Invasive breast cancers (IBCs) are heterogeneous, showing. Immunohistochemical Surrogates for Molecular Classification of Breast Carcinoma

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1 Immunohistochemical Surrogates for Molecular Classification of Breast Carcinoma A 2015 Update Ping Tang, MD, PhD; Gary M. Tse, MD Context. The pioneering works on molecular classification (MC) by Perou and Sorlie et al in the early 2000s using global gene expression profiling identified 5 intrinsic subtypes of invasive breast cancers (IBCs): luminal A, luminal B, normal breast-like, HER2-enriched, and basallike subtypes, each unique in incidence, survival, and response to therapy. Because the application of gene expression profiling in daily practice is not economical or practical at the present time, many investigators have studied the use of immunohistochemical (IHC) surrogates as a substitute for determining the MC of IBC. Objective. To discuss the continuing efforts that have been made to develop clinically significant and readily available IHC surrogates for the MC of IBC. Data Sources. Data were obtained from pertinent peer-reviewed English-language literature. Conclusions. The most commonly used IHC surrogates are estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), dividing IBC into luminal, HER2, and triple-negative subtypes. The addition of Ki-67, cytokeratin 5, and epidermal growth factor receptor (EGFR) separates luminal B from luminal A subtypes, and basal-like subtype from triple-negative breast cancer. More recently, biomarkers such as androgen receptor and p53 have been shown to further stratify these molecular subtypes. Although many studies of IHC-based MC have shown clinical significance similar to gene expression profiling defined MC, its critical limitations are: (1) a lack of standardization in terminology, (2) a lack of standardization in biomarkers used for each subtype, and (3) the lack of a uniform cutoff for each biomarker. A panel of IHC surrogates for each subtype of IBC is proposed. (Arch Pathol Lab Med. 2016;140: ; doi: / arpa ra) Invasive breast cancers (IBCs) are heterogeneous, showing distinct molecular and pathologic features and biologic behavior. 1 Morphologically, there are 21 distinct subtypes of IBC as defined by the World Health Organization classification. 2 Currently, morphologic classification, histologic grade, status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), along with tumor stage, are used to guide clinical management. The routine immunohistochemical (IHC) analysis for ER, PR, and HER2 provides critical prognostic and predictive information for IBC. 3 About 70% of IBCs are ER positive and are eligible for antiestrogen Accepted for publication December 9, From the Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York; and the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Shatin, Hong Kong. The authors have no relevant financial interest in the products or companies described in this article. Presented in part at the 2nd Princeton Integrated Pathology Symposium: Breast Pathology; February 8, 2015; Plainsboro, New Jersey. Reprints: Ping Tang, MD, PhD, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Ave, Box 626, Rochester, NY ( ping_tang@ urmc.rochester.edu). therapy. 4 PR is largely regulated by estrogen, 5 and PR negativity is associated with decreased response to tamoxifen therapy. 6 8 About 12% to 20% of IBCs show HER2 gene amplification and/or protein overexpression, and are associated with poor prognosis and predictive of response to anti-her2 targeted therapy. 9,10 Approximately 10% to 15% of IBCs are ER, PR, and HER2 negative (triple-negative breast cancer [TNBC]), and these tumors currently lack any targeted therapy. 11,12 The pioneering works on molecular classification (MC) for IBC by Perou 13 and Sorlie et al 14,15 with global gene expression profiling (GEP) for IBC identified 5 intrinsic subtypes of IBC: luminal A, luminal B, normal breast-like, HER2-enriched, and basal-like, with differing clinical outcomes and responses to neoadjuvant chemotherapy, 16 a bimodal age distribution, 17 and different risk factors. 18 Since then, several additional subtypes have been proposed: 1. Claudin-low subtype (CLBC), characterized by low-toabsent expression of luminal markers and enrichment of epithelial-to-mesenchymal transition markers, immune response genes, and cancer stem cell like features. 19,20 This subtype is also TNBC, showing poor prognosis and enriched for metaplastic carcinoma and carcinoma with medullary features. 806 Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse

2 2. Molecular apocrine subtype (MABC), which includes ERnegative, androgen receptor (AR) positive tumors with apocrine histology; they can be either triple negative (TN) or HER2 positive 21,22 and are associated with early recurrence A novel luminal-like subtype, which has a better prognosis than those of basal-like breast cancer (BLBC) and has a prognosis similar to those of the HER2- positive luminal B subtype and CLBC. 24 Although GEP was initially used, it is neither economical nor practical in daily practice. Many investigators have used IHC-based MC to study IBC and have shown predictive/ prognostic values comparable with that of GEP. Accurate IHC analyses for ER, PR, and HER2 are critical for IHCbased MC. 11 Semiquantitative evaluation of Ki-67 and PR is helpful for further typing of the luminal subtype, and evaluation of cytokeratin 5/6 (CK5/6) and epidermal growth factor receptor (EGFR) help to identify the BLBC among the TNBC. 28,29 In 2013, IHC-based MC was recommended in the St Gallen guidelines for clinical decision making. 30 Recently, several other biomarkers, including p53, AR, p16, and folate receptor a (FRA), have been shown to be significant in IBC as well. Positivity for p53 is associated with a worse prognosis and diminished response to therapy. 31,32 Status of p53 has been proposed to divide TNBC into 2 biologically distinct subgroups: a p53-negative normal breast-like TN subgroup, and a p53-positive basal-like subgroup with worse overall and event-free survival. 31,33,34 AR is expressed in up to 90% of breast cancers and up 50% of TNBCs. 35 AR expression is associated with better overall survival and disease-free survival, irrespective of coexpression of ER. 21,36,37 The positivity of p16, a tumor suppressor, is associated with better clinical outcome in some studies 38,39 but worse prognosis in others. 40,41 FRA, expressed in up to 80% of TN breast cancers, is associated with poor prognosis. 42,43 Addition of these molecules may further stratify these subtypes, and, more importantly, AR and FRA are both potential molecules for targeted therapy as well Although IHC-based MC has been used extensively in breast cancer research, there are 3 major limitations preventing it from being used more extensively in routine clinical practice: (1) confusing terminology, as exemplified by BLBC versus TNBC, and luminal HER2 versus luminal B HER2-positive IBC; 2) no standardization in the selection of biomarkers used for each subtype for example, using only ER, PR, and HER2 versus the addition of Ki-67, CK5, and EGFR; and (3) no uniform cutoff for each biomarker for example, 1% versus 10% for ER, or 14% versus 20% for Ki ,49 To address these issues, this review focuses on the clinical utility of these IHC surrogates in each of the following subtypes: hormone receptor positive luminal subtypes (luminal A and B subtypes), HER2-positive subtypes (luminal HER2 and HER2-enriched subtypes), TN subtypes (BLBC and unclassified subtypes), MABCs, CLBC, and normal breast-like subtype. IHC ANALYSIS AND SCORING FOR EACH BIOMARKER Although commonly used, IHC analysis is subjected to preanalytic, analytic, and postanalytic variables, especially in tissue handling and fixation, antibody sources and clones, and evaluation by pathologists. In recent years, standardization in IHC analysis for breast cancer has improved significantly, especially with the publication of the guidelines for ER, PR, and HER2 testing. 50,51 Immunohistochemistry procedures and scoring methods for each biomarker are briefly described here. ERa ER is a nuclear protein with 1 DNA-binding domain and 2 activation function domains. A total of 65% to 75% of IBCs express ER, with ER being a major contributor to its development. Clinically, ER-expressing IBCs are usually better differentiated, have a favorable prognosis, and respond to hormonal therapy. The American Society of Clinical Oncology/College of American Pathologists (AS- CO/CAP) guideline recommends that ER be considered positive if 1% or more of tumor cells have nuclear staining of any intensity. 50 Allred scoring and H-scoring are two other commonly used systems for ER and PR evaluations. 52,53 PR Similar to ER, PR is also a transcription factor, largely controlled by ER and to some degree by growth factors as well, and is expressed in 55% to 65% of IBC. In most cases, PR coexpresses with ER. Clinically, loss of PR suggests a more aggressive behavior and less response to hormonal therapy. The ASCO/CAP guideline recommends that PR be considered positive if 1% or more of tumor cells have nuclear staining of any intensity. 50 HER2 HER2 encodes a transmembrane tyrosine kinase receptor that binds to its extracellular signals and initiates a signaling cascade mediating cell proliferation and survival. About 12% to 20% of IBCs either overexpress the HER2 protein and/or have HER2 gene amplification, resulting in aggressive tumor growth and poor clinical outcome. Its expression is also indicative of a potential response to anti-her2 therapy. The ASCO/CAP guideline recommends that HER2 be defined as positive if 10% or more of tumor cells exhibit strong uniform membrane staining. 51 Ki-67 Ki-67 is a nuclear marker for proliferation expressed in all phases of the cell cycle except G 0. Clinically, higher Ki-67 expression is usually associated with a higher grade and more aggressive behavior. Nuclear staining of any intensity is evaluated. Currently, there is no standardized cutoff value for Ki A meta-analysis of 46 studies with patients has shown that high Ki-67 labeling correlated with increased relapse and decreased survival. However, the threshold designated as high Ki-67 labeling ranged from 3.5% to 35%. 57 A 13.25% of Ki-67 labeling was proposed as the cutoff to separate luminal B tumors from luminal A tumors based on PAM50-defined subtyping in 357 patients, even though the misclassified rate was as high as 25%. 25 Although a 14% cutoff was endorsed in St Gallen in 2011, 58 most of the panel voted for a cutoff of greater than 20% as the new cutoff in the 2013 St Gallen conference. 30 Also, because there is great variability within any given tumor for Ki-67 labeling, the actual score largely depends on the areas sampled and analyzed. CK5/6 CK5 is a high molecular weight cytokeratin and is expressed in normal myoepithelial cells. Its expression in Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse 807

3 breast cancer is associated with expression of EGFR, Ki-67, p53, and increased cytogenetic abnormalities 59 ; CK5/6 is often expressed in BRCA1-related breast cancers. 60 Nielsen et al 28 report that a panel of 4 markers (ER, HER2, CK5/6, and EGFR) accurately identifies BLBC. We have demonstrated that CK5/6 gave a higher detection rate than CK14 for BLBC. 61 The cutoffs for its positivity in the literature range from any positive cytoplasmic staining to 20% of tumor cells. 62 EGFR EGFR, also known as HER1, is a member of the HER family. Although the IHC expression of EGFR is not indicative of eligibility for current EGFR-targeted therapy, it has enhanced the identification of BLBC significantly because of its higher expression and easier scoring than that of CK5. 28 AR AR is a steroid nuclear receptor and is expressed more frequently than ER in breast cancer. 63 Although the precise role of AR in breast carcinogenesis is not clear, the possibility of it serving as a therapeutic target, especially in TNBC, is of significant interest. 35 Clinically, AR positivity is associated with better prognosis and, more recently, 21 associated with a lower recurrence score using the 21-gene test. 64 p53 By controlling the cell cycle and inducing apoptosis when cell damage is beyond repair, p53 is a vital regulator of genomic stability. In normal cells, p53 protein has a very short half-life, with degradation in minutes. However, a missense mutation of p53 results in protein stabilization and accumulation in the nucleus. 65 Expression of p53 has been shown to be associated with poor outcome for patients with IBC in some studies, but has not been shown in others, particularly when it is in combination with other markers, such as bcl-2 69 or HER2. 70 The different antibodies used in studies seem to produce different results as well. 71 Currently, most investigators use 10% of nuclear staining in tumor cells as the cutoff for p53 positivity. IHC-BASED MC ER-Positive BC Most ER-positive tumors fall into the luminal A and luminal B subtypes; however, they do not capture all of the biologic complexity of these tumors. 72,73 One recent study suggests that there are 6 distinct subgroups of ER-positive IBC, indicating the highly heterogeneous nature of these tumors. 74 Luminal A Subtype. Luminal A breast cancers account for about 30% to 40% of all IBC. They demonstrate the highest level of expression for ERa gene, GATA-binding protein 3 (GATA3), X-box binding protein 1, trefoil factor 3, hepatocyte nuclear factor 3, and estrogen-regulated LIV They do not overexpress HER2, and about 13% of luminal A tumors have a p53 mutation and 45% show a PIK3CA mutation ,75 The GATA3 mutation mainly occurs in this subtype and is associated with better prognosis. 76 Morphologically, most luminal A tumors are well differentiated carcinomas of no special type, tubular carcinomas, classical lobular carcinomas, mucinous carcinomas, and neuroendocrine carcinomas. 77 The 2013 St Gallen guidelines propose that a low recurrence score from the Oncotype DX test also suggests a luminal A subtype. 30 Luminal A tumors often have an IHC profile of high ER and PR expression, negative HER2, and low Ki-67. The original IHC surrogates were ER and/or PR positive, and HER2 negative. 11,78 Subsequent studies showed that a Ki-67 of 14% was the cutoff point to separate luminal A from luminal B subtypes. 25 More recently, a 20% cutoff point of PR to separate luminal A and B subtypes has been proposed, 26 and this was substantiated in a separate study. 79 Based on currently available data, the most commonly used IHC surrogates for luminal A subtype are ER þ, PR of 20% or greater, HER2, and Ki-67 of less than 14%. Luminal B (Luminal B HER2-Negative) Subtype. Luminal B subtype accounts for about 20% to 30% of all IBC. It demonstrates low to moderate expression of the luminal-specific genes, including the ER cluster, but not HER2. Compared with luminal A subtype, it shows a higher frequency of p53 mutations (32%) and a lower frequency of PIK3CA mutations (32%) ,75 Morphologically, this group of tumors is less well differentiated and consists mostly of invasive ductal carcinomas of no special type, and also some invasive micropapillary carcinomas. 77 By IHC analysis, these tumors show a lower level of ER expression, a lower level of or negative PR expression, and a higher level of Ki-67 labeling. However, molecular studies have shown that luminal B is not simply a more proliferative variant of luminal A, because both luminal A and B cancers have their own specific oncogenic drivers. 80 The IHC definition for this subgroup is less well defined. Originally, ER þ and PR, 81 or ER þ and HER2 þ, 82 was used, but later most investigators used ER þ and HER2 to describe this subtype if the score of Ki-67 was 14% or higher, 25,52 or higher than 20%. 30 More recently, a cutoff of less than 20% for PR 26 has been proposed. Cancello et al 83 reported that loss of PR increases the risk of relapse for patients with luminal B tumors. Prior studies also showed that the loss of PR is associated with poor prognosis, decreased responsiveness to endocrine therapy, a higher recurrence score with the Oncotype DX test, and a greater chance of bone metastasis. 6 8,84 86 Indeed, the latest St Gallen guideline proposed that a high Oncotype DX recurrence score is suggestive of luminal B like subtype. 30 More recent studies using IHC4 assay (ER, PR, HER2, and Ki-67) and Magee Equations have also emphasized the important role of PR levels in ERpositive breast cancer. 87,88 Most recently, data from a study with more than 9000 patients with an 8-year follow up indicated that combining Ki-67 and PR levels could better separate luminal B subtype from luminal A subtype. 28 The authors defined luminal A as ER þ, HER2, and Ki-67 of less than 14% with any PR, or Ki- 67 of 14% to 19% with PR greater than 20%; and luminal B as ER þ, HER2, and Ki-67 of greater than 14% with any PR, or Ki-67 of 14% to 19% and PR less than 20%. This proposal was supported by another large study of ER þ, HER2 breast cancers, in which PR negativity and high Ki-67 level predicted early relapse. 89 Based on currently available data, the most commonly used IHC surrogates for luminal B subtype are ER þ, HER2, and PR less than 20% or Ki-67 14% or greater. HER2-Positive BC This subgroup consists of 12% to 20% of all IBC. HER2- positive tumors are very heterogeneous at the molecular 808 Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse

4 level. 9,90 One study showed that HER2/HER3 heterodimers and p21 expression predicted response to adjuvant trastuzumab in HER2-positive tumors. 91 Among HER2-positive tumors, hormone receptor status is the most important factor affecting patients survival and treatment response. A recent study from Fountzilas et al 92 clearly demonstrated that luminal HER2 (ER þ,pr þ, HER2 þ ) and HER2-enriched subtypes (ER, PR, HER2 þ ) are clinically distinct, with different survival curves and metastatic patterns. Thus, HER2-positive tumors should be divided into two subtypes: luminal HER2 subtype (ER and/or PR positive/her2 positive) and HER2-enriched subtype (ER and/or PR negative/her2 positive) Luminal HER2 (Luminal B, HER2-Positive) Subtype. About half of HER2-positive IBCs fall into this luminal HER2 subtype. They are ER positive, although often at lower levels compared with HER2-negative tumors Tumors from this subtype have a higher expression of such genes as GATA3, BCL2, and ESR1, and a higher frequency of GATA3 mutations ,75 Morphologically, they are mostly grade 2 to 3 tumors (often with low or negative PR), relapse early, show frequent nodal metastasis, and have a diminished response to endocrine therapy. 98,99 One large study showed that tumors from luminal HER2 subtype have more frequent locoregional recurrence compared with hormone receptor negative, HER2-enriched subtype (9.8% versus 3.8%). 93 Preclinical evidence seems to confirm that the crosstalk between HER2 and ER signaling pathways may contribute to the resistance to endocrine therapy. 100,101 Simultaneous inhibition of both HER2 and ER pathways seems to be more effective than ER inhibition. 102,103 The luminal HER2 subtype can be further divided into two phenotypes based on PR expression: ER þ /PR þ /HER2 þ (triple-positive cancer) and ER þ /PR /HER2 þ, each having distinct clinical properties. 104 PR expression correlated inversely with HER2 overexpression, suggesting the loss of PR in ER-positive tumors may be a marker for an activated EGFR/HER2 pathway. 6,105 Patients with ER þ /PR / HER2 þ tumors have a reduced breast cancer specific survival compared with patients with ER þ /PR þ /HER2 þ. 83 A recent study showed that the survival of patients with ER þ /PR þ / HER2 þ tumors was superior to those with ER þ /PR /HER2 þ tumors across all stages, confirming that loss of PR is an unfavorable event. 106 These results highlighted the clinical importance of separating ER þ /PR þ /HER2 þ tumors from ER þ / PR /HER2 þ tumors. Based on currently available data, the most commonly used IHC surrogates for luminal HER2 subtype are ER and/or PR þ /HER2 þ, which may be further divided into ER þ /PR þ /HER2 þ and ER þ /PR /HER2 þ subtypes. HER2 þ -Enriched Subtype. By GEP, this subtype is characterized by low to absent gene expression of ER and several additional transcriptional factors expressed in the luminal/er þ cluster. It has high expression of several genes in the ERBB2 amplicon at 17q22.24, including ERBB2 and GRB7. About 71% of the tumors in this subtype have a p53 mutation and activation of receptor tyrosine kinase pathways, such as FGFR4, EGFR, and HER2; and 39% of tumors have a PIK3CA mutation ,75 There is an incomplete overlap between the HER2 groups as defined by molecular (GEP-based) and clinical (IHC-based or fluorescence in situ hybridization based) criteria. 107 One study with GEP for clinically HER2-positive tumors identified 3 distinct subtypes with mixed stage, histologic grade, and ER status. 108 Morphologically, besides invasive ductal carcinoma of no special type, special types of ER-negative tumors, including some of the apocrine carcinomas and pleomorphic invasive lobular carcinomas, also belong to this subtype. Based on currently available data, the most commonly used IHC surrogates for HER2-enriched subtype are ER,PR, HER2 þ. Triple-Negative Breast Cancer Triple-negative breast cancer is defined by the lack of expression for ER, PR, and HER2 by IHC analysis, 109 and has significant overlap with BLBC as defined by GEP. However, the concordance is not perfect. TNBC patients are usually younger, 110 with higher-grade tumors 111 and a higher risk of distant recurrence and death within the first 3 to 5 years after diagnosis. 112 Most importantly, there is no targeted therapy available for TNBC at the present time. Morphologically, most TNBCs are ductal carcinoma of no special type; others include special types of ER-negative tumors, such as adenoid cystic carcinoma, secretory carcinoma, metaplastic carcinoma, and carcinoma with medullary features, each with a distinctive morphology and clinical behavior. BL Subtype. About 15% of all IBCs belong to this subtype, and there is a great diversity in the histologic features, mutation profiles, response to chemotherapy, metastatic behavior, and survival. 113 Basal-like breast cancer, defined by GEP, shows high expression of many of the genes characteristic of breast basal epithelial cells, such as keratins 5 and 17, laminin, and fatty acid binding protein 7. Basal-like breast cancer can also be defined using IHC surrogates, including ER, PR, HER2, CK5, and EGFR. There have been p53 mutations reported in 82% of these BLBCs, and most are positive for keratins 5/6 and 17. Morphologically, BLBCs are frequently high grade and large size, with pushing borders, with a poor Nottingham Prognostic Index, and a high rate for local recurrence and distant visceral organ metastasis, especially within the first 5 years. Notably, there is a small group of BLBCs that are low grade and of good prognosis, including adenoid cystic carcinoma and secretory carcinoma with characteristic molecular changes. Secretory carcinoma has the t(12;15)(p13; q25) ETV6- NTRK3 fusion gene, and adenoid cystic carcinoma has the t(6;9)(q22-23; p23-24) MYB-NFIB fusion gene. Further study on TNBC has confirmed the heterogeneous nature of this group. Lehmann et al 12 further divided TNBC cancer into 6 different subgroups based on a detailed GEP study, namely: basal-like 1 and 2 subtypes; immunomodulatory subtype; mesenchymal and mesenchymal stem like subtypes; and luminal androgen receptor subtype. Each subtype has its specific gene expression pattern and response to its specific targeted therapy. 12 Responses to neoadjuvant chemotherapy are also different, with basallike 1 having the highest complete pathologic response rate (52%) and basal-like 2 and luminal androgen receptor tumors having the lowest complete pathologic response rates (0% and 10%, respectively). 114 Triple negativity is the most commonly used IHC surrogate for basal-like tumors. 11 Immunohistochemical analysis of BLBC cases that had been defined by GEP demonstrated that added IHC testing for CK5/6 and EGFR was better able to define this subtype than triple negativity alone. 28,115 Based on currently available data, the most commonly used IHC surrogates for BLBC are ER, PR, HER2, CK5 þ, and/or EFGR þ. Nonclassified TN Subtype. There is a small group of TNBCs that are also negative for CK5 and EGFR. 116 Rakha et al 117 showed that this subgroup of tumors is less likely to be Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse 809

5 Subtypes LA Immunohistochemical Surrogates for Molecular Classification of the Breast Luminal BC HER2-Positive BC TNBC Ki-67 14% LB PR,20% Luminal HER2 PR þ Luminal HER2 PR HER2 Enriched BLBC NCBC ER, % þ þ þ þ þ PR, % þ.20,20 þ HER2 þ þ þ Ki-67, %,14 14 Any Any Any Any Any Any CK þ EGFR þ Abbreviations: BC, breast cancer; BLBC, basal-like subtype; CK5, high molecular weight cytokeratin expressed in normal myoepithelial cells; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, Human Epidermal Growth Factor Receptor-2; LA, luminal A subtype; LB, luminal B subtype; NCBC, nonclassified subtype; PR, progesterone receptor; TNBC, triple-negative subtype. associated with a BRCA1 mutation, and they have better breast cancer specific survival and disease-free survival compared with BLBC. Molecular Apocrine BC. Molecular apocrine breast cancers are ER-negative, AR-positive tumors with apocrine histology; they can be either TN or HER2 positive 21,22 and are associated with early recurrence. 23 The commonly used IHC surrogates are ER,PR, and AR þ. 36 Recently it was shown that although all MABCs are ER,AR þ, and FOXA þ by GEP, only 93%, 58%, and 90% of these tumors are ER, AR þ and FOXA þ, respectively, by IHC analysis. 118 Because 67% and 57% of MABCs are positive for HER2 and GCDFP15, respectively, 94% of all MABCs can be identified by these two markers in ER-negative tumors. Thus, a modified IHC surrogate panel may be ER,PR,AR þ plus either HER2 þ and/or GCDFP15 þ. Clearly more studies need to be done to further characterize this subtype of tumors. Claudin-Low BC. Claudin-low BC has low-to-absent expression of luminal markers and enrichment of epithelialto-mesenchymal transition markers. 19,20 This subtype is usually TN with poor prognosis and enriched for metaplastic carcinoma and carcinoma with medullary features. An IHCbased study showed that CLBCs (defined by low expression of claudins 1, 3, 4, 7, and 8) are found in 77% of BLBCs, 20% of HER2-positive cancers, and 3% of luminal cancers. 116 Also, in a large study using GEP, 36% of CLBCs were ER positive. 119 These studies suggest that this subtype consists of a group of heterogeneous tumors. Normal Breast-like BC. The normal breast-like subtype of breast cancer, as identified in the original GEP intrinsic subtypes, has the same signature as fibroadenomas and normal breast samples. 13 They express genes characteristic of adipose tissue and lack of expression of ER and HER2. The clinical significance of these tumors remains to be determined because of the lack of studies on this subtype. In fact, in a study using the supervised risk predictor (PAM50) of breast cancer intrinsic subtypes, the normal breast-like subtype was not included. 120 Also, no single case of normal breast-like subtype was found in a large series of grade 3 breast cancer samples, in which each case had undergone microdissection and was made up of more than 90% of tumor cells. These data suggest that this normal breast-like subtype may be a technical artifact because of the presence of a high volume of normal breast tissue in the original 2000 study. 121 Thus, we decided not to include this subtype in the Table and the Figure. OTHER ISSUES REGARDING IHC-BASED MC 1. Immunohistochemistry-based MC is not equivalent to intrinsic subtypes as defined by GEP. Although IHCbased MC could separate breast cancer into subgroups that have similar clinical outcomes compared with those defined by GEP, they are not identical. 107,122 After comparing a large database with more than 1500 cases, Cheang et al 122 demonstrated that although there were overlaps between subtypes defined by GEP and IHC analysis, they were not identical. For example, only 73% of IHC-based TNBCs belonged to intrinsic basal-like subtypes, whereas another 17% belonged to intrinsic HER2-enriched subtype. Likewise, only 65% of clinically HER2-positive tumors belonged to intrinsic HER2- enriched subtype, whereas the others belonged to intrinsic luminal B (20%), basal-like (14%), and luminal A (7%) subtypes Issues with cutoff for ER and Ki-67: A. ER: Several recent studies suggested that IBCs with low ER expression (1% 10%) were more similar molecularly and biologically to BLBC than to luminal cancers, 123,124 whereas other authors reported that only 18% of low ER expression (1% 9%) tumors belonged to BLBC, with the remaining tumors belonging to intrinsic luminal A or B subtypes (44%). 122 Indeed, IBCs with low ER expression were uncommon, accounting for 2% in our cohort 125 and displaying pathologic features and clinical outcomes intermediate between ER-negative expression (,1%) and higher ER expression (11% 70%) tumors. 125,126 So far, the behavior of the low-er (1% 10%) IBC is far from clear, with somewhat conflicting results in the literature. Thus, using the current 1% cutoff as endorsed by ASCO/CAP for IHC-based MC appears to be sensible at the present time. B. Ki-67: The cutoffs for Ki-67 in the literature range from 3.5% to 35%. 57 A cutoff of 13.25% of Ki-67 labeling was proposed to separate luminal B BC from luminal A BC based on data from intrinsic subtyping. 25 The St Gallen breast expert panel had endorsed the 14% cutoff for Ki-67 in However, most of the panel voted for a cutoff of greater than 20% as the new cutoff in the 2013 conference. 30 One study proposed the IHC surrogates for luminal A tumors as ER þ, HER2, and Ki-67 less than 14% with any PR, or Ki-67 14% to 19% with PR greater than 20%; and luminal B tumors as ER þ, HER2, and Ki-67 greater 810 Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse

6 Samples of hematoxylin-eosin (H&E) and immunohistochemical patterns for 7 molecular subtypes. Abbreviations: BC, breast cancer; BLBC, basallike subtype; CK5, high molecular weight cytokeratin expressed in normal myoepithelial cells; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2; NCBC, nonclassified subtype; PR, progesterone receptor; TNBC, triple-negative breast cancer. than 14% with any PR, or Ki-67 14% to 19% and PR less than 20% Awareness of intratumor heterogeneity in breast cancer: Although in most situations, one can assign an IBC into one of the subgroups, there is still considerable intratumoral heterogeneity. 127,128 One recent preclinical study showed that ER-positive tumors admixed with a hormone receptor negative, CK5-positive basal-like subpopulation will respond better to a combination of endocrine and EGFR inhibitors. 129 Understanding the intratumor heterogeneity of BC will be critical in understanding the MC of IBC. CONCLUSIONS Clearly, MC of IBC has enhanced our understanding of breast carcinogenesis significantly in recent years. The capacity to define subtypes of breast cancer provides a framework for understanding the mechanism of breast carcinogenesis, and opportunities for improving therapeutic intervention. The IHC surrogates have been shown to be useful for advancing the understanding of the prognostic and predictive values of MC. The most current understanding of MC is summarized in the Table and illustrated in the Figure. Much more work needs to be done, especially in the standardization of IHC analysis and scoring for each biomarker, standardization of the definition for each classification, and the continued addition of newly discovered biomarkers, before MC can be used routinely in the clinical setting. We wish to thank Ms. Mary Jackson for her excellent assistance in editing this paper. References 1. Zardavas D, Irrthum A, Swanton C, Piccart M. Clinical management of breast cancer heterogeneity. Nat Rev Clin Oncol. 2015;12(7): Lakhani SR, Ellis IO, Schnitt SJ, et al. World Health Organization Classification of Tumours of the Breast. Lyon, France: IARC Press; Rakha EA, Reis-Filho JS, Ellis IO. Combinatorial biomarker expression in breast cancer. Breast Cancer Res Treat. 2010;120(2): Jensen EV, Jordan VC. The estrogen receptor: a model for molecular medicine. Clin Cancer Res. 2003;9(6): Horwitz KB, Koseki Y, McGuire WL. Estrogen control of progesterone receptor in human breast cancer: role of estradiol and antiestrogen. Endocrinology. 1978;103(5): Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse 811

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