myofibroblastoma of breast (A) typically shows the presence of desmin (B) and muscle-specific actin (HHF-35) (C).

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 on 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 a 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 1 to 2 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 (see Fig. 19.31B and C ). Occasionally, cytokeratin stains would identify macrometastases not readily apparent on H&E stain. Cserni and colleagues 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.

Small foci of tumor deposits in the lymph node from a primary ductal-type breast carcinoma can be identified with relative ease on hematoxylin and eosin (H&E) stain alone (A). Immunohistochemical stains are generally not required. In contrast to metastases from ductal carcinomas, a micrometastasis or even a macrometastasis from a primary lobular type carcinoma is sometimes difficult to identify on H&E stain (B). These types of metastases are best identified by cytokeratin stains, which can also help in estimating the correct size of metastatic focus (C).

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 and colleagues 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.

Possible “benign transport.” (A) Sentinel lymph node sinus with giant cells, macrophages, broken red blood cells, and epithelioid cells. (B) Low magnification of keratin-positive cells involving the lymph node sinus. (C) Higher magnification showing mixture of keratin-positive cells with debris of macrophages and degenerated red blood cells.

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, New Jersey), which was approved by the US 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, California). 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. Because 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 a 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 are 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.

A benign epithelial glandular inclusion in an axillary lymph node as shown here may result in a false-positive molecular test for assessing micrometastatic disease.

Fibroadenoma and Phyllodes Tumor

Phyllodes tumors are biphasic neoplasms that are distinguished from fibroadenomas mainly on morphologic grounds. Unlike fibroadenoma, phyllodes tumors are relatively large, heterogeneous neoplasms, and histologically show a “leaf-like” architecture and periglandular stromal condensation. The variably cellular spindled-cell stroma has been proven by clonal analysis to be the neoplastic compartment. Immunohistochemical reports have shown the majority, regardless of grade, to express CD34 in the stromal compartment, consistent with fibroblastic and/or myofibroblastic differentiation, which is supported by ultrastructural studies. The World Health Organization (WHO) has adopted a three-tiered classification of benign, borderline, and malignant phyllodes tumors. Benign and borderline tumors can recur (especially with positive or close margins); metastases are generally seen only with malignant phyllodes tumor. Benign phyllodes tumors are histologically akin to cellular intracanalicular fibroadenomas but with stromal heterogeneity, leaf-like architecture, periglandular stromal condensation, and/or 1 to 3 mitoses per 10 high-power field (HPF). Due to low proliferation activity (1 to 3 mitotic figures per 10 HPF), the benign phyllodes tumors often need to be distinguished from fibroadenomas. There is a great deal of morphologic overlap between cellular fibroadenomas and benign phyllodes tumor, but unfortunately, immunohistochemical stains are also of limited value in this differential diagnosis. Nevertheless, investigators have tried proliferation markers for this distinction. Jacobs and colleagues found significantly higher stromal proliferation indices, such as Ki-67 (marker of all phases of cell cycle) and topoisomerase II-α (marker of G2-M phase), in phyllodes tumors compared with fibroadenomas on core needle biopsy. In this report, however, Ki-67 index ranged from 0.4% to 4.4% (average 1.6%) in fibroadenomas and from 0% to 18% (average 6%) in benign phyllodes tumors. Thus the margin of error in determination of the proliferation index is relatively small, and given the subjectivity involved in its estimation, may not be entirely reliable to distinguish between the two lesions ( Fig. 19.35 ).

Proliferative activity in fibroadenomas versus benign phyllodes tumors. The fibroadenoma depicted in A and B shows no Ki-67–positive stromal cells, whereas that depicted in C and D shows focal (~1%) Ki-67 stromal cell immunoreactivity. Note the numerous positive ductal epithelial cells. Similarly, benign phyllodes tumor may show minimal to no proliferative activity with Ki-67, as shown in E to H.

(Photomicrographs courtesy Dr. Nicole N. Esposito, Tampa, Florida.)

Malignant phyllodes tumors are akin to sarcomas with stromal expansion, pleomorphism, and mitotic rates often greater than 10 per 10 HPF, and borderline tumors lie somewhere in between benign and malignant phyllodes tumor. Numerous immunohistochemical studies have shown correlation of biomarker expression with tumor grade. Studies targeting Ki-67 with the monoclonal antibody MIB-1 have shown progressively increased expression with tumor grade. Ki-67 labeling indices range from 1% to 5% in benign tumors, 6% to 16% in borderline tumors, and 12% to 50% in malignant tumors in published reports ( Fig. 19.36 ). Similarly, tumor suppressor gene p53 is increasingly expressed with tumor grade, though less consistently than Ki-67. More recently, the expression of proteins with targeted therapy implications in phyllodes tumors have been explored. Chen and colleagues first reported c-kit expression in the stroma of phyllodes tumors in 2000, and found c-kit expression to be preferentially expressed in histologically malignant phyllodes tumors. Since then, several additional studies have reported increased c-kit expression in malignant phyllodes tumors compared with benign and/or borderline tumors. However, whether c-kit expression in these tumors infers susceptibility to the KIT-receptor tyrosine kinase inhibitor imatinib mesylate is doubtful, as activating c-kit mutations have yet to be reported. Of interest is the study by Djordjevic and Hanna that suggests c-kit expression in fibroepithelial tumors to be related to the presence of mast cells. The authors have argued against any appreciable true stromal cell c-kit staining in fibroepithelial tumors. EGFR has also been studied in phyllodes tumors, with most reports correlating increased stromal expression with tumor grade as well as chromosome 7 polysomy ( Fig. 19.37 ). Prognostication solely based on histologic categories, however, has proved problematic in some cases, as histologically benign phyllodes tumors may recur locally as higher-grade tumors with subsequent metastases, and many histologically malignant tumors neither recur nor metastasize. The primary aim of most immunohistochemical studies has thus been to correlate biomarker expression with recurrence and/or patient outcome rather than diagnostic category. Unfortunately, most reports have failed to do so and are conflicting. For example, one study reported an inverse relationship between Ki-67 expression and overall survival in multivariate analysis of 117 phyllodes tumors, whereas others have not corroborated this finding. Similarly, although p53 expression appears to correlate with tumor grade as noted previously, it has failed to consistently predict tumor recurrence. The potential prognostic value of EGFR is yet to be determined, given the modest body of literature that exists on the subject to date.

Ki-67 expression in benign (A and B), borderline (C and D), and malignant (E and F) phyllodes tumors of the breast. Labeling indices as demonstrated by Ki-67 expression generally correlate linearly with tumor grade.

(Photomicrographs courtesy Dr. Nicole N. Esposito, Tampa, Florida.)

Epidermal growth factor receptor (EGFR) expression in two malignant phyllodes tumors (A and B). EGFR expression has been shown to be more commonly expressed in malignant phyllodes tumors and usually corresponds to polysomy 7 rather than EGFR amplification.

(Photomicrographs courtesy Dr. Nicole N. Esposito, Tampa, Florida.)

Overall, studies aimed at developing better prognostic markers in phyllodes tumors have suffered from low sample size, a lack of patient follow-up data, and lack of reproducibility. The most significant consistently reported variables in prediction of phyllodes tumor behavior remains histologic characteristics, in particularly the presence of stromal overgrowth, and adequate surgical resection margins. The Singapore General Hospital nomogram for phyllodes tumor is a useful tool for estimating recurrence based on four key morphologic features (AMOS criteria)—namely, stromal atypia (A), stromal mitotic activity (M), stromal overgrowth (O), and surgical margin status (S).

Periductal Stromal Tumor

Periductal stromal tumor was initially described by Burga and Tavassoli as a distinct entity from phyllodes tumors, though histologically identical, except lacking the intracanalicular or “leaf-like” pattern. Like phyllodes tumors, however, the stromal cells express CD34, and thus some have proposed they are best regarded as a phyllodes tumor variant that lacks the classic leaf-like architecture rather than a distinct entity.

Other Fibroepithelial Lesions

The so-called myoid hamartoma can also be considered a fibroepithelial lesion, as it has both stromal and epithelial components. Some authors regard it as a variant of fibroadenoma, or as adenosis tumor with myoid metaplasia, which may be regarded as a variant of mammary hamartoma. These tumors show positive stromal immunoreactivity for SMA, desmin, myosin heavy chain, and vimentin ( Fig. 19.38 ), and are negative for S100.

A well-circumscribed biphasic lesion with stromal smooth muscle metaplasia is consistent with the diagnosis of myoid hamartoma (A and B). The stromal smooth muscle metaplasia is positive for actin (C), h-caldesmon (D), and smooth muscle myosin heavy chain (E). Note that actin and myosin heavy chain also stain the blood vessels and myoepithelial cells around the ducts.

Hormone Receptors

Recognition that estrogen ablation had an impact on groups of patients with breast cancer and that clinical responsiveness correlated with the expression of the estrogen receptor were seminal events in the treatment of patients with breast cancer.

ER and PR bind hormones that exert their effects in the nucleus. Nuclear immunostaining for both receptor proteins can be demonstrated in normal breast acini, which serve as internal controls for the testing procedure. In general, approximately 15% to 20% of the luminal epithelial cells in a duct or lobule stain with ER and PR. However, nuclear staining in normal breast tissue is heterogeneous and may vary with the menstrual cycle. One of the effects of estrogen is to induce the PR, and thus the coordinate expression of both hormone receptors in the same cell reflects the fidelity of the ER/PR axis in the cell. In carcinomas of the breast, most PR+ tumors are also ER+, and ER−, PR+ tumors account for less than 1% of all breast cancers. In general, patients with positive PRs have a significantly longer disease-free survival than patients who are PR−.

Since the early 1990s, the immunohistochemical assay determination of ER/PR levels has replaced the dextran-coated charcoal (DCC) method, also called the LBA. The DCC method, the gold standard for many years, suffered significant drawbacks—namely, (1) tumor sampling error; (2) heavy reliance on obtaining tissue immediately on termination of the blood supply to the tumor, usually in the operating room; (3) normal tissue contamination; and (4) analytic error. Some of the advantages of the IHC method include (1) histologic documentation of the tumor tissue to be assayed; (2) appreciation of the heterogeneity of ERs/PRs in tumor cell nuclei; (3) rapid assessment of the tissue for ERs/PRs by semiquantitative method; (4) rapid turnaround time; (5) lower cost; and (6) the ability to use minute quantities of tissue.

Despite the widespread use of IHC for HR determination, the lack of standardization of preanalytic and analytic variables, scoring schemes, and threshold for determining HR positivity remain a concern.

Postanalytic

An equally important component for HR assays is the postanalytical (interpretation) portion of the tests; however, scoring schemes and threshold for determining HR positivity remain a concern. A critically important statement from the NIH Consensus Conference of December 2000 suggested that “any nuclear expression of HRs should be regarded as a positive result and render a patient eligible for hormonal therapy.” This statement was supported by the studies performed by Pertschuk and colleagues and Cheang and colleagues, where both documented a greater than 1% of cells cut off for the 1D5 and SP1 ER antibody clones, respectively. The current CAP/ASCO guidelines on HR testing also recommend ≥1% of positive cells as a positive result for ER and PR. ASCO/CAP also recommends that the pathologist quantify the HR test results using one of several methods.

Quantitative results of the IHC method correlate closely with biochemical results and are predictive of prognosis. Veronese and colleagues, in a study using ER1D5, found that immunostaining was predictive of response to tamoxifen in 65 homogeneously treated patients, and was a discriminator for relapse-free and overall survival. Barnes and coworkers and Goulding and colleagues confirmed that the ER by IHC correlated better than the DCC method, and the results were strongly related to patient outcome, regardless of the method of immunoscoring.

The effect of quantitation and establishing that ER is a continuous variable (and not bimodal) was clearly shown by Harvey and colleagues in 1999 using the Allred score ( Fig. 19.39 ) in their series of 1982 primary breast cancer cases. An Allred score of 0, 2, 3, 4, 5, 6, 7, 8 was seen in 26%, 3%, 6%, 10%, 16%, 19%, 16%, and 4% cases, respectively. The authors demonstrated that there was a linear correlation between the Allred score and ER content, as measured by LBA. This study also showed differences in disease-free survival based on the Allred score. Even the current RT-PCR assay for HR has shown a broad dynamic range of HRs that are present in tumor cells. These tests do not demonstrate a bimodal distribution of HR results, as suggested by a few studies. Although the data from the NSABP-B14 clinical trial showed that the level of ER expression (as measured by RT-PCR) has little prognostic significance in the absence of endocrine therapy, the expression level has clinical significance when patients are treated with tamoxifen. When the tamoxifen-treated cohort was examined, patients in the top two tertiles showed benefit, whereas the lower tertile showed the same outcome as the placebo control group. The results suggest that the benefit from tamoxifen was limited to the patients with the higher level of expression. The degree of ER and PR staining has consistently been shown to identify groups of patients with significantly different risks of overall survival, time to recurrence, and treatment response to hormonal therapy. Moreover, a combination of standard histologic and semiquantitative immunohistochemical results for prognostic/predictive markers in breast cancer can help prognosticate individual patient risk and also has the potential to predict for standard chemotherapy benefit. Therefore ER and PR results should be reported using a semiquantitative method. At our institution, we prefer the histochemical score (H-score) method of reporting, as it has a broad dynamic range compared with the Allred method of reporting. H-score is given as the sum of the percent staining multiplied by an ordinal value corresponding to the intensity level (0 = none, 1 = weak, 2 = moderate, 3 = strong). With four intensity levels, the resulting score ranges from 0 (no staining in the tumor) to 300 (diffuse intense staining of the tumor). We also recently showed excellent interobserver concordance for H-score among pathologists. The reason for good concordance is likely the way the H-score is calculated and the slightly increased time spent by the pathologist evaluating the slide to calculate the score ( Fig. 19.40 ). Because the likelihood of benefit correlates with the amount of HR protein in tumor cells, we recommend using a semiquantitative criterion in addition to a positive or negative result that clearly states the percentage and intensity of ER and PR staining within tumor cells.

Semiquantitative scoring method for hormone receptors known as the Allred score.

(Modified from Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol . 1999;17:1474–1481.)

In this example of an estrogen receptors (ER+) positive case, 10% cells are completely negative, 50% cells stain with 1+ intensity, 35% stain with 2+ intensity, and 5% stain with 3+ intensity. Therefore, H-score is 135 and calculated as follows—ER H-score = (0 × 10) + (1 × 50) + (2 × 35) + (3 × 5) = 135.

Routine H-scoring for HRs definitely has advantages but also poses some challenges in terms of pathologist training and performance review. Automated image analysis systems have been suggested as an alternative to human scoring. However, more performance studies are still needed. The image analysis systems that are currently used for analyzing immunohistochemical ER/PR slides are incapable of reporting H-score results on the entire tumor section. Most systems provide quite accurate assessment of percentage positive cells in the field of view, which is generally a very small area of the tumor unless the whole slide is scanned and interpreted. Whole slide scanning takes time, and even if it is performed, the final onus is on the pathologist to make sure the invasive tumor cells are counted by the computer for reporting. Improvement in image analysis technology may overcome some of these aspects. Image analysis systems using fluorescent probes have also been described as a method of identifying immunoreactive tumor cells with an increased interobserver reproducibility. However, the challenge of correctly identifying invasive carcinoma for reporting under dark field microscopy and increased time in doing such analysis can further impede incorporation into diagnostic pathology. Furthermore, there has been an effort to use mRNA expression levels of ER/PR through quantitative reverse transcription polymerase chain reaction as an alternative to IHC. The increased use of onco type DX and MammaPrint (TargetPrint) diagnostic testing, as ordered by clinicians, may increase the popularity of these methods. However, as noted by the ASCO/CAP committee, there is a paucity of data on the concordance between mRNA-based assays and IHC-based clinical validation studies. In regard to this issue, we have reported good agreement between IHC semiquantitative H-score results and Genomic Health onco type DX quantitative ER/PR quantitative reverse transcription polymerase chain reaction results, with IHC being slightly more sensitive than polymerase chain reaction for both ER and PR. There is no reason to replace IHC with polymerase chain reaction, and moreover, due to the speed, higher sensitivity, preservation of morphology, and widespread use in every pathology laboratory, IHC is more desirable.

As far as PR IHC is concerned, Press and colleagues compared 12 different PR antibodies and found that PgR636 and PgR1294 stained the highest percentage of breast carcinomas known to be positive by the biochemical assay (95%–98%), and they exhibited the highest concordance with the biochemical assay (88%–90%). Later, Mohsin and colleagues performed clinical validation of the progesterone receptor by IHC. Using a positive result of 1% of cells with PR clone 1294 (Dakocytomation, Carpinteria, California) and the Allred scoring system, it was demonstrated that PR was better than the LBA to predict disease-free survival and overall survival.

Progesterone nuclear staining by the IHC method is more heterogeneous than ER and may be a cause of false-negative results, especially in core biopsies or needle aspirates. We have seen similar results on some core biopsies; as a result, if a negative ER and PR result on a core biopsy specimen is obtained, the test is repeated on the excisional breast specimen, particularly when the HRs are negative in tumors that are expected to be positive, such as grade 1 and 2 ductal cancers or lobular, tubular, and mucinous carcinomas.

The sensitivity and specificity of other PR antibodies, namely 1A6 and the more recently available rabbit monoclonal antibodies (1E2), are similar to the previously used antibodies. There are two isoforms for PR (PRA and PRB), and both isoforms are recognized by the commercially available antibodies. Our in-house data indicate that there are far fewer ER-negative/PR-positive cases with the 1E2 clone (1% of cases) compared with clone 1A6 (5%–6%).

Although less importance is given to PR compared with ER in the clinical literature, it is increasingly recognized that the amount of PR expression in ER+ tumors provides significant prognostic and predictive information. In a comparative/correlative study by Clark and colleagues, semiquantitative PR expression had a huge impact on the onco type DX recurrence score that was independent of grade. Cheang and colleagues also showed the significance of PR and even proposed to modify the definition of luminal A tumors based on the amount of PR expression. Therefore it is critical that laboratories perform and interpret PR testing in a manner similar to ER testing.

Key Diagnostic Points: Hormone Receptors

• •
  

  Preanalytic, analytic, and postanalytic factors can significantly alter hormone receptor results.

  
  
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  There are clinically validated studies for ER clones SP1, 1D5, and 6F11, all of which demonstrate a positive cutoff value of greater than 1% of cells staining, linked to response to tamoxifen.

  
  
• •
  

  Some form of semiquantitation (H-score, Q score, Allred score) should be performed on all positive cases, as semiquantitation is therapeutically important.

  
  
• •
  

  Hormone receptors should be repeated on resection specimen if tumor was double negative on core biopsy sample and the tumor histology suggests a positive result.

HER-2/neu

The ERBB2 ( HER2 ) gene was originally called NEU , as it was first derived from rat neuro/glioblastoma cell lines. Coussens and colleagues named it HER2 because its primary sequence was very similar to human epidermal growth factor receptor ( EGFR or ERBB or ERBB1 ). Semba and colleagues independently identified an ERBB-related but distinct gene, which they named as ERBB2 . DiFiore and colleagues indicated that both NEU and HER2 were the same as ERBB2 . Akiyama and colleagues precipitated the ERBB2 gene product from adenocarcinoma cells and demonstrated it to be a 185-kD glycoprotein with tyrosine kinase activity. In 1987, 2 years after its discovery, clinical significance of HER2 gene amplification was shown in breast cancer. We now know that approximately 15% to 20% of breast cancers demonstrate HER2 gene amplification and/or protein overexpression. In absence of adjuvant systemic therapy, HER2-positive breast cancer patients have a worse prognosis (i.e., a higher rate of recurrence and mortality), clearly demonstrating its prognostic significance. An even more important aspect of determining HER2 status is its role as a predictive factor. HER2 positivity is predictive of response to anthracycline and taxane-based therapy, whereas the benefits derived from nonanthracyclines and nontaxane therapy may be inferior. It is also important to note that HER2 positive tumors generally show relative resistance to all endocrine therapies; however, this effect may be more toward selective endocrine receptor modulators like tamoxifen and less likely toward estrogen depletion therapies like aromatase inhibitors. Most important, the availability of HER2 targeted therapy brought this biomarker at the forefront of theranostic testing for breast cancer. Trastuzumab is a humanized monoclonal antibody to HER2 that was approved by the US FDA in 1998 for use in metastatic breast cancer. Trastuzumab improves response rates, time to progression, and survival when used alone or in combination with chemotherapy in the treatment of metastatic breast cancer. Although approved for use in metastatic cancer, several prospective randomized clinical trials have shown large therapeutic benefits from trastuzumab in early stage breast cancers. The same paradigm has also shifted to neoadjuvant chemotherapy using trastuzumab in HER2 positive tumors.

Given the enormous therapeutic benefit derived from trastuzumab and other anti-HER2 therapies in HER2 positive tumors, it is absolutely critical that an accurate determination of HER2 status be made on each case. Due to its prognostic and predictive value, HER2 status should be determined on all newly diagnosed invasive breast cancers, which is also recommended in the ASCO/CAP guidelines. These guidelines provide a detailed review of literature and recommendations for optimal HER2 testing. The issues ensuring reliable HER2 testing can be divided in three categories: preanalytical, analytical, and postanalytical. All three issues are equally important, and just like HR testing require a commitment to continuous quality improvement.

Postanalytic

This involves interpretation criteria, reporting methods, and quality assurance measures, including competency of the interpreting pathologist. The literature regarding HER2 IHC testing would suggest that 2+ score is the most problematic ; however, we believe that it is the 1+ and 3+ scores which are often misinterpreted that has grave clinical consequences. Almost all laboratories would do FISH for HER2 gene copy number assessment when the IHC score is 2, but would skip HER2 FISH testing for 0, 1+, or 3+ scores. There are ample data that HER2 FISH has great correlation with response to trastuzumab treatment. Therefore, a 2+ HER2 IHC score (seen in approximately 25% cases) coupled with HER2 FISH has no adverse clinical consequences. In contrast, a false-positive HER2 IHC 3+ score would result in inappropriate (ineffective, expensive, and potentially harmful) therapy. Similarly, a false-negative HER2 IHC 1+ score would not be tested by FISH. We urge that utmost care should be exercised in interpreting HER2 IHC results. The best strategy is to keep a low threshold for scoring a case as 2+ and a high threshold for scoring a case as 3+. Some laboratories perform only FISH testing, but there are drawbacks of performing FISH as the only test, because assessment of heterogeneity is better appreciated at low power on bright field microscopy rather than at high power examination that is required for FISH.

Last but not the least, a quality assurance program should be in place for the laboratories that perform HER2 testing. Quality control procedures for HER2 IHC should include the laboratory statistics of percentage of positive cases and the percentage of IHC cases that are amplified by FISH. Periodic laboratory assessment of these correlations is essential for quality reporting. Rigorous adherence to quality, tissue fixation times, control tissues/cell lines, and improved interobserver interpretation agreement or image assisted analysis is preferable. The CAP/ASCO guidelines recommend participation in proficiency testing program specific to each method used.

Key Diagnostic Points: HER2 Immunohistochemistry

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