Low ER+ Breast Cancer. Is This a Distinct Group? Nika C. Gloyeske, MD, David J. Dabbs, MD, and Rohit Bhargava, MD ABSTRACT

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1 Low ER+ Breast Cancer Is This a Distinct Group? Nika C. Gloyeske, MD, David J. Dabbs, MD, and Rohit Bhargava, MD From the Magee-Womens Hospital of University of Pittsburgh Medical Center, Pittsburgh, PA. Key Words: Low ER+/HER2 ; Morphology; Response to neoadjuvant chemotherapy Am J Clin Pathol May 2014;141: ABSTRACT Objectives: Estrogen receptor (ER) level can be semiquantified by immunohistochemistry (IHC) using the H-score. The score, given as the sum of the percent staining multiplied by the intensity level, ranges from 0 to 300. Methods: Forty-nine ER+/HER2 invasive tumors with low ER expression (H-scores of 1-100, representing approximately 5% of all tumors) were studied for various morphologic parameters, progesterone receptor (PR), and Ki-67 IHC. Results: Eighteen of 49 patients received neoadjuvant chemotherapy. The morphologic analysis showed that these tumors are often grade 3 and frequently demonstrate a sheet-like growth pattern, an intratumoral lymphocytic inflammatory infiltrate, and necrosis. Eighty percent of tumors showed a Ki-67 proliferation index of more than 50%, and 94% were PR. Of the 18 patients who received neoadjuvant chemotherapy, six (33%) achieved pathologic complete response. Conclusions: The low ER+/HER2 cases have morphologic features and a response to the chemotherapy rate that are more similar to triple-negative tumors than the usual type of ER+ tumors. On average, approximately 70% to 75% of breast cancers are estrogen receptor (ER) positive, with a slightly higher positive rate in whites and older patients and a slightly lower rate in nonwhite and younger people. 1 Most tumors that are ER+ are generally diffusely and strongly positive by immunohistochemistry (IHC). However, approximately 15% to 20% of ER+ tumors demonstrate heterogeneous expression (ie, a variable number of cells showing weak- to moderate-intensity staining). 2 Unfortunately, this detailed information is sometimes lost for the purposes of treatment and in clinical trials, where all ER+ breast cancers are grouped together regardless of their level of ER expression as detected by IHC. Although the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) recommend that ER status should be considered positive if 1% or more of tumor cells demonstrate positive nuclear staining with an IHC assay, they also recommend semiquantifying the results. 3 Typically, ER+ breast cancers are treated with endocrine-based therapies, and ER breast cancers are treated with adjuvant or neoadjuvant chemotherapy (NACT). Only a small percentage of patients with ER+ tumors are offered chemotherapy, and that decision is based on tumor biology. Not uncommonly, expensive multigene assays are used to make this decision, not realizing that similar information can be gained using tumor morphology and a tumor receptor profile. Recent studies have shown the clinical significance of semiquantitative hormone receptor levels, 4 and it is possible that breast cancers expressing ER at low levels, or low ER+ tumors, are different from the usual ER+ tumors. To obtain a better understanding of this rare subset of breast cancers, we characterized these tumors morphologically to determine Am J Clin Pathol 2014;141:

2 Gloyeske et al / Low ER+ Breast Cancer if these tumors are more similar to usual ER+ tumors or to ER tumors. Materials and Methods At our institution, hormone receptor results are semiquantified on IHC using the H-score method. The score is given as the sum of the percent staining multiplied by the intensity level (0 = absent, 1 = weak, 2 = moderate, and 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). IHC for ER and progesterone receptor (PR) was performed at the time of initial diagnosis using the SP1 and 1E2 antibodies, respectively, and IVIEW detection on the BenchMark XT (Ventana Medical Systems, Tucson, AZ). Low ER expression was defined as an H-score of 1 to 100. A search for invasive breast carcinoma cases for 2010 with an ER H-score of 1 to 100 yielded 38 ER+/ HER2 cases. These cases represented 5% of the total 731 cases of invasive breast carcinomas assessed for receptors at our institution. An additional 11 ER+/HER2 cases randomly selected from 2011 were also included, resulting in a total of 49 low ER+ cases for morphologic analysis. HER2/neu protein was analyzed and scored using the 4B5 antibody and basic 3,3 -diaminobenzidine detection on the BenchMark XT (Ventana Medical Systems). HER2 IHC was scored according to the 2007 ASCO/CAP criteria. All cases with an HER2 IHC score of 2+ were analyzed by fluorescence in situ hybridization (FISH; PathVysion Vysis dual-color FISH, Vysis, Downers Grove, IL) and classified as negative, equivocal, or positive for HER2 (ERBB2) gene amplification using the ASCO/CAP criteria. 5 All HER2+ cases (ie, IHC score of 3+ and cases with an IHC score of 2+ and unequivocally amplified by FISH) were excluded from this study. A total of 49 ER+/HER2 cases were analyzed for various morphologic parameters, tumor type (ductal vs lobular), Nottingham tumor grade, and type of growth pattern (infiltrative vs pushing). The following features were considered present if identified in more than 10% of the tumor: sheetlike growth pattern, spindle cells, apocrine differentiation, intratumoral/peritumoral lymphocytic infiltrate, and necrosis. Nucleoli were considered prominent if visible at a 10 objective, and substantial apoptosis was considered present if easily visible at 10. The presence of in situ carcinoma and lymphovascular space invasion was also recorded. Cell proliferative activity was assessed by counting the absolute number of mitotic figures in 10 high-power fields and also by the Ki-67 labeling index. IHC for Ki-67 was performed at the time of initial diagnosis using the 30-9 antibody and IVIEW detection on the BenchMark XT (Ventana Medical Systems). Of the 49 cases included in this study, 18 received NACT. Since many of these cases had either no residual tumor or minimal residual disease/tumor alteration in the posttherapy resection specimen, the tumor morphologic features in these cases were assessed in the pretherapy core biopsy specimens. Inclusion of cases that received NACT provided us an opportunity to assess the pathologic complete response (pcr) rate in low ER+ tumors to the first-line chemotherapy regimen used at our institution (Adriamycin and cyclophosphamide followed by taxane, commonly referred to as the AC followed by T regimen). pcr was defined as the absence of invasive carcinoma in the posttherapy resection specimen and in the lymph nodes. Percentage tumor volume reduction was also calculated based on pretherapy size and a detailed evaluation of the resection specimen. At our institution, this calculation requires knowledge of the pretherapy tumor size, posttherapy gross tumor bed size, and microscopic cellularity of the tumor bed compared with the pretherapy core biopsy specimen. The details of this procedure have been published previously. 6 Although the focus of this study was to describe morphologic characteristics of tumors with ER H-scores of 1 to 100, these tumors were further subdivided into two groups: tumors with an ER H-score of 1 to 10 and tumors with an ER H-score of 11 to 100. These two groups were compared for all variables to see if the definition of low ER+ tumors could be further refined. Statistical analysis was performed using GraphPad QuickCalcs online software (GraphPad Software, La Jolla, CA). For comparison of means, independent sample t tests were performed. Univariate analysis was performed using c 2 and Fisher exact tests to compare the differences in percentages between groups. A P value less than.05 was considered significant. Results All clinical and pathologic data on these 49 cases are shown in Table 1. Twenty-six (53%) cases showed ER H-scores from 1 to 10, and 23 (47%) had ER H-scores of more than 10. Data were analyzed between these two groups, with none of the variables significantly different between them (P >.05 for all variables). Of the 18 patients who received NACT, six (33%) showed pcr. Four of the six cases that achieved pcr had ER H-scores of more than 10. Mean tumor volume reduction in 18 cases that received NACT was 82%. Mean tumor volume reduction was 72% in 12 cases that failed to achieve pcr. Due to the small number of patients who received NACT, statistical analysis was not meaningful. However, the two features that were almost universally present in cases with pcr were sheetlike growth pattern (six of six cases) and intratumoral/peritumoral lymphocytic infiltrate (five of six cases). 698 Am J Clin Pathol 2014;141: Downloaded 698 from

3 Discussion Breast cancers are heterogeneous at the morphologic, immunohistochemical, and molecular levels. This heterogeneity persists even within the apparently homogeneous groups of ER+ and ER tumors. In the past few years, ER+ tumors have been subjected to multigene prognostic/predictive assays to identify more aggressive types of ER+ tumors that are expected to benefit from additional chemotherapy and to define risk of recurrence in an individual patient Immunohistochemically, these high-risk ER+ tumors have been reported to have lower hormone receptor expression and a high proliferation index using the Ki-67 antibody. 11,12 The current study was undertaken to specifically examine the tumors that have low ER expression. The detailed morphologic analysis of low ER+ tumors in our study showed that these tumors are often grade 3, have a very high proliferation index, and frequently demonstrate a sheet-like growth pattern, an intratumoral lymphocytic inflammatory infiltrate, and necrosis Image 1. All of these features have been described for basal-like and triple-negative tumors. 2,13 These morphologic features are generally uncommon in typical strongly ER+ tumors. Therefore, the morphologic features suggest that low ER+ tumors have a histologic kinship with triple-negative tumors rather than with typical ER+ tumors. The inclusion of 18 cases that were subjected to NACT in our study provided us the opportunity to assess response to NACT in these low ER+ tumors. Six cases (33%) achieved pcr, and mean estimated tumor volume reduction was 82%. This result is strikingly different from that observed in strongly ER+/HER2 tumors, in which the pcr rate has been reported below 5% and mean tumor volume reduction below 30%. 14 The pcr rate in the current study is similar to what is observed in triple-negative or basal-like tumors The ASCO/CAP guidelines on hormone receptor testing have recommended considering a tumor ER+ (or PR+) if 1% or more tumor cells stain on IHC testing. This recommended cutoff is based on the premise that patients derive benefit from endocrine therapy even with minimal receptor reactivity. This benefit of endocrine therapy in very weakly ER+ tumors is debatable. Unfortunately, our current study did not address this issue. However, our study does shed light on chemotherapy benefit in these low ER+ tumors. Using a single variable of the ER semiquantitative H-score, one can be certain of tremendous chemotherapy benefit in terms of tumor resolution in the neoadjuvant setting in low ER+ tumors. Whether this will result in sustained disease-free survival and overall survival, although expected, remains to be seen. The results of our study are quite comparable to a handful of studies that have evaluated low ER+ tumors. In a gene expression profiling study of 26 low ER+ tumors (defined as tumors having 1%-10% immunoreactive cells), 88% were Table 1 Clinical and Pathologic Characteristics of Low ER+ Tumors (n = 49) a Characteristic Value Age, y Mean/median (range) 57/56 (32-88) (41) Tumor type Ductal 48 (98) Lobular 1 (2) Tumor size, mean/median (range), cm 2.8/2.5 ( ) Positive nodal status, b excluding isolated tumor cells 14 (35) Nottingham grade Grade 2 4 (8) Grade 3 45 (92) PR Positive 3 (6) H-scores on PR+ cases, mean/median (range) 36/35 (3-70) HER2 status IHC score 0 or (57) IHC score 2+, FISH not amplified 17 (35) IHC score 2+, FISH equivocal 4 (8) Ki-67 labeling index Mean/median (range), % 73/80 (11-100) Index >50% 39 (80) Absolute mitotic count/10 hpf, mean/median (range) 26/22 (3-100) Tumor borders Circumscribed/pushing 20 (41) Infiltrative 29 (59) Sheet-like growth pattern 35 (71) Spindle cells in tumor 15 (31) Apocrine differentiation 17 (35) Prominent nucleoli 26 (53) Intratumoral/peritumoral lymphoid cells 29 (59) Necrosis 22 (45) Apoptosis 10 (20) Ductal carcinoma in situ 25 (51) Lymphovascular space invasion 12 (24) ER, estrogen receptor; FISH, fluorescence in situ hybridization; hpf, high-power field; IHC, immunohistochemistry; PR, progesterone receptor. a Values are presented as number (%) unless otherwise indicated. b Data not available for nine cases. classified as basal-like or HER2 enriched. 18 In another similar study of 465 breast carcinomas by Iwamoto and colleagues, 19 the 25 tumors expressing 1% to 9% ER+ cells showed molecular features similar to basal-like tumors. Our study included cases that showed H-scores of 1 to 100, which represents the lowest third of ER expression level. Although an H-score of 100 indicates a weak ER expression pattern, our study allowed slightly higher expression compared with the studies reported by Deyarmin et al 18 and Iwamoto et al. 19 Nevertheless, we compared the two groups (ie, tumors with an ER H-score of 1-10 and an ER H-score of ) in our data to see if any differences exist. Interestingly, no differences were identified with respect to any of the morphologic features or variables. Moreover, the pcr rate and tumor volume reduction were quite similar between the two groups. Another interesting aspect of the low ER+ tumors in our study was the lack of PR expression in more than 90% of the cases. The prognostic and predictive value of PR Am J Clin Pathol 2014;141:

4 Gloyeske et al / Low ER+ Breast Cancer A B C D Image 1 Examples of low estrogen receptor positive invasive ductal carcinoma demonstrating high-grade nuclei (A, 200), intratumoral lymphocytic inflammatory infiltrate (B, 100), sheet-like growth pattern (C, 40), and necrosis (D, 20). expression has been challenged from time to time. 20 However, the importance of PR expression becomes evident in the studies that have used PR scores along with routine histopathologic variables in predicting prognosis or comparison with multigene tests. 4,12,21,22 Even the initial pioneering studies by Clark and McGuire 23,24 showed the prognostic significance of PR. Some recent studies have also shown that semiquantified PR scores further provide additional prognostic/predictive information. 4,12 PR expression in ER+ tumor cells indicates integrity of the ER pathway. The lack of PR expression in low ER+ tumors may indicate upregulation of growth factor receptors, which makes these tumors highly proliferative and amenable to suppression by standard chemotherapeutic agents. It will be of clinical value to know how much benefit these low ER+ tumors derive from endocrine therapy. Unfortunately, our study is unable to answer the latter question. This study is one of the few to evaluate the morphologic characteristics of low ER+ breast cancers. Low ER+ breast cancers have morphologic characteristics more akin to those of triple-negative breast cancers than those of the usual ER+ breast cancers. We also found that pcr rates to NACT are also more similar to those of triple-negative breast cancers than those of strongly ER+ breast cancers. We conclude that low ER+ breast cancers are a distinct subset of ER+ tumors. Patients with low ER+ tumors should be offered chemotherapy due to the likelihood of deriving substantial benefit. At the current time, there is a lack of evidence for not treating these 700 Am J Clin Pathol 2014;141: Downloaded 700 from

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