Estrogen Receptor Expression Is High but Is of Lower Intensity in Tubular Carcinoma Than in Well-Differentiated Invasive Ductal Carcinoma

Transcription

1 Estrogen Receptor Expression Is High but Is of Lower Intensity in Tubular Carcinoma Than in Well-Differentiated Invasive Ductal Carcinoma Julie M. Jorns, MD; Dafydd G. Thomas, MD; Patrick N. Healy, MS; Stephanie Daignault, MS; Tammi L. Vickery, BS; Jacqueline E. Snider, BS; Elaine R. Mardis, PhD; Sherri R. Davies, PhD; Matthew J. Ellis, MD, PhD; Daniel W. Visscher, MD Context. Tubular carcinoma (TC) is a rare, luminal A subtype of breast carcinoma with excellent prognosis, for which adjuvant chemotherapy is usually contraindicated. Objective. To examine the levels of estrogen receptor (ER) and progesterone receptor expression in cases of TC and well-differentiated invasive ductal carcinoma as compared to normal breast glands and to determine if any significant differences could be detected via molecular testing. Design. We examined ER and progesterone receptor via immunohistochemistry in tubular (N ¼ 27), mixed ductal/tubular (N ¼ 16), and well-differentiated ductal (N ¼ 27) carcinomas with comparison to surrounding normal breast tissue. We additionally performed molecular subtyping of 10 TCs and 10 ductal carcinomas via the PAM50 assay. Results. Although ER expression was high for all groups, TC had statistically significantly lower ER staining percentage (ER%) (P ¼.003) and difference in ER expression between tumor and accompanying normal tissue (P ¼.02) than well-differentiated ductal carcinomas, with mixed ductal/tubular carcinomas falling between these 2 groups. Mean ER% was 79%, 87%, and 94%, and mean tumor-normal ER% differences were 13.6%, 25.9%, and 32.6% in tubular, mixed, and ductal carcinomas, respectively. Most tumors that had molecular subtyping were luminal A (9 of 10 tubular and 8 of 10 ductal), and no significant differences in specific gene expression between the 2 groups were identified. Conclusions. Tubular carcinoma exhibited decreased intensity in ER expression, closer to that of normal breast parenchyma, likely as a consequence of a high degree of differentiation. Lower ER% expression by TC may represent a potential pitfall when performing commercially available breast carcinoma prognostic assays that rely heavily on ER-related gene expression. (Arch Pathol Lab Med. 2014;138: ; doi: /arpa OA) Tubular carcinoma (TC) of the breast is uncommon, Favorable prognosis, however, appears limited to pure comprising fewer than 2% of invasive breast carcinoma or nearly pure tumors, defined as those tumors with 90% cases. Histologically, TC is infiltrative but well differentiated, or more classic histologic pattern. 1 4 Like other welldifferentiated breast carcinomas, TC is characteristically forming evenly spaced, small, often angulated tubules that are lined by a single layer of banal columnar epithelial cells estrogen receptor (ER) and progesterone receptor (PR) that invade breast stroma, eliciting a desmoplastic response. positive. 5,6 Early investigation via gene expression profiling has shown that most TCs fall into the luminal A (LumA) When compared to invasive ductal carcinoma of no special subgroup, which is composed of low-grade tumors with type (IDC NST), TCs are more likely to be smaller, have less good prognosis and high expression of ER-related genes. nodal involvement, and have better overall prognosis. 1 However, this intrinsic subtype also includes well-differentiated invasive ductal and lobular carcinomas, and a definitive gene set has yet to be described that distinguishes Accepted for publication January 24, From the Department of Pathology (Drs Jorns and Thomas) and TC from other LumA breast carcinomas, which may Comprehensive Cancer Center (Mr Healy and Ms Daignault), correlate with its exceptionally favorable prognosis even University of Michigan, Ann Arbor; the Genome Institute (Ms among this molecular subgroup. 7,8 Vickery and Dr Mardis) and Division of Oncology (Ms Snider and Fitting with their low-grade features and superior Drs Davies and Ellis), Washington University School of Medicine, St prognosis, we had anecdotally noted that many TCs have Louis, Missouri; and the Department of Laboratory Medicine and Pathology (Dr Visscher), Mayo Medical Laboratories, Rochester, heterogeneous ER/PR staining patterns as compared to the Minnesota. strong ER/PR positivity typically seen in well-differentiated Dr Ellis receives licensing and patent income from Bioclassifier IDC NST. Heterogeneity of ER/PR expression becomes LLC, PAM50 (St Louis, Missouri). The other authors have no relevant clinically relevant in cases sent for Oncotype DX analysis. financial interest in the products or companies described in this Oncotype DX (Genomic Health, Redwood City, California) article. Reprints: Julie M. Jorns, MD, Department of Pathology, University is a commercially available reverse transcription polymerase of Michigan,1500 E Medical Center Dr, 2G332, Ann Arbor, MI chain reaction (RT-PCR) assay performed on formalin-fixed, ( jjorns@umich.edu). paraffin-embedded (FFPE) tissue, which analyzes a 21-gene Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al 1507

2 panel composed of 16 cancer-related and 5 reference genes categorized into estrogen, HER2/neu, proliferation, invasion, other, and reference groups. A recurrence score (RS) is generated that stratifies patients into a low, intermediate, or high category for 10-year risk of distant recurrence. Oncotype DX is approved for use in patients with lowstage, ER-positive, and lymph node negative breast cancer, features that typify TC Contrary to the known favorable prognosis, we have seen cases of TC sent for Oncotype DX analysis that paradoxically have been returned with intermediate risk scores. This has important implications in future treatment decisions. Therefore, we aimed to further explore ER/PR expression in TC by standard immunohistochemical methods in concordance with molecular analysis in a subset of cases via PAM50, a quantitative real-time PCR assay that uses RNA extracted from FFPE tissue and a 50- gene signature to classify tumors into their respective molecular subtypes and generate risk of relapse (ROR) scores MATERIALS AND METHODS Case Selection Following institution review board approval, cases were identified from the University of Michigan Hospital and Health Systems (Ann Arbor, Michigan) files by querying an electronic medical database from Two board-certified pathologists with expertise in breast pathology (J.M.J. and D.W.V.) independently reviewed and classified the tumors. All patients included were confirmed to have modified Bloom-Richardson grade 1 invasive breast carcinoma. Cases were stratified into 3 groups tubular (T) (N ¼ 27), mixed ductal/tubular (M) (N ¼ 15), and ductal (D) (N ¼ 27) by percentage of tubular features (defined as open, angulated tubules, cytoplasmic luminal tufting or snouts, and low-grade histologic pattern), with at least 90%, 25% to 89%, and less than 25% tubular features for T, M, and D groups, respectively. For those tumors for which classification was not initially agreed upon, consensus was later reached between the 2 pathologists. Additional clinicopathologic features including age at diagnosis, tumor size, lymph node status, final surgery, previous Oncotype DX assay results (if performed), and patient outcome were also assessed by reviewing electronic medical records. Immunohistochemical Staining Estrogen receptor and PR status were assessed by immunohistochemistry (IHC) using a refined labeled streptavidin-biotin technique (Dakocytomation, Carpinteria, California) with monoclonal antibodies against ER (1:100; clone SP1, Ventana Medical Systems, Tucson, Arizona) and PR (1:400; clone IE2, Dakocytomation). The IHC laboratory has Clinical Laboratory Improvement Amendments of 1988 certification, and ER/PR IHC was validated per College of American Pathologists (CAP) and American Society of Clinical Oncology (ASCO) guidelines before the start of this study. Continued CAP proficiency testing and involvement in other quality assurance programs by the IHC laboratory is ongoing. Staining was performed with the Ventana BenchMark XT platform and results were obtained via Ventana Image Analysis System (VIAS). This quantitative image analysis system is approved by the US Food and Drug Administration, validated and performed by specially trained pathologists. A percentage positive score is established by colorimetric and morphometric analysis of at least 4 images with analysis of segmented objects (eg, nuclei, cytoplasm), based on an adaptive threshold in the score formula. Patient slides with representative tumor were stained for ER and PR and percentage of staining both in tumor and in normal ducts and lobules (within the same slide) were assessed by the same VIAS-trained pathologist (J.M.J.) before histologic classification. Additionally, 12 benign samples from macromastia cases served as normal controls. Gene Expression Profiling Intrinsic subtyping was performed on 10 tumors from each of the T and D groups by using the RT-PCR PAM50 technique previously described by Parker et al. 12 Briefly, RNA was extracted from 231- mm-diameter cores obtained from available FFPE blocks. After removal of contaminating DNA and NanoDrop ND100 (NanoDrop Products, Wilmington, Delaware) quantification, a reverse transcriptase reaction was performed by using random hexamers and specific primers. Aliquots of the products were subjected to the 50- gene quantitative PAM50 PCR assay and the results normalized to the expression of several housekeeping genes. Intrinsic subtype call and ROR analysis were performed by using these data and the algorithm described by Parker et al. 12 Statistical Analysis Characteristics of T, M, and D groups were described by using means for continuous covariates and frequencies for categorical covariates. Analysis of variance models were used to test continuous covariates for differences between the clinical covariates. Categorical covariates were tested with Fisher exact test. The Jonckheere-Terpstra test was used to test the ordinal Oncotype DX results between groups. The difference between normal and tumor ER and PR expression was compared between the 3 carcinoma groups by using a 1-way analysis of variance adjusted for multiple comparisons using the Tukey-Kramer method. Additionally, Wilcoxon rank sum tests were used to compare tumor ER staining percentage (ER%) and PR staining percentage between T and D groups. Box plots of the differences between normal and tumor ER and PR expression among the 3 groups were constructed. SAS 9.2 (SAS Institute, Cary, North Carolina) was used for all statistical analyses. RESULTS Patients There was no significant difference in age at diagnosis, tumor size, lymph node status, specimen type used for IHC, final surgery, follow-up availability, recurrence, and Oncotype DX score distribution for T, M, and D groups. All patients had T1 tumors that were (either clinical or pathologic) lymph node stage pn0, with only 1 patient in the T group having a positive lymph node in the form of a single focus of isolated tumor cells seen in only 1 level of a sentinel lymph node (pn0(iþ)(sn)). Follow-up information was available for most patients, with recurrence occurring in only 1 patient in the M group. This patient had recurrence of a mammographically identified, 0.6-cm, morphologically similar carcinoma within the ipsilateral breast 24.8 months following initial diagnosis, at which time she had been receiving hormonal therapy. Oncotype DX had been previously performed and the result was associated with low risk (RS ¼ 16). Oncotype DX had been previously performed for a limited number of patients and yielded predominantly low-risk RS, but notably intermediate-risk RS was noted for 2 patients from both the T and D groups. There was statistically significant reduction in mean follow-up time for patients in the D group as compared to T and M groups, likely due to the ease in identifying these more common cancer subtype cases. Complete clinicopathologic information is provided in Table 1. Estrogen Receptor/PR Immunohistochemistry There was no significant difference in ER/PR expression in the normal glands among the T, M, and D tumor groups, and a similar range of ER/PR staining was seen in the normal glands of macromastia controls. There was not a 1508 Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al

3 Table 1. Clinicopathologic Features of Tubular, Mixed Ductal/Tubular, and Ductal Carcinoma Groups Tubular Carcinoma (N ¼ 27) Mixed Ductal/Tubular Carcinoma (N ¼ 15) Ductal Carcinoma (N ¼ 27) P Value Age at diagnosis, y Mean a Range Tumor size, cm Mean a Range Lymph node status Pathologically positive b Pathologically negative 25 c Clinically negative (no surgery) Immunohistochemistry specimen Core b Resection Resection surgery Breast-conserving therapy b Mastectomy Unknown Recent follow-up Available b Unavailable d Follow-up time, mo Mean a Range Recurrence Yes b No Oncotype DX results (if previously performed) Low risk e Intermediate risk High risk a Analysis of variance test. b Fisher exact test. c One patient had isolated tumor cells. d Recent follow-up is defined as unavailable if the patient transferred care for treatment or if 5 or more years elapsed since last follow-up. e Jonckheere-Terpstra test. significant relationship between tumor type and PR expression by IHC staining. However, the T group had a statistically significant decrease in tumor ER% (P ¼.003) and difference between tumor and normal ER expression (P ¼.02) when compared to grade 1 IDC NST. Mixed tubular/ductal carcinomas had intermediate values for both parameters. Mean ER% was 79%, 87%, and 94% in T, M, and D groups, respectively. Mean tumor-normal ER% differences were 13.6%, 25.9%, and 32.6% in T, M, and D groups, respectively. Box plots showing tumor ER% and difference between tumor and normal tissue are shown in Figures1and2.ExamplecasesofTCandIDCNSTwith hematoxylin-eosin and ER IHC staining are shown in Figure 3, A through D. Molecular Subtyping by PAM50 Assay Despite small sample size and, in some cases, old material (FFPE blocks up to 10 years old), PAM50 successfully subtyped 19 of 20 tumors sent for gene expression analysis. The analysis revealed that 9 of 10 tumors and 8 of 10 tumors were of the LumA intrinsic subtype in T and D groups, respectively. One case in both T and D groups was normal breast like. The remaining case from the D group yielded insufficient RNA for analysis. Analysis of the raw data revealed no significant differences in gene expression between the 2 groups. Summarized PAM50 results with correlating clinicopathologic features are shown in Table 2. COMMENT Tubular carcinoma is a rare subtype of breast carcinoma known to have an exceptionally favorable prognosis. Superior prognosis, however, appears restricted to those patients with tumors of pure or nearly pure composition. Chemotherapy is typically not recommended to patients with TC given the excellent prognosis, low risk for recurrence, and negative side effects that significantly outweigh the small benefit chemotherapy may have. Thus, correct classification is very important or there is risk of unnecessary and potentially hazardous treatment. Conversely, some patients with well-differentiated IDC NST with tubular features, so-called mixed ductal/tubular carcinoma, may derive clinical benefit from chemotherapy and could be denied appropriate treatment with incorrect tumor classification as TC. Despite well-defined morphologic criteria, some cases of TC can still be difficult to distinguish from mixed ductal/tubular carcinomas via histologic examination alone. Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al 1509

4 Figure 1. Box plots comparing estrogen receptor (ER) expression among ductal (D), mixed ductal/tubular (M), and tubular (T) groups. Recent advances in gene expression profiling have shown promise in the classification of breast carcinoma and identification of prognostic markers. In this study PAM50 classified all TCs and well-differentiated IDCs NST as lowrisk molecular subtype (17 LumA and 2 normal breast like), fitting of the indolent clinical courses observed in these patients. However, PAM50 generated a medium ROR-S score (subtype weighted) in 5% (1 of 20) of patients and a medium ROR-P score (proliferation weighted) in 35% (7 of 20) of patients. Although Oncotype DX is usually not performed for patients with TC, some previously had tumor blocks sent for this test because the designation as TC had gone unrecognized by ordering support staff and because other tumor characteristics (ie, ER-positive, lymph node negative status) fit submission criteria. Like PAM50, Oncotype DX also failed to distinguish the 2 groups and yielded intermediate RS in 28.6% (4 of 14) of patients. A noted limitation is small sample size, which is due to both the rarity of TC and small tumor sizes in all groups. Figure 2. Box plots comparing the difference (Diff) between (B/T) tumor and normal gland expression of estrogen receptor (ER) among ductal (D), mixed ductal/tubular (M), and tubular (T) groups Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al

5 Figure 3. Tubular carcinoma (A) with decreased estrogen receptor (ER) staining intensity (B) when compared to invasive ductal carcinoma (C) with strong ER staining intensity (D) (hematoxylin-eosin, original magnification 320 [A and C]; original magnification 320 [B and D]). Case No. Table 2. Results From 20 Cases Sent for PAM50 Analysis and Corresponding Clinicopathologic Features Morphologic Subtype Molecular Subtype ROR-S (Subtype) ROR-P (Subtype þ Proliferation) Tumor Size, cm Lymph Node Status Outcome Follow-up Time, mo 1 T LumA Low Low 0.6 pn0 NR T LumA Low Low 0.6 pn0 NR T LumA Low Low 0.6 pn0 NR T LumA Low Low 0.8 pn0 NR T LumA Low Low 1.0 pn0 NR LTF (84.6) 6 T LumA Low Low 1.4 pn0 NR T LumA Low Low 1.4 pn0 NR T LumA Low Med 0.2 pn0 NR T LumA Low Med 1.2 pn0 NR T Normal Med Med 0.3 pn0 NR D LumA Low Low 0.2 pn0 NR D LumA Low Low 0.5 pn0 NR LTF (2.5) 13 D LumA Low Low 1.1 pn0 NR D LumA Low Low 1.3 pn0 NR D LumA Low Low 1.4 pn0 NR D LumA Low Med 0.7 pn0 NR D LumA Low Med 0.9 pn0 NR D LumA Low Med 1.5 pn0 NR D Normal Low Med 0.8 pn0 NR D QNS N/A N/A 0.2 pn0 NR 45.8 Abbreviations: D, ductal carcinoma; LTF, lost to follow-up (5 years since last follow-up); LumA, luminal A intrinsic subtype; Med, medium; N/A, not applicable; Normal, Normal breast like intrinsic subtype; NR, no recurrence; pn0, negative by pathologic assessment; QNS, quantity insufficient for analysis; ROR-P, risk of relapse (low, medium, or high) based on intrinsic subtype and proliferation; ROR-S, risk of relapse (low, medium, or high) based on intrinsic subtype; T, tubular carcinoma. Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al 1511

6 Tubular carcinoma and well-differentiated ductal and lobular carcinoma subclassification as LumA has previously been well described. Although TC typically is most morphologically similar to well-differentiated IDC NST, it shows a closer resemblance to low-grade invasive lobular carcinoma (ILC) than to IDC NST at the molecular level. 7,8 It has been suggested that TC may be further distinguished from ILC in that TC has lower expression of proliferation and cell cycle genes than ILC, and ILC exhibits downregulation of cell adhesion/extracellular matrix related genes. 15 These subtle differences are relevant in that they support the biological distinction of TC from ILC and other well-differentiated breast carcinomas. Unfortunately, however, these differences cannot yet be exploited to reliably distinguish these well-differentiated carcinomas in the clinical setting, as commercially available assays examining molecular subtype and recurrence risk usually fail to separate them. Microscopic analysis therefore remains the mainstay of TC classification. In difficult cases other features can often be helpful in supporting the diagnosis of TC, such as small size, negative axillary lymph node status, and association with flat epithelial atypia, which frequently accompanies it and has been speculated to be the precursor lesion of TC. 16,17 Additionally, we have found that careful evaluation of ER% may also be helpful in supporting morphologic impression, as ER expression by IHC is of decreased intensity in patients with TC as compared to welldifferentiated IDC NST, which it most closely resembles histologically. Additionally, ER% is significantly more akin to that within the patient s normal breast glands in TC as compared to well-differentiated IDC NST. Estrogen receptor staining is still quite strong as previously reported, 18 with mean ER% of 79% in this series, but it is significantly lower than the very strong ER staining typically seen in well-differentiated IDC NST (mean ER% of 94% in this series). We believe the lower level of ER expression in TC may be a consequence of the high degree of differentiation and further supports its biological distinction. A similar relationship between moderately high ER expression and superior outcome has been previously described. Several recent publications outline the utility of the IHC4 score, which analyzes ER, PR, and HER2/neu expression and Ki-67 proliferation index via IHC, to potentially provide breast cancer predictive information with similar efficacy but at lower cost than molecular gene expression assays. One study by Dowsett et al 20 compares IHC4 and Oncotype DX RS predictive value in the translational arm of the ATAC (anastrozole or tamoxifen alone or combined) trial (TransATAC). As expected, patients with lowest ER and PR (first quartile) expression had the least favorable prognosis, with greater rate of recurrence and shorter time to recurrence. However, those with highest (fourth quartile) ER expression had higher recurrence than those with slightly lower ER (third quartile) expression, a finding most evident in the anastrozole arm. 20 These findings support our study because the most favorable breast carcinomas, which undoubtedly included TC, had high, but not the highest, level of ER expression. Unfortunately, however, this presents a significant pitfall for assays that are heavily weighted toward ER-related gene expression, potentially resulting in higher-than-expected estimate of recurrence risk, which can complicate clinical decision making. A noted limitation of this study is lack of cold ischemia time data, as these cases occurred before documentation of this variable. Recent studies 22,23 have shown that ER/PR expression can be significantly reduced with prolonged cold ischemia time, or time from tissue removal and permeation of the tissue by formalin. The effects of prolonged cold ischemia time are largely restricted to excision specimens, as core biopsy specimens are quickly immersed in formalin and have rapid tissue permeation. The tumors in this study were small and thus IHC was often performed on diagnostic excisional biopsies. However, there was no difference in the distribution of specimen type for which IHC was performed between cancer subgroups, therefore minimizing sample selection bias and staining variation due to cold ischemia time alone. In this study all cases were stained in a single large batch in compliance with CAP/ASCO ER/PR staining guidelines; thus, staining differences between groups due to technical issues were also minimized. Appropriate classification of TC is imperative as it has important prognostic and treatment implications. Our findings suggest that careful examination of ER staining in tumor cells with comparison to staining in adjacent normal glands may aid in establishing the diagnosis of TC in more challenging cases. Clinical follow-up and molecular PAM50 data further support the favorable outcome of TC and support the notion that ancillary prognostic tests likely are unnecessary for this rare subset of breast carcinoma with excellent prognosis. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St Louis, Missouri, for the use of the Tissue Procurement Core, which provided sample accessioning, distribution, and quality assurance services. The Siteman Cancer Center is supported in part by a National Cancer Institute Cancer Center Support Grant (No. P30 CA91842). References 1. Rosen P. Tubular carcinoma. In: Rosen s Breast Pathology. Philadelphia, PA: Lippincott Williams & Wilkins; 2009: Cooper HS, Patchefsky AS, Krall RA. Tubular carcinoma of the breast. Cancer. 1978;42(5): Rakha EA, Lee AH, Evans AJ, et al. Tubular carcinoma of the breast: further evidence to support its excellent prognosis. J Clin Oncol. 2010;28(1): Rosen PR, Groshen S, Saigo PE, Kinne DW, Hellman S. A long-term followup study of survival in stage I (T1N0M0) and stage II (T1N1M0) breast carcinoma. J Clin Oncol. 1989;7(3): Diab SG, Clark GM, Osborne CK, et al. Tumor characteristics and clinical outcome of tubular and mucinous breast carcinomas. J Clin Oncol. 1999;17(5): Fasano M, Vamvakas E, Delgado Y, et al. Tubular carcinoma of the breast: immunohistochemical and DNA flow cytometric profile. Breast J. 1999;5(4): Geyer FC, Marchio C, Reis-Filho JS. The role of molecular analysis in breast cancer. Pathology. 2009;41(1): Weigelt B, Horlings HM, Kreike B, et al. Refinement of breast cancer classification by molecular characterization of histological special types. J Pathol. 2008;216(2): Cronin M, Pho M, Dutta D, et al. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol. 2004;164(1): Cronin M, Sangli C, Liu ML, et al. Analytical validation of the Oncotype DX genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node-negative, estrogen receptor-positive breast cancer. Clin Chem. 2007;53(6): Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004;351(27): Parker JS, Mullins M, Cheang MC, et al. Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol. 2009;27(8): Kelly CM, Bernard PS, Krishnamurthy S, et al. Agreement in risk prediction between the 21-gene recurrence score assay (Oncotype DX(R)) and the PAM50 breast cancer intrinsic Classifier in early-stage estrogen receptor-positive breast cancer. Oncologist. 2012;17(4): Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al

7 14. Nielsen TO, Parker JS, Leung S, et al. A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor-positive breast cancer. Clin Cancer Res. 2010;16(21): Lopez-Garcia MA, Geyer FC, Natrajan R, et al. Transcriptomic analysis of tubular carcinomas of the breast reveals similarities and differences with molecular subtype-matched ductal and lobular carcinomas. J Pathol. 2010; 222(1): Aulmann S, Elsawaf Z, Penzel R, Schirmacher P, Sinn HP. Invasive tubular carcinoma of the breast frequently is clonally related to flat epithelial atypia and low-grade ductal carcinoma in situ. Am J Surg Pathol. 2009;33(11): Kunju LP, Ding Y, Kleer CG. Tubular carcinoma and grade 1 (welldifferentiated) invasive ductal carcinoma: comparison of flat epithelial atypia and other intra-epithelial lesions. Pathol Int. 2008;58(10): Masood S, Lu L, Rodenroth N. Potential value of estrogen receptor immunocytochemical assay in formalin-fixed breast tumors. Mod Pathol. 1990; 3(6): 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 the Genomic Health recurrence score in early breast cancer. J Clin Oncol. 2011;29(32): Dowsett M, Salter J, Zabaglo L, et al. Predictive algorithms for adjuvant therapy: TransATAC. Steroids. 2011;76(8): Dowsett M, Sestak I, Lopez-Knowles E, et al. Comparison of PAM50 risk of recurrence score with Oncotype DX and IHC4 for rredicting risk of distant recurrence after endocrine therapy. J Clin Oncol. 2013;31(22): Neumeister VM, Anagnostou V, Siddiqui S, et al. Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues. J Natl Cancer Inst. 2012;104(23): Yildiz-Aktas IZ, Dabbs DJ, Bhargava R. The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol. 2012;25(8): Arch Pathol Lab Med Vol 138, November 2014 Estrogen Receptor Expression in Tubular Carcinoma Jorns et al 1513