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

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;140:806 814; doi: 10.5858/ arpa.2015-0133-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, 2015. 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 14642 (email: 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. 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. 23 3. 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, 25 27 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. 44 47 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- 67. 48,49 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-67. 54 56 A meta-analysis of 46 studies with 12 155 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

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, 66 68 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- 1. 13 15 They do not overexpress HER2, and about 13% of luminal A tumors have a p53 mutation and 45% show a PIK3CA mutation. 13 15,75 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%). 13 15,75 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

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). 92 94 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. 95 97 Tumors from this subtype have a higher expression of such genes as GATA3, BCL2, and ESR1, and a higher frequency of GATA3 mutations. 13 15,75 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. 13 15,75 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

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 CK5.................. 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. 107 2. 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 2011. 58 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

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%. 28 3. 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):381 394. 2. Lakhani SR, Ellis IO, Schnitt SJ, et al. World Health Organization Classification of Tumours of the Breast. Lyon, France: IARC Press; 2012. 3. Rakha EA, Reis-Filho JS, Ellis IO. Combinatorial biomarker expression in breast cancer. Breast Cancer Res Treat. 2010;120(2):293 308. 4. Jensen EV, Jordan VC. The estrogen receptor: a model for molecular medicine. Clin Cancer Res. 2003;9(6):1980 1989. 5. Horwitz KB, Koseki Y, McGuire WL. Estrogen control of progesterone receptor in human breast cancer: role of estradiol and antiestrogen. Endocrinology. 1978;103(5):1742 1751. Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse 811

6. Arpino G, Weiss H, Lee AV, et al. Estrogen receptor-positive, progesteronereceptor negative breast cancer: association with growth factor receptor expression and tamoxifen resistant. J Natl Cancer Inst. 2005;97(17):1254 1261. 7. Cui X, Schiff A, Arpino G, et al. Biology pf progesterone receptor loss in breast cancer and its implication for endocrine therapy. J Clin Oncol. 2005; 23(30):7721 7735. 8. Rakha EA, El-Sayed ME, Green AR, et al. Biologic and clinical characteristics of breast cancer with single hormone receptor positive phenotype. J Clin Oncol. 2007;25(30):4772 4778. 9. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177 182. 10. Yu DH, Hung MC. Overexpression of ErbB2 in cancer and ErbB2-target strategies. Oncogene. 2010;19(53):6115 6121. 11. Carey LA, Perou CM, Livasy CA, et al. Race, breast cancer subtypes, and survival in the Carolina breast cancer study. JAMA. 2006;295(21):2492 2502. 12. Lehmann BD, Bauer JA, Chen X, et al. Identification of human triplenegative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121(7):2750 2767. 13. Perou CM, Sorlie T, Eisen MB, et al. Molecular portrait of human breast tumours. Nature. 2000;406(6797):747 752. 14. Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001;98(19):10869 10874. 15. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003;100(14):8418 8423. 16. Rouzier R, Perou CM, Symmans WF, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res. 2005;11(16):5678 5686. 17. Anderson WF, Resonberg PS, Prat A, et al. How many etiological subtypes of breast cancer: two, three, four, or more? J Natl Cancer Inst. 2014;106(8):dju 165. 18. Anderson KN, Schwab RB, Martinez ME. Reproductive risk factors and breast cancer subtypes: a review of the literature. Breast Cancer Res Treat. 2014; 144(1):1 10. 19. Herschkowitz JI, Simin K, Weigman VJ, et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol. 2007;8(5):R76. 20. Prat A, Parker JS, Karginova O, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 2010;12(5):R68. 21. Vera-Badillo FE, Templeton AJ, de Gouveia P, et al. Androgen receptor expression and outcomes in early breast cancer: a systematic review and metaanalysis. J Natl Cancer Inst. 2014;106(1):djt319. 22. Farmer P, Bonnefoi H, Becette V, et al. Identification of molecular apocrine breast tumours by microarray analysis. Oncogene. 2005;24(29):4660 4671. 23. Guedj M, Marisa L, de Reynies A, et al. A refined molecular taxonomy of breast cancer. Oncogene. 2012;31(9):1196 1206. 24. Dvorkin-Gheva A, Hassell JA. Identification of a novel luminal molecular subtype of breast cancer. PLoS One. 2014;9(7):e103514. 25. Cheang MC, Chia SK, Voduc D, et al. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst. 2009; 101(10):736 750. 26. Prat A, Cheang MC, Martin M, et al. Prognostic significance of progesterone receptor positive tumor cells within immunohistochemically defined luminal A breast cancer. J Clin Oncol. 2013;31(2):203 209. 27. Maisonneuve P, Disalvatore D, Rotmensz N, et al. Proposed new clinicopathological surrogate definitions of luminal A and luminal B (HER2- negative) intrinsic breast cancer subtypes. Breast Cancer Res. 2014;16(3):R65. 28. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004;10(16):5367 5374. 29. Cheang MC, Voduc D, Bajdik C, et al. Basal-like breast cancer defined by five biomarkers has superior prognostic value than tripe-negative phenotype. Clin Cancer Res. 2008;14(5):1368 1376. 30. Goldhirsh A, Winer EP, Coates AS, et al. Personalizing the treatment of women with early breast cancer: highlights of the St. Gallen International Expert Consensus on the primary therapy of early breast cancer 2013. Ann Oncol. 2013; 24(9):2206 2223. 31. Bigarzoli E, Coradini D, Ambrogi F, et al. p53 status identifies two subtypes of triple negative breast cancers with distinct biologic features. Jpn J Clin Oncol. 2011;41(2):172 179. 32. Coradini D, Biganzoli E, Ardoino I, et al. p53 status identifies triplenegative breast cancer patients who do not respond to adjuvant chemotherapy. Breast. 2015;24(3)294 297. 33. Green AR, Powe DG, Rakha EA, et al. Identification of key clinical phenotypes of breast cancer using a reduced panel of protein biomarkers. Br J Cancer. 2013;109(7):1886 1894. 34. Rakha EA, Soria D, Green AR, et al. Nottingham prognostic index plus (NPIþ): a modern clinical decision making tool in breast cancer. Br J Cancer. 2014;110(7):1688 1697. 35. Qi J, Yang YL, Zhu H, et al. Expression of the androgen receptor and its correlation with molecular subtypes in 980 Chinese breast cancer patients. Breast Cancer (Auckl). 2012;6:1 8. 36. Lakis S, Kotoula V, Eleftheraki AG, et al. The androgen receptor as a surrogate for molecular apocrine breast cancer subtyping. Breast. 2014;23(3): 234 243. 37. Sutton LA, Cao D, Sarode V, et al. Decreased androgen receptor expression is associated with distant metastases in patients with androgen receptor-expressing triple negative breast cancer. Am J Clin Pathol. 2012;138(4): 511 516. 38. Peurala E, Koivunen P, Haapasaari KM, Bloigu R, Jukkola-Vuorinen A. The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer. Breast Cancer Res. 2013;15(1):R5. 39. Bogina GS, Lunardi G, Marcolini L, et al. P16 but not retinoblastoma expression is related to clinical outcome in no-special-type triple-negative breast carcinomas. Mod Pathol. 2014;27(2):204 213. 40. Hui R, Macmillan RD, Kenny FS, et al. INK4a gene expression and methylation in primary breast cancer: overexpression of p16ink4a messenger RNA is a marker of poor prognosis. Clin Cancer Res. 2000;6(7):2777 2787. 41. Abou-Bakr AA, Eldweny HI. p16 expression correlates with basal-like triple-negative breast carcinoma. Ecancermedicalscience. 2013;7:317. 42. Hartmann LC, Keeney GL, Lingle C, et al. Folate receptor overexpression is associated with poor outcome in breast cancer. Int J Cancer. 2007;121(5):938 942. 43. Zhang Z, Wang J, Tacha DE, et al. Folate receptor alpha associated with triple negative breast cancer and poor prognosis. Arch Pathol Lab Med. 2014; 138(7):890 895. 44. Proverbs-Singh T, Feldman JL, Morris MJ, et al. Target the androgen receptor in prostate and breast cancer: several new agents in development. Endocr Relat Cancer. 2015;22(3):R87 R106 45. Ledermann JA, Canevari S, Thigpen T. Targeting the folate receptor: diagnostic and therapeutic approaches to personalize cancer treatments. Ann Oncol. 2015;26(10):2034 2043. 46. Assaraf YG, Leamin CP, Reddy JA. The folate receptor as a rational therapeutic target for personalized cancer treatment. Drug Resist Updat. 2014; 17(4 6):89 95. 47. Konner JA, Bell-McGuinn KM, Sabbatini P, et al. Farletuzumab, a humanized monoclonal antibody against folate receptor alpha, in epithelial ovarian cancer: a phase I study. Clin Cancer Res. 2010;16(21):5288 5295. 48. Tang P, Wang J, Bourne P. Molecular classification of breast carcinoma with similar terminology and different definitions: are they the same? Hum Pathol. 2008;39(4):506 513. 49. Tang P, Skinner K, Hicks D. Molecular classification of breast carcinomas by immunohistochemical analysis, are we ready? Diagn Mol Pathol. 2009;18(3): 125 132. 50. Hammond ME, Hayes DF, Dowestt M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48 e72. 51. Wolff AC, Hammond EA, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997 4013. 52. Harvey JM, Clark CM, Osborne K, Allred DG. 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(5):1474 1481. 53. McCarty KS, Miller LS, Cox EB, et al. Estrogen receptor analysis, correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med. 1985;109(8):716 721. 54. Dowsett M, Nielsen TO, A Hern R, et al. Assessment of Ki67 in breast cancer: recommendations for the international Ki67 in breast cancer working group. J Natl Cancer Inst. 2011;103(22):1656 1664. 55. Polley MYC, Leung SCY, McShane LM, et al. An international Ki67 reproducibility study. J Nat. Cancer Inst. 2013;105(24):1897 1906. 56. Polley MYC, Leung SCY, Gao D, et al. An international study to increase concordance in Ki67 scoring. Mod Pathol. 2015;28(6):778 786. 57. De Azarmbuja E, Cardosi F, de Castro G, et al. Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients. Br J Cancer. 2007;96(10):1504 1513. 58. Goldhirsch A, Wood WC, Coates AS, et al. Strategies for subtyped of dealing with the diversity of breast cancer highlights of the St. Gallen International Expert Consensus on the primary therapy of early breast cancer 2011. Ann Oncol. 2011;22(8):1736 1747. 59. Korsching E, Packeisen J, Agelopoulos K, et al. Cytogenetic alterations and cytokeratin expression patterns in breast cancer: integrating a new model of breast differentiation into cytogenetic pathways of breast carcinogenesis. Lab Invest. 2002;82(11):1525 1533. 60. Foulkes WD, Stefansson IM, Cheappuis PO, et al. Germline BRCA1 mutations and basal epithelial phenotype in breast cancer. J Natl Cancer Inst. 2003;95(19):1482 1485. 61. Shao MM, Chan SK, Yu AMC, et al. Keratin expression in breast cancers. Virchows Arch. 2012;461(3):313 322. 62. Korsching E, Jeffrey SS, Meinerz W, et al. Basal carcinoma of the breast revisited: an old entity with new interpretations. J Clin Pathol. 2008;61(5):553 560. 812 Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse

63. Tsang JY, Ni YB, Chan SK, et al. Androgen receptor expression shows distinctive significance in ER positive and negative breast cancers. Ann Surg Oncol. 2014;21(7):2218 2228. 64. Vera-Badillo F, Chang MC, Kuruzar C, et al. Association between androgen receptor expression, Ki-67 and the 21-gene recurrence score in nonmetastatic, lymph node-negative, estrogen receptor-positive and HER2-negative breast cancer. J Clin Pathol. 2015;68(10):839 843. 65. Dai MS, Sun XX, Lu H. Aberrant expression of nucleostemin activates p53 and induces cell cycle. Mol Cell Biol. 2008;28(13):4365 4376. 66. Lowe SW, Bodis S, McClatchey A, et al. p53 status and the efficacy of cancer therapy in vivo. Science. 1994;266(5186):807 810. 67. Overgaard J, Yilmaz M, Gulberg P, et al. TP53 is an independent prognostic marker for poor outcome in both node-negative and node-positive breast cancer. Acta Oncol. 2000;39(9):327 333. 68. Rozan S, Vincent-Salomon A, Zafrani B, et al. No significant predictive value of C-erbB-2 or p53 expression regarding sensitivity to primary chemotherapy or radiotherapy in breast cancer. Int J Cancer. 1998;79(1):27 33. 69. Rolland P, Spendlove I, Madjid Z, et al. The p53 positive Bcl-2 negative phenotype is an independent marker of prognosis in breast cancer. Int J Cancer. 2007;120(6):1131 1137. 70. Yamashita H, Nishio M, Toyama T, et al. Co-expression of Her-2 overexpression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer. Breast Cancer Res. 2004;6(1):R24 R30. 71. Lara JF, Thor AD, Dressler LG, et al. p53 expression in node positive breast cancer patients: results from the cancer and leukemia group B (CALGB) 9344 trials (159905). Clin Cancer Res. 2011;17(15):5170 5178. 72. Ignatiadis M, Sotiriou C. Luminal breast cancer: from biology to treatment. Nat Rev Clin Oncol. 2013;10(9):494 506. 73. Gatza ML, Silva GO, Parker JS, et al. An integrated genomics approach identifies drivers of proliferation in luminal-subtype human breast cancer. Nat Genet. 2014;46(10):1051 1059. 74. Hallett RM, Hassell JA. Estrogen independent gene expression defines clinically relevant subgroups of estrogen receptor positive breast cancer. BMC Cancer. 2014;14:871 75. Cancer Genome Atlas Network (TCGA). Comprehensive molecular portraits of human breast tumors. Nature. 2012;490(7418):61 70. 76. Jiang YZ, Yu KD, Zuo WJ, et al. GATA3 mutations define a unique subtype of luminal-like breast cancer with improved survival. Cancer. 2014;120(9):1329 1337. 77. Vuong D, Simpson PT, Green B, et al. Molecular classification of breast cancer. Virchows Arch. 2014;465(1):1 14. 78. Subik K, Lee JF, Baxter L, et al. The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer (Auckl). 2010;4(1):35 41 79. Tang P, Wei B, Wang J., et al. Pathological features of invasive ductal carcinoma of the breast according to the expression of progesterone receptor. Mod Pathol. 2015;28(suppl 2):70A. 80. Ades F, Zardavas D, Bozovic-Spasojevic I, et al. Luminal B breast cancer: molecular characterization, clinical management, and future perspectives. J Clin Oncol. 2014;32(25):2794 2803. 81. Coldstein NS, Decker D, Severson D, et al. Molecular classification system identifies invasive breast carcinoma patients who are most likely and those who are least likely to achieve a complete pathologic response after neoadjuvant chemotherapy. Cancer. 2007;110(8):1687 1696. 82. Carey LA, Dees EC, Sawyer L, et al. The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res. 2007;13(8): 2329 2334. 83. Cancello G, Maisonneuve P, Rotmansz N, et al. Progesterone receptor loss identifies luminal B breast cancer subgroups at higher risk of relapse. Ann Oncol. 2013;24(3):661 668. 84. Bardou VJ, Arpino G, Eledge RM, et al. Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. J Clin Oncol. 2003;21(10):1973 1979. 85. Tang P, Wang J, Hicks DG, et al. A lower Allred score for progesterone receptor is strongly associated with a higher recurrence score of 21-gene assay in breast cancer. Cancer Invest. 2010;28(9):978 982. 86. Wei B, Wang J, Bourne P, et al. Bone metastasis is strongly associated ER positive/pr negative breast carcinomas. Hum Pathol. 2008;39(12):1809 1815. 87. Cuzick J, Dowsett M, Pineda S, et al. Prognostic value of a combined estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor 2 immunohistochemical score and comparison with Genomic Health recurrence score in early breast cancer. J Clin Oncol. 2011;29(32):4273 4278. 88. Klein ME, Dabbs DJ, Shuai Y, et al. Prediction of the Oncotype DX recurrence score: use of pathology-generated equations derived by linear regression analysis. Mod Pathol. 2013;26(5):658 664. 89. Zong Y, Zhu L, Wu J, et al. Progesterone receptor status and Ki-67 index may predict early relapse in luminal B/HER2 negative breast cancer patients: a retrospective study. PLoS One. 2014;9(8):e95629. 90. Dandachi N, Dietze O, Hauser-Kronberger C. Chromogenic in situ hybridization: a novel approach to a practical and sensitive method for the detection of HER2 oncogene in archival human breast carcinoma. Lab Invest. 2002;82(8):1007 1014. 91. Green AR, Barros FFT, Abdel-Fatah TMA, et al. HER2/HER3 heterodimers and p21 expression are capable of predicting adjuvant trastuzumab response in HER2þ breast cancer. Breast Cancer Res Treat. 2014;145(1):33 44. 92. Fountzilas G, Dafni U, Bobos M, et al. Differential response of immunohistochemically defined breast cancer subtypes to anthracycline-based adjuvant chemotherapy with or without paclitaxel. PLoS One. 2012;7(6):e34946. 93. Kneubil MC, Brollo J, Botteri E, et al. Breast cancer subtype approximations and loco-regional recurrence after immediate breast reconstruction. Eur J Surg Oncol. 2013;39(3):260 265. 94. Vici P, Pizzuti L, Natoli C, et al. Triple positive breast cancer: a distinct subtype? Cancer Treat Rev. 2015;41(2):69 76. 95. Konecny G, Pauletti G, Pegram M, et al. Quantitative association between HER2/neu and steroid hormone receptors in hormone receptor-positive primary breast cancer. J Natl Cancer Inst. 2003;95(2):142 153. 96. Lal D, Tan LK, Chen B. Correlation of HER2 status with estrogen and progesterone receptors and histologic features in 3655 invasive breast carcinomas. Am J Clin Pathol. 2005;123(4):541 546. 97. Wu VS, Nanaya N, Lo C, et al. From bench to bedside: what do we know about hormone receptor-positive and human epidermal growth factor receptor 2 positive breast cancer. J Steroid Biochem Mol Biol. 2015;153:45 53. 98. De Laurentiis M, Arpino G, Massarelli E, et al. A meta-analysis on the interaction between HER2 expression to endocrine treatment in advanced breast cancer. Clin Cancer Res. 2005;11(13):4741 4748. 99. Dowsett M, Allred C, Knox J, et al. Relationship between quantitative estrogen and progesterone receptor expression and human epidermal growth factor receptor-2 (HER-2) status with recurrence in the Arimidex, Tamoxifen, Alone or in Combination trial. J Clin Oncol. 2008;26(7):1059 1065. 100. Osborne CK, Schiff R. Mechanisms of endocrine resistance in breast cancer. Annu Rev Med. 2011;62:233 247. 101. Shou J, Massarweh S, Osborne CK, et al. Mechanisms of tamoxifen resistance: increased estrogen receptors-her2/neu cross-talk in ER/HER2 positive breast cancer. J Natl Cancer Inst. 2004;96(12):926 935. 102. Witters LM, Kumar R, Chinchilli VM. Enhanced anti-proliferation activity of the combination of tamoxifen plus HER2 neu antibody. Breast Cancer Res Treat. 1997;42(1):1 5. 103. Witters L, Engle L, Lipton A. Restoration of estrogen responsiveness by blocking the HER2/neu pathway. Oncol Rep. 2002;9(6):1163 1166. 104. Alqaisi A, Chen L, Romond E, et al. Impact of estrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2) co-expression on breast cancer disease characteristics: implications for tumor biology and research. Breast Cancer Res Treat. 2014;148(2):437 444. 105. Purdie CA, Quinlan P, Jordan LB, et al. Progesterone receptor expression is an independent prognostic variable in early breast cancer: a population-based study. Br J Cancer. 2014;110(3):565 572. 106. Parise CA, Caggiano C. Breast cancer survival defined by the ER/PR/HER2 subtypes and a surrogate classification according to tumor grade and IHC biomarkers. J Cancer Epidermiol. 2014;2014:469251. 107. Prat A, Carey L, Adamo B, et al. Molecular features and survival outcomes of the intrinsic subtypes within HER2-positive breast cancer. J Natl Cancer Inst. 2014;106(8). pii: dju152. 108. Staaf J, Ringner M, Vallon-CHristersson J, et al. Identification of subtypes in human epidermal growth factor receptor 2-positive breast cancer reveals a gene signature prognostic of outcome. J Clin Oncol. 2010;28(11):1813 1820. 109. Carey L, Winer E, Viale G, et al. Triple-negative breast cancer: disease entity or title of convenience? Nat Rev Clin Oncol. 2010;7(12):683 692. 110. Bauer K, Brown M, Cress R, et al. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple negative phenotype: a populationbased study from California cancer registry. Cancer. 2007;109(9):1721 1728. 111. Rakha EA, El-Sayed M, Green A, et al. Prognostic markers in triple negative breast cancer. Cancer. 2007;109(1):25 32. 112. Dent R, Trudeau M, Pritchard K, et al. Triple negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13(15, pt 1): 4429 4434. 113. Badve S, Dabbs DJ, Schnitt SJ, et al. Basal-like and triple negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Mod Pathol. 2011;24(2):157 167. 114. Masuda H, Baggerly KA, Wang Y, et al. Differential response to neoadjuvant chemotherapy among 7 triple-negative breast cancer molecular subtypes. Clin Cancer Res. 2013;19(19):5533 5540. 115. Livasy CA, Karaca G, Nanda R, et al. Phenotypic evaluation of the basallike subtype of invasive breast carcinoma. Mod Pathol. 2006;19(2):264 271. 116. Lu S, Singh K, Mangray S, et al. Claudin expression in high grade invasive ductal carcinoma of the breast: correlation with the molecular subtype. Mod Pathol. 2013;26(4):485 495. 117. Rakha EA, Elsheikh SE, Aleskandarany MA, et al. Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin Cancer Res. 2009;15(7):2302 2310. 118. Lehmann-Che J, Harny AS, Porcher R, et al. Molecular apocrine breast cancer are aggressive estrogen receptor negative tumors over-expression either HER2 or GCDFP15. Breast Cancer Res. 2013;15(3):R37. 119. Sabatier R, Finetti P, Guille A, et al. Claudin-low breast cancers: clinical, pathological, molecular and prognostic characterization. Mol Cancer. 2014;13: 228. Arch Pathol Lab Med Vol 140, August 2016 Molecular Classifications of Breast Carcinoma Tang & Tse 813