Comparison of risk classification between EndoPredict and Mammaprint in ER-positive/HER2-negative primary invasive breast cancer.

Alberto Pelez-Garcia, Laura Yebenes, Alberto Berjon, Antonia Angulo, Pilar Zamora, Jose Ignacio Snchez-Mendez, Enrique Espinosa, Andres Redondo, Victoria Heredia, Marta Mendiola, Jaime Feliu, David Hardisson

Corresponding Author: David Hardisson, MD, PhD; Department of Pathology; Hospital Universitario La Paz, IdiPAZ; Paseo de la Castellana, 261; 28046 Madrid, Spain.

ABSTRACT

Purpose

To compare the prognostic performance of the EndoPredict assay with the MammaPrint scores obtained for the same cancer samples on 40 estrogen-receptor positive/HER2-negative breast carcinomas.

Methods

Formalin-fixed, paraffin-embedded invasive breast carcinoma tissues that were previously analyzed with MammaPrint as part of routine care of the patients, andwere classified as high-risk (20 patients) and low-risk (20 patients), were selected to be analyzed by the EndoPredict assay, a second generation gene expression test that combines expression of 8 genes (EP score) with two clinicopathological factors (tumor size and nodal status, EPclin score).

Results

The EP score classified 15 patients as low-risk and 25 patients as high-risk. EPclin re-classified 5 of the 25 EP high-risk patients into low-risk, resulting in a total of 20 high-risk and 20 low-risk tumors. EP score and MammaPrint score were significantly correlated (p=0.008). Twelve of 20 samples classified as low-risk by MammaPrint were also low-risk by EP score (60%). 17 of 20 MammaPrint high-risk tumors were also high-risk by EP score. The overall concordance between EP score and MammaPrint was 72.5%.  EPclin score also correlated with MammaPrint results (p=0.004). Discrepancies between both tests occurred in 10 cases: 5 MammaPrint low-risk patients were classified as EPclin high-risk and 5 high-risk MammaPrint were classified as low-risk by EPClin (overall concordance 75%).

Conclusions

This study demonstrates a moderate concordance between MammaPrint and EndoPredict. Differences in results could be explained by the inclusion of different gene sets in each platform, and the inclusion of clinical parameters, such as tumor size and nodal status, in the EndoPredict test.

Keywords: Breast cancer prognosis; gene expression signatures; EndoPredict; MammaPrint

INTRODUCTION

Breast cancer is the most common cancer and the second most frequent cause of cancer death among women in developed countries. Approximately 231,840 new cases of invasive breast cancer and 40,290 deaths are expected among US women in 2015 [1].

Currently, the decision on adjuvant treatment for breast cancer patients is based on risk assessment using clinicopathological criteria, such as patient age, menopausal status, axillary lymph node status, tumor size, tumor grade, estrogen receptor (ER)/progesterone receptor (PgR) expression, HER2 status, and Ki67 score. However, decision making in adjuvant treatment of women with ER-positive/HER2-negative early breast cancer remains a difficult task. Routinely, all of these patients will receive adjuvant hormonal treatment. However, a substantial proportion of these patients are also treated with adjuvant chemotherapy, although a significant part of these will not achieve a further reduction of their risk of recurrence [2].Therefore, a major challenge for clinical oncologists is to identify those patients who will not benefit for adjuvant chemotherapy, and those who are more likely to develop recurrence, so that the most appropriate therapeutic regime can be administered [2, 3].

In recent years, molecular characterization of breast cancer has contributed to broaden our understanding of breast cancer as a heterogeneous disease, and led to the development of a variety of prognostic and predictive gene signatures [4]. Morever, these assays may also be useful in recurrence prediction and treatment decision making [5]. One of the most widely used tests is the MammaPrint (MP) assay (Agendia Laboratories, Amsterdam, The Netherlands), which is a prognostic score performed by a central laboratory that was cleared by the FDA in 2007. MP was initially limited by its requirement for fresh tissue, but it is now validated for formalin-fixed, paraffin-embedded (FFPE) tissue [6]. MP measures the expression of 70 genes using a microarray platform, and reports a binary risk classification (low-risk or high-risk) for recurrence without adjuvant chemotherapy. This information is intended to spare patients at low-risk of recurrence from receiving adjuvant chemotherapy, with its attendant morbidity. It is not intended to predict the response, per se, to chemotherapy; rather, it helps to select patients who are likely to benefit from chemotherapy from a prognostic point of view [7]. More recently developed, the EndoPredict assay (EP) (Sividon Diagnostics GmbH, Cologne, Germany), is a diagnostic test based on gene expression data in combination with clinicopathological risk parameters to assess the risk of distant metastasis in patients with ER-positive/HER2-negative primary breast cancer if treated with adjuvant endocrine therapy alone [8]. This test measures the expression of eight cancer-related genes of interest (BIRC5, UBE2C, DHCR7, RBBP8, IL6ST, AZGP1, MGP and STC2) and three reference genes (CALM2, OAZ1 and RPL37A) to calculate a molecular risk score (EP score). The molecular risk score is then combined with the nodal status and tumor size resulting in a molecular-clinicopathological hybrid score (EPclin score) with improved prognostic power. Using a predefined cutoff value, patients are stratified into low- or high-risk of distant recurrence. The test can be carried out on routinely processed and archived FFPE tissue, and is designed to be performed decentrally [9, 10]. EP was validated in three randomized endocrine phase III trials with patients with ER-positive/HER2-negative node negative and node positive breast carcinomas [5, 8]. The EP provided additional prognostic information to conventional risk factors such as grading, quantitative ER, or Ki67 and outperformed risk classification by clinical guidelines. Moreover, it could be demonstrated that EP is prognostic for early and late metastasis [5, 11].The EPclin score was also directly compared to purely clinical risk classifications (like St. Gallen, German S3, and NCCN) and found to be superior to these classifiers [11].

The objective of this study was to compare the concordance of EndoPredict results in 40 ER-positive/HER2-negative breast carcinomas which were previously tested with MammaPrint and categorized as low-risk (20 patients) or high-risk (20 patients). We further evaluate TargetPrint (Agendia Laboratories), a commercially available mRNA-based gene expression test that quantitatively determines gene expression levels of ER, PgR, and HER2.

MATERIALS AND METHODS

• Patients and tumor samples

This study involved 40 patients with ER-positive/HER2-negative early-stage breast carcinoma. All patients underwent surgery between March 2012 and December 2015 at the University Hospital La Paz, Madrid, Spain. Data on age and tumor characteristics were collected for all patients. The surgical specimens were fixed in 10% buffered formalin and embedded in paraffin. Four-µm thick sections were stained with hematoxylin-eosin for histological diagnosis. Sections (10µm) with at least 40% of tumor cellularity were selected for the study.

• Immunohistochemistry for ER/PR/HER2 and Ki67 and Fluorescence in situ Hybridization (FISH) for HER2

All cases were reviewed by two breast pathologist (DH and LY) to assess tumor grade (using the Nottingham histological three-tier grading system), tumor size, nodal status, ER, PgR, HER-2, and Ki67 expression. The expression of ERα (clone EP1; Dako, Glostrup, Denmark, prediluted), PgR (clone PgR1294; Dako, prediluted), and Ki67 (clone MIB1; Dako, prediluted) were determined by immunohistochemistry (IHC) during routine pathologic examination. ER and PgR status was determined based on the percentage of positive nuclei in the invasive neoplastic compartment of the tissue. Tumors were classified as ER- or PgR-positive when ≥1% invasive tumor cells showed definite nuclear staining, regardless of staining intensity. Ki67 was evaluated as the percentage of positively stained nuclear cancer cells (regardless of staining intensity). HER2 expression was evaluated with the HercepTest kit (Dako) and scored as 0, 1+, 2+, or 3+, according to the FDA scoring system. Tumors scored as 2+ were re-tested with FISH using the HER2 IQFISH PharmDx kit (Dako).

• Mammaprint Test

The MammaPrint test was performed on representative paraffin blocks at the centralized Agendia Laboratories (Amsterdam, The Netherlands) blinded for clinical and histological data as part of routine care of the patients included in this study. Additionally to MammaPrint, TargetPrint assay, an additional test that is an alternative measurement of ER, PgR, and HER2 to IHC/FISH assessment, was also performed.

• EndoPredict Test

The same tumor tissue block used for MammaPrint testing in each case was used for EP test. RNA extraction was performed as previously described [9]. Total RNA was extracted from one 10-µm whole formalin-fixed, paraffin-embedded tissue section using a silica-coated magnetic bead-based method with Tissue Preparation Reagents (Sividon Diagnostics). Expression of eight genes-of-interest (AZGP1, BIRC5, DHCR7, IL6ST, MGP, RBBP8, STC2, UBE2C), three normalization genes (CALM2, OAZ1, RPL37A) as well as the amount of residual genomic DNA (HBB) were assessed by the EP assay (Sividon Diagnostics). Gene expression was assessed by one-step RT-qPCR using the SuperScript III PLATINUM One-Step Quantitative RT-PCR System with ROX (Invitrogen, Karlsruhe, Germany) according to manufacturer’s instructions in a VERSANT® kPCR Molecular System (Siemens Healthcare Diagnostics, Erlangen, Germany). EP and EPclin scores were determined as published earlier [8, 9] using the EndoPredict Report Generator software which is available online (www1.endopredict.com). The predefined cut-offs for diagnostic decisions were applied to stratify patients into low- or high-risk groups: EP low-risk (<5), EP high-risk (≥5); EPclin low-risk (<3.3), EPclin high-risk (≥3.3). The EPclin cutoff value corresponds to a 10% distant recurrence rate at 10 years.

• Statistical analyses

The association between the clinicopathological features and EP and EPclin scores was analyzed using the Pearson correlation or Fisher’s exact test, as appropriate. The correlation between EP and MP was analyzed using Fisher’s exact test. The statistical significance between the Ki67 and EP and EPclin scores was examined using the Pearson correlation coefficient. Agreement measurements between binary (positive versus negative) TargetPrint mRNA and IHC classifications were based on two-way contingency table analysis and included overall concordance, positive agreement (defined as the number of samples classified positive by both IHC and mRNA divided by the number of positive samples using IHC), and negative agreement [12]. Statistical analysis was performed with the SPSS statistics 19 software (IBM, Armonk, NY, USA).

RESULTS

• Patient characteristics

The characteristics of the 40 patients included in this study are summarized in Table 1.

• MammaPrint test

MammaPrint test revealed low-risk in 20 patients and high-risk in 20 patients.

• EndoPredict Test

The EndoPredict test results in an EP score and an EPclin score. According to EP score 15 patients were classified as low-risk and 25 patients as high-risk. The EPclin score (combining EP score with tumor size and nodal status) re-classified 5 of the 25 EP high-risk patients into the low-risk group resulting in 20 patients with low- and 20 patients with high-risk of distant metastasis. The clinicopathological characteristics of the patients according to EPclin score are summarized in Table 2.

• Correlation and concordance between EP Score and MammaPrint test results

EP scores and MP scores were significantly correlated (p= 0.008). Twelve of 20 samples classified as low-risk by MP were also low-risk by EP score (60%). Seventeen of 20 MP high-risk samples were also EP high-risk (85%). The overall concordance between both risk classifications was 72.5% (Table 3).

• Correlation and concordance between EPclin Score and MammaPrint test results

The combined molecular-clinicopathological EPclin score also correlated with the MP results (p= 0.004). Fifteen of 20 samples classified as low-risk by MP were also low-risk by EPclin score (75%). Similarly, 15 of 20 MP high-risk patients were EPclin high-risk (75%). Overall concordance was 75% (Table 4).

• Correlation of Proliferation Index Ki67 to EP Score and EPclin score.

There was a statistically significant but moderate correlation between the EP score and Ki67 (Pearson coefficient = 0.535, p = 0.01) (Figure 1A). No significant correlation was found between EPclin score and Ki67 proliferation index (Pearson coefficient = 0.37, p = 0.05) (Figure 1B).

• Comparison of ER/PgR/HER2 Status with Conventional Immunohistochemistry/FISH and TargetPrint

Qualitative IHC/FISH (positive versus negative) showed a high concordance with TargetPrint readout (Table 5): of the 40 ER-positive cases by IHC, 39 (97.5%) were TargetPrint ER-positive. The only discordant case had ER positivity in approximately 85% of tumor nuclei on IHC but was assessed as negative with TargetPrint. This case was PgR+/HER2- with a proliferation index (Ki67) of 6%, and was classified as low-risk by both tests, MP and EPclin. For PgR, the positive agreement was 81.6%. Thus, 31 PgR-positive cases (77.5%) were also TargetPrint PgR-positive. Of the 9 discordant cases, 7 tumors were PgR-positive by IHC but were classified as PgR-negative by TargetPrint whereas 2 cases were PgR-negative by IHC but PgR-positive by TargetPrint. Of the 40 HER2-negative cases by IHC/FISH, 39 (97.5%) were TargetPrint HER2-negative. The only discordant case was HER2-negative by IHC and FISH, and corresponded to an ER+/PgR+ tumor with a proliferation index (Ki67) of 20%, and was classified as high-risk by both tests, MP and EP.

DISCUSSION   

In this study we compared retrospectively the correlation between EP scores (EP score and EPclin score) and MP scores in 40 ER-positive/HER2-negative early breast carcinomas. EPclin, as opposed to MP, includes information from clinical factors, making it more clinically useful but also making fair comparisons with MP complicated. We found a moderate concordance between EP, EPclin and MP-based risk classifications with an overall concordance of 72.5% and 75% between EP molecular score and MP, and EPclin score and MP, respectively.

To the best of our knowledge, our study reports the first direct comparison of the clinical performance of EndoPredict (EP/EPclin) and MammaPrint. Our results are similar to those reported in previous studies comparing different gene expression signatures in breast cancer. Comparison of the poor prognosis group of the MP and the intermediate- and high-risk groups from the Oncotype DX recurrence-score models showed that their sample predictions agreed in 77% of patients with ER-positive early-stage breast carcinomas. These analyses suggest that even though there was very little gene overlap (the MP and Oncotype DX recurrence-score profiles overlapped by only 1 gene, SCUBE2) and different algorithms were used, the outcome predictions for the majority of patients with breast cancer would be similar [13]. The Optimal Personalised Treatment of early breast cancer usIng Multiparameter Analysis preliminary study (OPTIMA prelim) compared predicted risk stratification and subtype classification of five multiparameter tests (Oncotype DX, Prosigna [PAM50], MammaPrint, MammaTyper,  and NexCourse Breast [IHC4-AQUA]) performed directly in a series of 313 women with early breast cancer that were randomized to standard (chemotherapy and endocrine therapy) or test-directed (chemotherapy if Oncotype DX recurrence score >25) treatment [14]. The five tests categorized comparable numbers of tumors into low- or high-risk groups but there was only moderate agreement between tests at an individual tumor level. Thus, only 119 (39.4%) tumors were classified uniformly as either low/intermediate-risk or high-risk, and 183 (60.6%) were assigned to different risk categories by different tests (kappa ranges 0.33-0.60), although 94 (31.1%) showed agreement between four of five tests. This study concluded that existing evidence on the comparative prognostic information provided by different tests suggests that current multi-parameter tests provide broadly equivalent risk information for the population of women with ER-positive breast cancers. However, for the individual patient, tests may provide differing risk categorization and subtype information. [14]. A similar finding has been reported in a comparison between PAM50 and Oncotype DX [15]. Varga et al reported that the concordance of classification in low- or high-risk between Oncotype DX (combining the Oncotype DX intermediate-risk and high-risk groups to one high-risk group) and EP risk score and EPclin score was 76% and 65%, respectively [16]. TransATAC, the translational study of the Arimidex, Tamoxifen, Alone or in Combination trial (ATAC), which served as a validation study for the Oncotype DX recurrence score (RS), PAM50 risk of recurrence (ROR), and Breast Cancer Index (BCI) scores, has recently assessed the prognostic value of EP and EPclin for 10-year distant recurrence risk in postmenopausal women with hormone receptor-positive, HER2-negative primary breast cancer, and compared their prognostic ability with that of the Oncotype DX RS [5]. This study confirmed the independent prognostic ability of EP and EPclin in this population of women with breast cancer. Moreover, EPclin provided more prognostic information than RS partly because of its integration with clinicopathological factors (nodal status and tumor size) but also because of a superior molecular component able to predict late events better than RS [5]. The importance of integrating clinical and molecular variables to create a more accurate prognostic index for RS has led Genomic Health to develop an online Recurrence Score Pathology-Clinical (RSPC) calculator to use in ER-positive/HER2-negative/node-negative breast cancer patients that combines RS with pathologic and clinical factors such as patient age, tumor size, tumor grade, and planned adjuvant hormonal therapy (tamoxifen or aromatase inhibitor). This online calculator is intended to help physicians understand how integrating clinical and pathological factors with the OncotypeDX Breast Cancer Recurrence Score result can enhance the understanding of the score [17]. Tang et al demonstrated that RSPC had significantly more prognostic value for distant recurrence than did RS and showed better separation of risk in the study population [18]. RSPC classified fewer patients as intermediate-risk (17.8% versus 26.7%, p<0.001) and more patients as lower risk (63.8% versus 54.2%, p<0.001) than did RS among 1,444 node-negative ER-positive breast cancer patients [18].

These results indicate that most of the gene signatures currently available provide similar outcome predictions, although significant differences across predictors are present at the individual level. Interestingly, our study shows that despite the discrepancies observed between EP and MP, both tests categorized the patients in the same proportion of cases of high- and low-risk categories (50% each group). Therefore, all tests, while of significant value in informing patient choice, are only modestly predictive of the risk of relapse at the individual patient level. Within the validation studies for each test there are patients assigned to low-risk groups who relapse and die of their disease, and in all cases the majority of patients who are high-risk do not in fact relapse and die from their disease [19]. These tests, therefore, represent a significant step towards personalized medicine but are not yet perfect. This is important since it implies that discordance in risk estimates between tests that measure risk in different ways may not be unexpected [19].

Multiple factors may contribute to the discrepancies between EP and MP scores. It may be due to the differences in weighting of main biological motives covered by the genes included in the test algorithms such as proliferation or ER signaling. Some differences could be explained by the coverage of other motives such as cell adhesion, invasion, or DNA repair, as it has been demonstrated in previous studies comparing EP and Oncotype DX [16]. Additionally, test training may also contribute to discrepancies, although most of the tests were developed in ER-positive patient populations treated with endocrine therapy but not chemotherapy [19]. This heterogeneity may also be attributed partially to the different methodologies that were used to build both classifiers (cDNA microarray and qRT-PCR for MP and EP, respectively), and the heterogeneity in the sample population used to develop the tests. Moreover, each technology has unique normalization methods. Thus, the discrepancies between the tests are likely to reflect the differences in both the specific genes and the number of genes assessed by the individual tests.

We observed a moderate statistically significant correlation between Ki67 and the EP score, but no correlation between Ki67 and the EPclin score. Similar results have been described by Varga et al which performed a direct comparison of the concordance between Ki67 as a continuous variable and the EP score in a series of 34 ER-positive/HER2-negative primary breast carcinomas [16]. The authors found a moderate statistically significant correlation between Ki67 and the EP score (Pearson coefficient 0.55, p<0.0001), but no significant correlation was observed between the EPclin score and Ki67 (Pearson coefficient 0.24, p=0.16).We agree with Varga et al that this may be at least partially due to the lack of standardization in assessing the Ki67 index in breast cancer [20]. Using the Ki67 cutoff of 14% Dubsky et al divided over 1,000 patients into luminal A and B subtypes and noted that the EPclin score can divide the patients of these subtypes into two additional groups regarding to a better or worse prognosis [11]. Similarly, Filipits et al demonstrated that the EP score provided independent prognostic information in a multivariate analysis with conventional clinicopathological variables including Ki67 (cutoff 11%) [8]. These data suggest that the EP molecular score will likely perform better than Ki67 alone for the prognosis of breast cancer patients, but the performance of the EPclin score versus Ki67 is less clear.

TargetPrint, a new diagnostic test providing the precise molecular readout of ER, PgR, and HER2 mRNA gene expression levels that can be used in conjunction with MP, was additionally analyzed in our series of patients. Our results showed a high concordance between TargetPrint and IHC/FISH for ER (97.5%) and HER2 (97.5%), and fair concordance for PgR (77.5%). These results are similar to those reported in other studies, with concordances of approximately 96% for ER, 95% for HER2, and 80% for PgR [12, 21].The concordance for mRNA and IHC assessment of PgR has been shown to be less than for ER in previous studies [22, 23]. However, mRNA-derived PgR status seems to be more strongly associated with clinical outcome, suggesting that mRNA may be a more reliable method for assessing receptor status [24]. The likely causes of the discordant results between mRNA readout and pathology assessment of ER, PgR, and HER2 have not been elucidated so far. Suggested possible causes such as intratumoral heterogeneity, have not yet been analyzed in a randomized patient cohort [21]. Definitely, TargetPrint assessment of ER and HER2, and to lesser extent PgR, status gives results comparable with IHC/FISH and provide an objective and quantitative assessment ER, PgR, and HER2 expression.

The main limitations of this study are related to the moderate sample size which may have limited the conclusions reached in the study. Thus, a further large-scale study including data on patients follow-up is necessary to validate our results.

In conclusion, this study is the first direct comparison of the clinical performance of EP/EPclin with MP. Our data show moderate concordance between EndoPredict and MammaPrint results on individual patients. Recent studies suggest that the addition of clinical parameters into molecular risk scores, such as EPclin, seems to improve their prognostic ability in breast cancer [25, 26]. The moderate concordance between tests reflect the fact that the tests are measuring different genes, using different technology and highlight the problems of predicting recurrence risk based on the biology and management of the tumor [19]. Further clinical studies evaluating large patient cohorts including follow-up data are needed to compare both tests.

ACKNOWLEDGMENTS

We wish to thank Inmaculada Briones and Isabel Moreno for excellent technical assistance.

AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: APG, AA, and DH

Performed the experiments: APG

Analyzed the data: APG, LY, AB, PZ, AA, JISM, VH, MM, DH

Contributed to the collection of clinical data: PZ, EE, AR, JF

Wrote the paper: APG, DH

All authors read and approved the final manuscript.

COMPLIANCE WITH ETHICAL STANDARDS

Conflict of interest

AA is employee of Myriad Genetics Iberia[m1]. All remaining authors declare that they have no competing interest. The study was supported by the company Myriad Genetics[m2].

Ethical standards

The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid, Spain (code HULP: PI-2146).

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