Tan et al. Breast Cancer Research (2016) 18:31 DOI 10.1186/s13058-016-0692-6 RESEARCH ARTICLE Open Access A five-gene reverse transcription-PCR assay for pre-operative classification of breast fibroepithelial lesions Wai Jin Tan1, Igor Cima1, Yukti Choudhury1, Xiaona Wei1, Jeffrey Chun Tatt Lim2, Aye Aye Thike2, Min-Han Tan1* and Puay Hoon Tan2,3* Abstract Background: Breast fibroepithelial lesions are biphasic tumors and include fibroadenomas and phyllodes tumors. Preoperative distinction between fibroadenomas and phyllodes tumors is pivotal to clinical management. Fibroadenomas are clinically benign while phyllodes tumors are more unpredictable in biological behavior, with potential for recurrence. Differentiating the tumors may be challenging when they have overlapping clinical and histological features especially on core biopsies. Current molecular and immunohistochemical techniques have a limited role in the diagnosis of breast fibroepithelial lesions. We aimed to develop a practical molecular test to aid in distinguishing fibroadenomas from phyllodes tumors in the pre-operative setting. Methods: We profiled the transcriptome of a training set of 48 formalin-fixed, paraffin-embedded fibroadenomas and phyllodes tumors and further designed 43 quantitative polymerase chain reaction (qPCR) assays to verify differentially expressed genes. Using machine learning to build predictive regression models, we selected a five-gene transcript set (ABCA8, APOD, CCL19, FN1,andPRAME) to discriminate between fibroadenomas and phyllodes tumors. We validated our assay in an independent cohort of 230 core biopsies obtained pre-operatively. Results: Overall, the assay accurately classified 92.6 % of the samples (AUC = 0.948, 95 % CI 0.913–0.983, p = 2.51E-19), with a sensitivity of 82.9 % and specificity of 94.7 %. Conclusions: We provide a robust assay for classifying breast fibroepithelial lesions into fibroadenomas and phyllodes tumors, which could be a valuable tool in assisting pathologists in differential diagnosis of breast fibroepithelial lesions. Keywords: Phyllodes tumors, Fibroadenomas, Fibroepithelial lesions, Prediction, Multigene assay, Core biopsy, FFPE Background recommended management for phyllodes tumor diagnosed Fibroadenomas and phyllodes tumors are fibroepithelial on core biopsy is wide excision without axillary staging lesions of the breast, characterized by proliferation of both regardless of grade [3]. Conversely, fibroadenomas are ob- epithelial and stromal components. Fibroadenomas are served conservatively or, if tumors are larger than 2 cm, more commonly encountered on core biopsies than the may be simply excised without achieving negative surgical rarer phyllodes tumors (approximately 20 % and <1 % margins [3]. This approach is due to the indolent behavior of breast core needle biopsies respectively) [1, 2]. The of fibroadenomas, despite sporadic reports of recurrences preoperative distinction between the two lesions has [4, 5], while phyllodes tumors have unpredictable outcomes significant impact on subsequent treatment. The current with malignant tumors potentially progressing to metastasis and mortality [6–10]. It has been challenging separating *Correspondence:[email protected]; [email protected] cellular fibroadenoma from benign phyllodes tumor due to 1Division of Biodevices and Diagnostics, Institute of Bioengineering and overlapping histological features, and this is particularly Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Republic of Singapore problematic on limited material of core biopsies, which 2Department of Pathology, Singapore General Hospital, 20 College Road, may lead to over- or under-treatment for some patients, Academia, Level 7, Diagnostics Tower, Singapore 169856, Republic of Singapore resulting in unnecessary anxiety and cost. Full list of author information is available at the end of the article © 2016 Tan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tan et al. Breast Cancer Research (2016) 18:31 Page 2 of 9 Several studies have proposed differentiating histological Table 1 Clinical features of the training cohort from 38 patients features such as stromal cellularity, stromal overgrowth, Features Fibroadenomas Phyllodes p value fragmentation, subepithelial condensation and presence of (n = 19) tumors (n = 19) adipose tissue within stroma on core biopsies being indica- Age tive of phyllodes tumor [11–13].However, interpretation of Median (range) 35 (17–80) 44 (18–64) 0.09 these parameters is subjective, with interobserver variation Size and only moderate reproducibility between pathologists Median (range) 25 (15–50) 65 (25–220) < 0.001 [11, 14]. Varied reports of immunohistochemical markers used in distinguishing phyllodes tumors from fibroaden- Ethnicity, n (%) 0.2 omas suggest a lack of consensus and objectivity in asses- Chinese 13 (68.4) 11 (57.9) sing the expression of these biomarkers. Some authors Malay 0 (0.0) 4 (21.0) reported Ki-67 expression to be helpful in diagnosing phyl- Indian 2 (10.5) 1 (5.3) – lodestumors[15 17] but there are reports to the contrary Others 4 (21.1) 3 (15.8) [18, 19]. Lin et al. suggested a combination immunoscore Histology of p16-INK4a and retinoblastoma-associated protein (pRB) a [20] while Maity et al. reported expression of collagen I, III Simple fibroadenoma 15 c and CD105-positive microvessel density as parameters to Complex fibroadenoma 4 differentiate the two lesions [21]. The vast majority of these Benign phyllodes tumor 13b studies were not conducted using pre-operative biopsies, Borderline phyllodes tumor 3c which is where key management decision is required. Malignant phyllodes tumor 3c We set out to identify a useful molecular signature to aFour paired core biopsies and surgical excisions help differentiate fibroadenomas from phyllodes tumors bThree paired core biopsies and surgical excisions using pre-operative core biopsies to improve prediction cOne paired core biopsy and surgical excision of the final diagnosis. same FFPE tumor block were obtained, deparaffinized Methods and macrodissected. RNA was extracted using the Training set for assay development RNeasy FFPE kit (Qiagen, Hilden, Germany) and quanti- The study received approval from the Centralized Institu- fied by Nanodrop Spectrophotometer (Thermo Fisher tional Review Board (CIRB 2005/002/F). As this was a Scientific, Waltham, MA, USA). A total of 100 ng was retrospective study with anonymized cases, no specific used for quality assessment by real-time amplification of patient consent was individually required. Forty-eight sam- the RPL13A gene (forward primer, 5’-CACTTGGGGA- ples (24 fibroadenomas and 24 phyllodes tumors) were first CAGCATGAG-3’, and reverse primer, 5’-GTAACCCCT employed as the training set for assay development. These TGGTTGTGCAT-3’) using the Power SYBR® Green included 10 paired core biopsies and surgical samples (20 RNA-to-CT™ 1-Step Kit (Life Technologies, Carlsbad, samples), and 28 independent core and excisional samples CA, USA) on a CFX96™ Real-Time PCR instrument from 38 patients (Table 1 and Additional file 1: Table S1). (Bio-Rad Laboratories, Hercules, CA, USA). Samples These formalin-fixed, paraffin-embedded (FFPE) samples with threshold cycle (Ct) below 29 were further sub- were randomly selected from cases diagnosed at the jected to quality assessment on a bioanalyzer. Eligible Department of Pathology, Singapore General Hospital samples were submitted for expression profiling on the from 2008 to 2012. Hematoxylin and eosin (H&E)-stained Whole-Genome DASL® HT Assay (Illumina, Inc., San slides were retrieved and reviewed. Phyllodes tumor was Diego, CA, USA) at the Biopolis Shared Facilities A*Star, defined when there were well-developed fronds accompan- Singapore. The assay interrogates 29,377 features using ied by increased stromal cellularity as opposed to fibro- the HumanHT-12 v4 BeadChip (Illumina, Inc.). Quantile- adenomas in which epithelial and stromal components normalized gene expression data pre-analyzed using were arranged in either intracanalicular or pericanalicular GenomeStudio® (Illumina, Inc.) was delivered. Data are patterns without fronds or stromal hypercellularity. available through GEO [GEO: GSE78071]. Differences in clinical features between fibroadenomas and phyllodes tumors were assessed with Mann–Whitney Selection of normalization genes and differentiating genes U test and Fisher’s exact test. Normalization genes were selected based on the smallest value of coefficient of variation among all samples. Dif- Expression profiling by Whole-Genome DASL® High ferentiating genes were selected using the Significance Throughput (HT) Assay Analysis of Microarrays package [22] and filtered based Representative tumor areas were identified of which on the following criteria: (1) q-value less than 0.05; (2) three to seven sections of 10-μm-thick sections from the mean difference of expression above 500; (3) R-fold Tan et al. Breast Cancer Research (2016) 18:31