The Oncologist, Vol. 13, No. 5, 477-493, May 2008; doi:10.1634/theoncologist.2007-0248
© 2008 AlphaMed Press
Commercialized Multigene Predictors of Clinical Outcome for Breast Cancer
Jeffrey S. Rossa,
Christos Hatzisb,
W. Fraser Symmansc,
Lajos Pusztaid,
Gabriel N. Hortobágyid
aDepartment of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, USA;
bNuvera Biosciences, Woburn, Massachusetts, USA;
cDepartment of Pathology and
dDepartment of Breast Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA
Key Words. Breast cancer • oncotype DX • MammaPrint • Rotterdam signature • Invasiveness signature • Two-gene expression ratio
Correspondence: Correspondence: Jeffrey S. Ross, M.D., Department of Pathology and Laboratory Medicine, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208, USA. Telephone: 518-262-5461; Fax: 518-262-3663; e-mail: rossj{at}mail.amc.edu
Received December 19, 2007;
accepted for publication March 10, 2008.
Disclosure: C.H. is employed by, owns stock in, and discloses receipt of intellectual property rights/patent holder in Nuvera Biosciences (Nuvoselect). G.N.H. discloses that except for oncotype DXTM and MammaPrint®, all predictors described in the article are investigational and the indications are evolving. L.P. is a founder and stockholder of Nuvera Biosciences (Nuvoselect test), has acted as a consultant for Roche, Dako, Bristol-Myers Squibb, and Pfizer, and discloses that the article discusses the Affymetrix U133 GeneChip® System (Affymetrix Corporation) for potential use as a diagnostic test. W.F.S. has ownership and intellectual property in Nuvera BioSciences (Nuvoselect test) and he discloses that the article discusses prognostic/predictive genomic signatures by Genomic Health, Inc, Agendia, and Nuvera Biosciences for breast cancer diagnostics.
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Learning Objectives
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After completing this course, the reader will be able to:- Assess the strengths and weaknesses of the four main techniques used to measure multiple gene expression using clinical breast cancer specimens.
- Compare the advantages and disadvantages of the oncotype DXTM and MammaPrint® multigene assays and compare the TAILORx and MINDACT clinical trials for the prediction of clinical outcome in breast cancer.
- Evaluate the costs versus benefits associated with the use of expensive multigene breast cancer predictors in the management of breast cancer.
This article is available for continuing medical education credit at CME.TheOncologist.com
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ABSTRACT
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In the past 5 years, a number of commercialized multigene prognostic and predictive tests have entered the complex and expanding landscape of breast cancer companion diagnostics. These tests have used a variety of formats ranging from the familiar slide-based assays of immunohistochemistry and fluorescence in situ hybridization to the nonmorphology-driven molecular platforms of quantitative multiplex real-time polymerase chain reaction and genomic microarray profiling. In this review, 14 multigene assays are evaluated as to their scientific validation, current clinical utility, regulatory approval status, and estimated cost–benefit ratio. Emphasis is placed on two tests: oncotype DXTM and MammaPrint®. Current evidence indicates that the oncotype DXTM test has the advantages of earlier commercial launch, wide acceptance for payment by third-party payors in the U.S., ease of use of formalin-fixed paraffin-embedded tissues, recent listing by the American Society of Clinical Oncology Breast Cancer Tumor Markers Update Committee as recommended for use, continuous scoring system algorithm, ability to serve as both a prognostic test and predictive test for certain hormonal and chemotherapeutic agents, demonstrated cost-effectiveness in one published study, and a high accrual rate for the prospective validation clinical trial (Trial Assigning Individualized Options for Treatment). The MammaPrint® assay has the advantages of a 510(k) clearance by the U.S. Food and Drug Administration, a larger gene number, which may enhance further utility, and a potentially wider patient eligibility, including lymph node–positive, estrogen receptor (ER)-negative, and younger patients being accrued into the prospective trial (Microarray in Node-Negative Disease May Avoid Chemotherapy). A number of other assays have specific predictive goals that are most often focused on the efficacy of tamoxifen in ER-positive patients, such as the two-gene ratio test and the cytochrome P450 CYP2D6 genotyping assay.
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INTRODUCTION
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In the past 10 years, the introduction of whole genome profiling technologies has greatly expanded our knowledge of the genes and genetic pathways associated with the development and progression of breast cancer [1–4]. More recently, a number of commercialized multigene prognostic and predictive tests have entered the complex and expanding landscape of breast cancer diagnostics [5]. These multigene predictors have been introduced using both familiar (immunohistochemistry [IHC] and fluorescence in situ hybridization [FISH]) and relatively unfamiliar (quantitative real-time polymerase chain reaction [qRT-PCR] and genomic microarrays) technologies and typically are launched as either diagnostic kits or centralized commercial laboratory assays with integrated unique statistical data analysis tools and algorithms designed to calculate the test results [5–7]. A number of these new molecular diagnostic assays for breast cancer use widely available starting material such as formalin-fixed paraffin-embedded (FFPE) tissue blocks and thick sections, whereas others require fresh-frozen tissue samples or unfrozen samples stored in an RNA-preserving solution [5]. Although several organizations have sought and achieved regulatory approval for their tests, others have not and offer their tests as "home brew" assays. A number of these commercialized new multigene assays have been tested against conventional diagnostic and prognostic tests in a variety of multivariate analysis models, with the results reported in peer-reviewed, well-respected scientific journals [5]. Several multigene predictor kits that are now on the market have essentially been tested only "in house" and do not have any accompanying medical literature to support their developers' claims of clinical utility and value.
The following review of multigene predictors is intended for the clinician whose daily practice includes the diagnosis and treatment of patients newly diagnosed with breast cancer. In the comparative evaluation of these assays, rather than focus on each test's technical platform or statistical algorithm, the review emphasizes practical issues such as the ease of use, level of clinical validation, and current and potential clinical utility of the assay, including its projected cost–benefit ratio.
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TEST PLATFORMS
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The three major breast cancer multigene predictor test platforms are summarized in Table 1. IHC is a widely used technique that has been a cornerstone for the management of breast cancer since the mid 1990s when the estrogen receptor (ER) test moved from a biochemical assay to a semiquantitative IHC procedure [8]. More recently, IHC has been used as the platform for multigene predictors in breast cancer, combining a series of antibodies with some form of digital image analysis slide scoring. Having the advantage of using a morphology-driven signal and thus not requiring tissue microdissection, IHC is nonetheless exposed to preanalytic tissue processing and antigen retrieval variables that can significantly impact the test results [8]. There are additional concerns about uneven performance of IHC reagents including both mouse and rabbit monoclonal antibodies and widespread variation in slide scoring techniques, which can significantly impact the determination of important prognostic and predictive biomarkers including the hormone receptor status (ER/progesterone receptor [PgR]) and the cell proliferation status (Ki-67) [8]. IHC is limited in the number of markers that can be used whether fluorescent or bright field signal development procedures are used, but this "limitation" also allows for a less complex statistical algorithm required for data analysis, reducing the likelihood of false biomarker discovery. However, the statistical analysis of IHC-based assays using multiple markers may become problematic as a result of the nonlinear nature of IHC staining, different subcellular localization of different markers, and the impact of different slide scoring thresholds for different immunostains. The stand-alone prognostic value of IHC in breast cancer is well established and includes testing of ER, PgR, human epidermal growth factor receptor (HER)-2, and the proliferation marker Ki-67 [9]. IHC is widely used to predict response to both hormonal and HER-2–targeted therapies [8, 9], but is not established as a predictor of either the efficacy or toxicity of cytotoxic drugs. One commercialized IHC-based multigene predictor is in late-stage clinical development [10] and another has recently entered the market as a centralized testing service [11]. In contrast to the higher cost of the molecular assays described below, the cost of commercialized IHC multigene predictors is likely to range from an estimated $300 to $600 per test.
The FISH method has been primarily used for determining the copy number of the HER-2 gene for purposes of selecting HER-2–targeted therapies such as trastuzumab and lapatinib [3]. Testing for HER-2 gene amplification using the FISH technique has now been widely used in prospective clinical trials evaluating HER-2–targeted therapies in both the adjuvant and neoadjuvant settings in both the U.S. and Europe. Although FISH technology has been used to measure a number of other prognostic factors, including chromosomal aneusomies and amplifications of cell proliferation–associated genes, until recently these assays were not formally commercialized and have not been widely used in clinical studies [3]. In 2007, a three-color FISH assay was commercialized to assess stand-alone prognosis in ER-positive and ER-negative stage I breast cancers [12].
qRT-PCR has enabled a rapid growth in gene-expression studies for both hematologic malignancies and solid tumors [13]. In multiplex qRT-PCR, housekeeping and gene-specific oligonucleotide primers and dye-conjugated probes are added to cDNA produced from RNA isolated from clinical samples and a quantitative level of mRNA expression is obtained by normalizing the amplification cycle time for the target gene against that of a housekeeping gene [13]. A variety of commercial closed system qRT-PCR technologies are used for clinical applications, with the TaqMan® system (Applied Biosystems Inc., Foster City, CA) and LightCycler® system (Roche Diagnostics Inc., Indianapolis, IN) used by the three commercialized breast cancer multigene predictors [14–24]. RT-PCR applications generally focus on the enhanced sensitivity associated with PCR-based strategies because of the ability to detect RNA over a seven-log range. Although RT-PCR is often considered the "gold" standard for the quantification of mRNA, it is far from being a standardized assay, demonstrating significant variability in the RNA templates and protocols used as well as in the inappropriate data normalization and data analysis methodologies employed [25]. RT-PCR procedures designed to predict outcome in breast cancer can be performed on either fresh or FFPE samples. Morphologic review of the tissue from which the mRNA will be extracted is recommended because, on occasion, some form of tissue microdissection may be needed to make certain that the mRNA extracted for analysis is highly enriched for invasive carcinoma and not excessively diluted with cells from benign tissues and in situ carcinoma areas [8, 15]. The heterogeneous expression of important mRNAs, such as ER, HER-2, and Ki-67, often reflected in the varying histologic grades seen in larger tumors can influence the predictive accuracy of transcriptional profiling measurements. In addition, the area sampled in a resection specimen must avoid including extracted mRNA from a zone of wound healing associated with a recent previous biopsy. Although the number of genes that can be simultaneously assessed by multiplex qRT-PCR is significantly greater than that for IHC, this requires a more complex statistical evaluation of the gene-expression profiles. However, RT-PCR does allow multiple biologic processes to be assessed simultaneously, including proliferation, hormone receptor, and HER-2 pathways [14]. The RT-PCR technique has been used to predict overall prognosis and response to both hormonal and cytotoxic therapies [14–24].
Microarray profiling has now been extensively used for the evaluation of breast cancer specimens [5]. Microarray-based gene-expression profiling has been used to define cellular functions, biochemical pathways, cell proliferation activity, and regulatory mechanisms [26]. A variety of commercial sources of microarray chips and slides has been used, with the Affymetrix U133 GeneChip® (Affymetrix Corporation, Santa Clara, CA) and the Agilent custom microarrays (Agilent Technologies, Santa Clara, CA) being the most prominent. These technologies have required the use of freshly prepared mRNA extracts and have not, to date, been fully adapted to FFPE tissues. Furthermore, the type of tissue sampling clearly has a major impact on profiling results because the transcriptional profiles are a composite of mRNA contributed by all tissue components of the biopsy or resection specimen. Tumor mRNA expression heterogeneity and the relative tumor cell versus benign tissue volumes in fine-needle aspirations or core-needle biopsies may give a significantly different transcriptional profile than that of the resected tumor [27]. Interpretation of microarray results is also very different from interpretation of conventional prognostic markers in that complex bioanalytic techniques are used, which have, to date, not been fully standardized. However, transcriptional profiling results can be compared with a pre-existing database of profiles for further confirmation of their validity. One microarray-based multigene predictor test for breast cancer has received U.S. Food and Drug Administration (FDA) clearance [2, 28–33]. Commercialized microarray-based multigene predictors have been developed as stand-alone prognostic biomarkers [34–38], predictors of response to hormonal therapy [2, 21–24, 28–33], and predictors of response to multiagent cytotoxic chemotherapy [39–48]. The FDA is currently leading an effort to further standardize microarray technology and the related analytical methodology to facilitate broader clinical adaptation [49]. The anticipated costs for microarray-based multigene predictors for breast cancer are in the $3,000 per test range. A potential promise of microarray-based tests is that multiple distinct predictions, including prognosis, ER and HER status, and sensitivity to various treatment modalities, may be generated from a single experiment.
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MOLECULAR CLASSIFICATION OF BREAST CANCER
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Molecular Portraits
The original Perou classification, "the molecular portrait" of breast cancer, based on gene-expression profiling, although widely cited, continues to evolve as further subdivisions of the original classification are proposed [1, 50]. The initial version of the molecular portraits included luminal, normal, HER-2 and basal-like subtypes of invasive breast cancer. The luminal group was subsequently subdivided into luminal-A and luminal-B (with a luminal-C group added and then removed). Luminal-A tumors have the highest ER expression level as well as high expression levels of GATA-binding protein 3, X-box binding protein 1, trefoil factor 3, hepatocyte nuclear factor 3
, and LIV-1 [50]. Luminal-B tumors have low to moderate expression levels of the luminal-specific genes, some of which are HER-2 positive. The frequency of mutations of the p53 tumor suppressor gene is lower in the luminal-A than in the luminal-B group (Fig. 1). The basal-like group has been associated with the so-called "triple-negative" breast cancer phenotype (ER negative, PgR negative, and HER-2 negative). Importantly, all of the luminal groups of breast cancers are ER positive and nearly two thirds of them are of low or intermediate histologic grade, whereas 95% of basal-like cancers are ER negative and 91% of these tumors are high grade [51]. Almost all (
80%–90%) triple-negative tumors cluster in the basal-like genotype, but the basal-like genotype as a whole is heterogeneous and can be divided into multiple additional subgroups [52–53]. The basal-like tumors lack ER and HER-2 expression and feature more frequent overexpression of basal cytokeratins, epidermal growth factor receptor and c-Kit [52]. Studies of clinical outcome based on the molecular subtypes have shown that the basal-like and HER-2–positive/ER-negative subtypes are more sensitive to anthracycline-based neoadjuvant chemotherapy than are the luminal breast cancers [54] and that the basal-like and HER-2 –positive subgroups are associated with the highest rates of pathologic complete response to neoadjuvant multiagent chemotherapy [51]. Several studies have compared and validated the molecular portraits classification with other published breast cancer gene-expression signatures using different transcriptional profiling platforms with uniform success [55, 56]. However, a recent statistical meta-analysis of previous studies concluded that only three subtypes are consistently identifiable across datasets—HER-2 positive, ER positive/HER-2 negative, and ER negative/HER-2 negative [57, 58]. In summary, although the original molecular classification of breast cancer into distinct portraits has mostly been validated using other profiling platforms and databases, it is currently held that the subgroups are heavily driven by the ER and HER-2 status and by the proliferative activity of the tumor, explaining why, despite having a greater response to chemotherapy, tumors in the basal-like subgroup continue to feature a relatively poor prognosis (if they fall into the chemotherapy-insensitive category), reflecting their higher histologic grade and inability to be impacted by hormonal-targeted therapies [59]. As further studies are published, it is likely that additional pathways important for the prediction of breast cancer response to specific types of chemotherapy will emerge as important new subclassifiers of the original molecular portrait system.
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Figure 1. Histologic versus molecular classification of breast cancer. In this figure, the molecular portrait subtypes of invasive breast cancer are listed to the left and examples of gene amplification (HER-2 detected by CISH) and protein overexpression (p53, cytokeratin 5, and EGFR by IHC) are shown to the right. The importance of the relationship is estimated by the thickness of the associated arrows.
Abbreviations: CISH, chromogenic in situ hybridization; EGFR, epidermal growth factor receptor; HER-2, human epidermal growth factor receptor 2; IHC, immunohistochemistry.
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Histologic Versus Molecular Tumor Grading
In a study using a discovery cohort of 189 breast tumors and an independent validation cohort of 597 tumors, a series of 97 genes was identified that was capable of reclassifying the traditional three histologic grades of breast cancer into only two distinct molecular grades [4]. In that study, the gene expression grade index ("molecular grade") was strongly associated with histologic grades 1 and 3, but not grade 2. For patients with histologic grade 2 tumors, a high Genomic Grade Index (GGI) was associated with a higher risk for recurrence than with a low GGI, suggesting the possibility that histologic grade 2 was actually a mixture of grade 1 and grade 3 tumors [4]. In other words, the histologic grade 2 tumors appeared to be similar to the grade 1 or grade 3 tumors in their molecular profiles, but lacked a distinctive profile of their own. One possible partial explanation for this observation is the so-called "3 + 3 + 1" tumor (Fig. 2), which features a solid growth pattern devoid of tubules (Bloom-Richardson score = 3), with severe nuclear anaplasia (Bloom-Richardson score = 3), but devoid of mitotic figures (Bloom-Richardson score = 1). In a subset of these cases it is possible that, after surgical removal or biopsy, the tumor sits at room temperature for a protracted length of time before it is immersed in formalin fixative, which allows for the mitotic figures that would have been observed to disappear as the tumor cells are able to complete their cell division. A Ki-67 immunostain used to assess the tumor's proliferative index could serve as a surrogate for mitotic figure counting in these cases and allow for them to be "unofficially" reclassified as grade 3 lesions [60]. Recently, the GGI was used to define two ER-positive molecular subgroups that were highly comparable with the luminal-A and luminal-B subtypes of the molecular portraits and were associated with a statistically distinct clinical outcome in both untreated and tamoxifen-treated patients [67]. The GGI was licensed to Ipsogen Inc, (Marseille, France) for commercial development.
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Figure 2. Histologic versus molecular grade. This photomicrograph of an invasive duct carcinoma in a 66-year-old woman shows a high degree of nuclear pleomorphism (Bloom-Richardson score = 3) and a completely solid growth pattern lacking tubule formation (Bloom-Richardson score = 3) but is devoid of mitotic figures (Bloom-Richardson score = 1). This tumor would total 3 + 3 + 1 = 7 in the Bloom-Richardson system, which is designated as grade 2 or intermediate grade. The Sotiriou molecular grade for this lesion would likely be high grade, and the lack of mitotic figures needed to achieve a Bloom-Richardson score of 8 or 9 (high histology grade) may reflect a delay in tumor fixation after surgical removal of the neoplasm. In such cases, a high Ki-67 labeling index of 60% by immunostaining, as shown for this case in the inset at the lower right, could be used as a surrogate of the mitotic figure count and "upscore" this tumor to high histologic grade. If this approach was generally adopted, the number of grade 2 cases would be reduced, the number of grade 3 cases would be increased, and the correlation between molecular and histologic grading would likely increase (hematoxylin and eosin, 200x; inset, immunoperoxidase, 200x).
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IHC-BASED MULTIGENE PREDICTORS
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There are two IHC-based multigene assays for the prediction of outcome for breast cancer; one is in development [10] and the other is now on the market [11]. IHC-based assays face numerous challenges for accuracy that include preanalytic facts such as the nature and duration of fixation, the type of tissue processing, and the type and intensity of the antigen retrieval technique used. Another major issue in IHC is the accuracy and reproducibility of the slide scoring system and the cutoffs used to determine positive and negative results [3, 8]. These issues have been widely discussed because they relate to the determination of ER, PgR, and HER-2 by IHC and the critical need to standardize these procedures to improve their accuracy, which is of critical importance for the selection of therapy for breast cancer.
ProExTM Br
The BD/TriPath (TriPath Oncology, Durham, NC) ProExTM Br is a five antibody/five separate slide purely prognostic IHC assay that uses an image analysis system–based slide scoring system [10]. The five antibodies in the panel are the E2F transcription factor, p21 Ras associated protein, Src kinase protein, secretory leukocyte peptidase inhibitor, and proteasome core subunit beta 1. Overexpression of two or more of these markers (ProExTM Br score 
2) has been associated with disease relapse in both lymph node–negative and lymph node–positive patient cohorts (Fig. 3). The test has not been directly linked to response to a specific therapy.
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Figure 3. The ProExTM BR assay. In this five-antibody image analysis–scored IHC-based assay, a 44-year-old white woman was treated for a 1.0-cm, ER-negative, PgR-positive, HER-2–negative invasive ductal carcinoma with mastectomy, local radiation, hormonal therapy, and cytotoxic chemotherapy. The tumor was Bloom-Richardson grade 2 (3 + 2 + 2) and had an aneuploid DNA content. In the five-marker ProExTM Br assay, immunostains for PSMB9 and p21 Ras were scored positive and immunostains for E2F, Src, and SLPI were negative. The ProExTM Br test result was "positive for high risk." The tumor recurred 25 months after completion of primary therapy and the patient died of her disease at 36 months (hematoxylin and eosin and multiple immunoperoxidase, 100x).
Abbreviations: ER, estrogen receptor; HER-2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; PgR, progesterone receptor; PSMB9, proteasome core subunit beta 9; SLPI, secretory leukocyte peptidase inhibitor.
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Mammostrat®
The Mammostrat® test (Applied Genomics Inc., Huntsville, AL) has been fully commercialized and is currently available via centralized testing at The Molecular Profiling Institute, Inc. (Phoenix, AZ). This standard purely prognostic IHC format test uses five antibodies—p53 tumor suppressor protein, N-myc downstream regulated gene 1, carcinoembryonic antigen cell adhesion molecule 5, solute carrier family 7 cationic amino acid transporter, y+ system member 5, and HpaII tiny fragments locus 9C—with routine slide scoring to divide cases of ER-positive, lymph node–negative tumors treated with tamoxifen alone into low-, moderate-, and high-risk groups. In a multivariate analysis study, the calculated risk for recurrence for Mammostrat® was independent of stage, grade, and lymph node status [11]. In that this test uses standard IHC procedures, it is currently billed and reimbursed as five separate charges using the 88342 Current Procedural Terminology (CPT) code. Although the reimbursement for this procedure varies by insurance carrier and geographic location, the global (technical preparation plus professional interpretation) reimbursement for five 88342 codes is approximately $450–$500.
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FISH-BASED PREDICTORS
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The FISH technique can also be influenced by preanalytic factors and is best performed on FFPE tissue sections. The length of the probe used for hybridization can significantly influence the results by altering the sensitivity and specificity of the assay. Although DNA is a more stable target than protein under these conditions, appropriate controls must be used to confirm accurate hybridization, and a sufficient number of nuclei must be scored for the markers to allow for tumor heterogeneity, tissue section thickness, and uneven hybridization.
eXagenBCTM
Although FISH-based testing in breast cancer has largely been focused on the assessment of the copy number of the HER-2 gene and focused on the selection of anti-HER-2–targeted therapies, a new multicolor FISH assay, the eXagenBCTM (eXagen Diagnostics, Inc., Albuquerque, NM) has been developed as a pure prognostic test to predict breast cancer outcome in node-positive or node-negative patients. Performed on FFPE surgical pathology slides, this assay employs fluorescent-labeled DNA probes to measure the copy numbers of three genes for ER-positive tumors—cytochrome p450 family 24 (CYP24), programmed cell death 6 interacting protein (PDCD6IP), and baculoviral IAP repeat-containing 5 (BIRC5 [survivin])—and three genes for ER-negative tumors—nuclear receptor subfamily 1, group D, member 1 (NR1D1), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1 (SMARCE1), and BIRC5 [12]. Using a proprietary algorithm to predict the prognostic index (PI), a single published study has found that, in an independent test validation cohort stratified by PI, recurrence rates were significantly higher among high-risk patients than among low-risk patients for both ER/PgR-positive (odds ratio, 9.52; p = .0024) and ER/PgR-negative (odds ratio, 12.3; 95% confidence interval, >1.45; p = .0188) cancers. However, both the discovery and validation datasets included patients who either received systemic adjuvant therapy (hormonal therapy, chemotherapy, or both) or were not treated, and this could complicate the interpretation of the test results. This test has been submitted to the FDA for 510(k) clearance. It is likely that, when fully marketed, this test will be decentralized and performed on site by laboratories familiar with the FISH technique. The ultimate cost to patients for this procedure will likely reflect three individual 88365 global CPT codes or approximately $700–$1,000.
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RT-PCR–BASED MULTIGENE PREDICTORS
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oncotype DXTM
The oncotype DXTM is a 21-gene multiplex prognostic and predictive RT-PCR assay performed on primary FFPE breast cancer samples by a Clinical Laboratory Improvement Act (CLIA) and State of California licensed centralized laboratory at the developer's headquarters (Genomic Health, Inc., Redwood City, CA). The original 16 informative genes that calculate the recurrence score (RS) were discovered on archived FFPE samples by transcriptional profiling and then converted to the FFPE RT-PCR assay [14]. oncotype DXTM determines the 10-year risk for disease recurrence in patients with ER-positive, lymph node–negative tumors using a continuous variable algorithm and assigning a tripartite RS (
17, low risk; 18–30, intermediate risk; >30, high risk). Of the multiple pathways assessed by the assay, the proliferation and ER pathways are the most influential on RS calculation followed by the HER-2 pathway. High relative levels of ER mRNA and low levels of Ki-67 proliferation gene mRNA have a low RS. Low levels of ER mRNA and high levels of Ki-67 mRNA have a high RS. The other 14 informative mRNA levels play their greatest roles in determining the RS in tumors with intermediate ER and Ki-67 mRNA levels. It should also be noted that oncotype DXTM is best suited for detecting breast cancers with a low potential for recurrence. Thus, lymph node–negative, ER-positive tumors that are HER-2 positive will generally feature a high histologic grade and will uniformly be referred for adjuvant treatment with chemotherapy and, most often, with anti-HER-2–targeted therapy as well.
The oncotype DXTM discovery cohort consisted of 447 stored samples from three sources, including 233 available samples from the tamoxifen-treated arm of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-20 clinical trial. The test was then validated on 668 ER-positive, lymph node–negative cases of tamoxifen-only treated breast cancer patients of various ages who were enrolled in the NSABP B-14 trial [14]. In this validation cohort, 51% of patients had tumors with a low RS, and 6.8% of these recurred at 10 years. In the high RS group (27% of cases), 30.5% recurred at 10 years [14]. The assay was not prognostic in a different single-center study of untreated, node-negative patients, although neither was tumor grade [62]. A subsequent analysis of the available samples from the NSABP B-20 trial demonstrated that the assay predicted benefit from tamoxifen in those with a low or intermediate (but not high) RS and benefit from chemotherapy in those with a high RS [17]. However, it must be noted that the patients in the tamoxifen treatment arm from that analysis of the NSABP B-20 study were the same patients from which the RS was developed and the thresholds for the three RS groups were defined [17]. In addition, the adjuvant cytotoxic drug regimen cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) in the NSABP B-20 trial was administered concurrently with tamoxifen treatment, which can stop proliferation in endocrine-sensitive tumor cells [17]. The RS is strongly weighted by the quantification of gene expression that is related to ER, HER-2, and proliferation, and so it is intuitive that it should be informative of the sensitivity to endocrine therapy versus chemotherapy, in general. However, the test's ability to function as a predictive factor of therapy response must continue to be explored in other cohorts [63]. For example, the impact of the RS was further validated in a community hospital–based study of 790 patients from 14 Northern California Kaiser Permanente Hospitals [64]. The oncotype DXTM assay has been studied in lymph node–positive patients with promising results, which has led the developer to expanded clinical trials for the test in this setting [65]. In a recently presented study of oncotype DXTM in ER-positive tamoxifen-treated node-positive patients, the findings confirmed, to a large extent, the results obtained in node-negative tumors, including the prediction of chemotherapy benefit [66]. In that study, a high RS was also predictive of added benefit of the cyclophosphamide, doxorubicin, and 5-fluorouracil chemotherapy regimen in these node-positive patients [66]. Finally, in a recently published study of 93 patients in one community and three academic oncology practices, the RS was found to impact the treatment plan in 31.5% of cases [67].
To date, oncotype DXTM, which is approved by the California State Licensing Agency for Laboratories, has not been submitted to the FDA for formal approval. As a centralized home brew assay, it is currently exempt from the standard review FDA requires for diagnostic kits. The original discovery and validation of the test were conducted on archived NSABP clinical samples [20], and the test has been successfully marketed without classic prospective validation. According to the Genomic Health Website, since January 2004, >6,000 physicians have ordered the oncotype DXTM test for >33,000 patients. The test is accepted by a wide variety of third-party payors, including the Centers for Medicare and Medicaid Services, and is endorsed by the most recent American Society of Clinical Oncology (ASCO) tumor marker guidelines. The current list price for the test is $3,460.
The TAILORx Clinical Trial
A significant interest in molecular profiling has led the National Cancer Institute to sponsor a prospective clinical trial using the oncotype DXTM to guide treatment selection. Known as the Trial Assigning Individualized Options for Treatment (TAILORx), the accrual of patients began in May 2006 [67]. This trial is being conducted by the North American Breast Cancer Intergroup, which includes all of the major National Cancer Institute–funded cooperative groups in the U.S. and Canada and is coordinated by the Eastern Cooperative Oncology Group. The TAILORx plans to enroll at least 10,000 women with ER- or PgR-positive, HER-2–negative, lymph node–negative breast cancer at 900 sites in North America. The main goal of the trial is to determine if ER-positive patients with an intermediate RS benefit from chemotherapy or not. Of note is the fact that the RS criteria were changed for the TAILORx, with the 18–30 intermediate RS range of the current clinically available assay changed to 11–25 for the trial [68]. Patients with an RS 10 receive hormonal therapy alone, patients with an RS 26 receive hormonal therapy and chemotherapy, and patients with an RS of 11–25 are randomized into either hormonal therapy alone or hormonal therapy plus chemotherapy. Accrual to this trial appears to be progressing at a rapid rate, with a higher than predicted proportion of patients in the intermediate group, but the trial results will not be known for a number of years and likely not until at least 2013.
Breast Cancer Two-Gene Expression Ratio (H/ITM)
The H/ITM is a six-gene multiplex prognostic RT-PCR assay that uses FFPE tissues and is based on the original report of the impact of the ratio of the relative mRNA expression of two genes—the homeobox gene-B13 (HOXB13) and the interleukin-17B receptor gene (IL17BR)—to predict recurrence in patients with ER-positive, lymph node–negative primary breast cancer [21]. The HOXB13 gene is located in a region on chromosome 17 and is expressed exclusively in neoplastic breast tissue, whereas expression of the IL17BR gene is frequently lost in invasive tumors [21–24]. The prognostic significance of this test has been found to show significance in both tamoxifen-treated [23] and untreated [22] patients, although the significance of the test in the tamoxifen-treated group has been challenged. The original background research for this test was developed at the Massachusetts General Hospital/Harvard Medical School and licensed to AviaraDx (AviaraDx, Inc., Carlsbad, CA). In December of 2006, the test was made commercially available by Quest Diagnostics (Quest Diagnostics, Lyndhurst, NJ) as a centralized test performed in their esoteric testing laboratories in San Juan Capistrano, CA. The cost of the test uses the following CPT codes: 83891, 83896x6, 83898x6, 83902, and 83912, which total approximately $1,400.
Celera Metastasis ScoreTM
This purely prognostic 14-gene multiplex RT-PCR test is also indicated for ER-positive, lymph node–negative tumors treated with tamoxifen and is performed on FFPE tissues. The original geneset was discovered by the Celera Corporation (Celera, Inc, Rockville, MD). In their preliminary studies based in multiple European institutions, the Metastasis ScoreTM for breast cancer predicted a 3.5-fold difference in risk between the 20% of women at the highest risk and the 20% of women at the lowest risk for disease recurrence [69]. This test has been licensed for commercialization to the Laboratory Corporation of America (Lab Corp, Burlington, NC) [69]. The launch date for testing, FDA status, billing strategy, and cost of this test are not currently available.
The Breast BioClassifier
The Associates in Regional and University Pathologists (ARUP, Salt Lake City, UT) Breast BioClassifier is a real-time qRT-PCR assay that can identify the different biological subtypes of breast cancer (luminal-A, luminal-B, HER-2, and basal-like) and provide a prognostic risk assessment [70]. The test consists of 50 classifier genes and five control genes that are measured simultaneously using a 384-well format in the LightCycler® 480 system. Previous studies have shown that the biological subtypes of breast cancer can be recapitulated using a qRT-PCR assay [62] and that the assay can be performed using FFPE tissues [71]. The Breast BioClassifier provides prognosis within different molecular subtypes of ER-negative and ER-positive breast cancer, and identifies groups of patients that may potentially benefit from personalized therapy. This assay will be available at the ARUP central testing laboratory in mid-2008. The test has been submitted to the FDA for 510(k) clearance and, at the present time, the cost is not published.
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GENOMIC MICROARRAY–BASED MULTIGENE PREDICTORS
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MammaPrint®
The MammaPrint® assay (Agendia BV, Amsterdam, The Netherlands) was the first fully commercialized microarray-based multigene assay for breast cancer. This test is currently designed as a pure prognostic assay and has received 510(k) clearance from the FDA, and is offered as a prognostic test for women under the age of 61 with either ER-positive or ER-negative, lymph node–negative breast cancer. The test was also the first assay to be approved by the FDA's new in vitro diagnostic multivariate index assay classification. Unlike oncotype DXTM, this test cannot currently be performed on FFPE tissues and requires either fresh-frozen tumor samples or tissues collected into an RNA preservative solution. The commercial significance of the requirement for fresh or stored frozen tissue on the general acceptance of the test is not currently known. The test is not yet marketed in the U.S. The test was originally developed at the Netherlands Cancer Institute in Amsterdam as a single site using stored frozen samples from breast cancer patients under the age of 53 years and using the Rosetta Inpharmatics DNA microarray system (Merck and Co., Whitehouse Station, NJ) and then commercialized on the Agilent microarray platform (Agilent Technologies, Wilmington, DE). The 70 genes that comprise the MammaPrint® assay are focused primarily on proliferation, with additional genes associated with invasion, metastasis, stromal integrity, and angiogenesis. The discovery of the MammaPrint® 70-gene prognostic assay was originally criticized for including some patients in both the discovery and validation cohorts [28]. However, the test was subsequently validated by the TRANSBIG Consortium of European Cancer Centers on a separate cohort that used the gene signatures to classify patients into the low-risk group when the test algorithm determined that they had a >90% chance of being free from disease for a minimum of 5 years [32]. The TRANSBIG Consortium also found that the MammaPrint® signature could further risk stratify patients within the Adjuvant! Online clinicopathologic risk categories [32, 72]. Moreover, when compared with the St. Gallen prognostic criteria, high-risk patients identified by MammaPrint® have a higher rate of distant metastases than the high-risk patients identified by the St. Gallen criteria and the MammaPrint® low-risk patients have a higher likelihood of metastasis-free survival than those classified as low risk using the St. Gallen criteria. The MammaPrint® assay is at its best when identifying cases at the extremes of the spectrum of disease outcome—the identification of patients with a very good or very poor prognosis. It has not yet been studied if the assay can also predict sensitivity to various treatment modalities. The MammaPrint® test is offered as a centralized assay for lymph node–negative patients independent of hormone receptor status in Europe where the extracted mRNA from primary breast cancers is shipped for the custom microarray profiling. The test results are dichotomous, with reports indicating either a high risk or low risk of disease recurrence. The cost of the test is not published.
MINDACT Trial
The Microarray in Node-Negative Disease May Avoid Chemotherapy (MINDACT) trial is sponsored by the European Organization for Research and Treatment of Cancer and opened in August of 2007; to date, it has accrued 93 patients from five European countries. In this prospective trial of primary lymph node–negative (and more recently, lymph node–positive) breast cancer, all patients are assessed by the standard clinicopathologic prognostic factors included in Adjuvant! Online and by the 70-gene MammaPrint® assay [73]. If both the traditional and molecular assays predict a high-risk status, the patient receives adjuvant cytotoxic chemotherapy and also hormonal therapy if ER positive. If both assays indicate a low risk, no chemotherapy is given and ER-positive patients are given adjuvant hormonal therapy only. When there is a discordance between the traditional clinicopathologic prognostic factor prediction of risk and the MammaPrint® prediction of risk, the patients are randomized to receive treatment based on either the genomic or the clinical prediction results.
Comparison of oncotype DXTM and MammaPrint®
Given that the oncotype DXTM and MammaPrint® assays are fully commercialized and represent two distinct test platforms, a comparison of the two tests is summarized in Table 2. At first glance, given that the two assays share only one overlapping gene, it would appear that the two assays have little in common in terms of genomic content. However, the priority gene lists are consistent in that three biologic pathways are emphasized in both: proliferation, ER and HER-2. In that virtually all newly diagnosed breast cancers in the U.S. and Europe are fully processed for microscopic examination, it provides an ease of use for FFPE-based assays. However, the method of tissue processing and preservation is a choice of the pathologist and is guided by the clinical value of and need for a particular test. The 70-gene MammaPrint® assay could conceivably provide a greater opportunity to assess additional pathways and potentially provide additional pharmacogenomic information to what the 21-gene oncotype DXTM assay can provide. The MammaPrint® test also has a wider indication than oncotype DXTM by including both ER-positive and ER-negative patients, which also allows for the inclusion of a greater number of younger patients. Based on currently published studies, the oncotype DXTM test has been validated as a stand-alone prognostic test and has been interpreted as a predictive test for response to tamoxifen and to the CMF adjuvant chemotherapy regimen (although concurrent with tamoxifen). On the other hand, the MammaPrint® assay is validated as a prognostic test only and has not been formally tested as a predictive test for specific endocrine or cytotoxic therapy regimens. In the marketplace, it would appear that each test has current competitive advantages and disadvantages. The MammaPrint® assay has received 510(k) clearance by the FDA, whereas oncotype DXTM has been exempt. Genomic Health Inc.'s central testing laboratory has been approved by the U.S. CLIA to offer the oncotype DXTM test as a home brew assay. More recently, oncotype DXTM was designated as "recommended for use" by the ASCO Breast Cancer Tumor Markers Update Committee, whereas the MammaPrint® assay was classified by the group as "under investigation" [74].
The two prospective trials designed to determine the potential clinical value of the molecular stratification of early-stage breast cancers have different designs, as described in Table 2. Both trials are attempting to prospectively evaluate the clinical value of these novel multigene molecular signatures. The TAILORx is designed to determine whether intermediate risk ER-positive patients benefit from adjuvant chemotherapy or not, whereas the MINDACT trial is designed to determine if a molecular test is more accurate than Adjuvant! Online to identify low-risk patients who can be spared from cytotoxic therapy. Neither trial is focused on lymph node–positive tumors, although the MINDACT trial recently allowed accrual of node-positive cases. Although the MINDACT trial allows for a wider patient eligibility, including both ER-positive and ER-negative patients, the accrual rate to date appears to be greater for the TAILORx. The TAILORx has been criticized for "performing a randomized comparison in a stratum of intermediate-risk patients" according to the RS that was modified from the current test indication (low score, <10 versus low score, <18) without any effort being made to compare this with a traditional clinical prediction [72]. The oncotype DXTM assay was shown to be cost-effective in one pharmacoeconomic study [73], whereas a similar study for MammaPrint® has not been published. However, it is important to understand that cost-effectiveness results are highly dependent on assumptions of how physicians use the test results and alter their treatment plan based on molecular data. Finally, it should be noted that it will take at least five more years before either trial can be fully evaluated for clinical significance and thus impact the true assessment of the cost-effectiveness of either assay.
The Rotterdam Signature
The Rotterdam signature, also known as the 76-gene assay, was developed as a pure prognostic assay at the Erasmus University Cancer Center in Rotterdam, The Netherlands, and is being commercially developed by the Veridex Corp. (Warren, NJ). Sharing no genes in common with either oncotype DXTM or MammaPrint®, and run on the Affymetrix U-133 GeneChipTM System (Affymetrix, Inc., Santa Clara, CA), this assay is validated for lymph node–negative patients independently of hormone receptor status [34–35]. The most recent validation of this assay was performed in four European cancer centers, achieving high stand-alone prognostic significance and the ability to predict recurrence in ER-positive patients treated with tamoxifen alone [35–37]. The gene list for this assay is heavily weighted toward proliferation genes. This assay requires fresh/frozen extracted mRNA and, similar to MammaPrint®, has not been validated for use on FFPE tissues or core biopsies. The test has not been commercially launched, and its FDA approval status and cost have not been announced.
Invasiveness Gene Signature
The invasiveness gene signature (IGS) is a prognostic assay that also uses the Affymetrix U-133 GeneChip® System and is currently being developed as a stand-alone prognostic test (OncoMed Pharmaceuticals, Redwood City, CA) [38]. This assay is designed for both node-negative and node-positive and both ER-negative and ER-positive patients. The IGS consists of 186 genes that may be related to tumor stem cells, and appears to also predict prognosis for lung and prostate cancers as well as medulloblastoma. The launch date, FDA status, and cost of this assay are not currently available.
NuvoSelectTM
The NuvoSelectTM assay is a combination of several pharmacogenomic genesets obtained from resected tumor specimens (SET index) or from fine-needle aspiration specimens (chemotherapy response predictor). Rather than serving as a stand-alone prognostic assay, NuvoSelectTM is primarily a predictive test for guiding selection of therapy. One gene set (30 genes) predicts complete response to preoperative paclitaxel, 5-fluorouracil, doxorubicin, and cyclophosphamide (TFAC) chemotherapy and the other (200 genes) predicts outcome after 5 years of endocrine therapy [42, 43]. This program also identified the overexpression of mRNA of the biomarker microtubule-associated protein tau as a major predictor of resistance to the TFAC regimen, but at the same time a predictor of greater sensitivity to endocrine therapy among ER-positive patients [44]. The test has also been proven for routine ER and HER-2 status determination based on mRNA levels of these two genes [75]. This assay is being developed by Nuvera Bioscience, Inc., (Woburn, MA) in collaboration with the University of Texas M.D. Anderson Cancer Center (Houston, TX). The FDA approval strategy, commercial launch date, and cost for this test have not been released.
Cytochrome p450 CYP2D6 Genotyping
A wide variety of drugs are metabolized by a group of liver enzymes known as cytochrome P450 or CYP enzymes. The CYP2D6 enzyme activates tamoxifen by metabolizing it to endoxifen, the potent antiestrogen. It is now known that a subset of patients with low or completely deficient levels of CYP2D6 fail to activate tamoxifen and thus are unable to benefit from its antitumor effects [76–78]. Thus, it has been suggested that, for patients deficient in CYP2D6 who have been diagnosed with ER-positive breast cancer, aromatase inhibitors may be the preferred hormonal therapeutics rather than tamoxifen. The test platforms used for determining the CYP2D6 genotype include PCR with restriction fragment length polymorphism assay, a proteomics approach using gas chromatography and mass spectroscopy, and the FDA-approved genomic microarray approach. The Roche AmpliChip® technology (Roche Diagnostics Inc., Indianapolis, IN) has been customized to detect CYP2D6 deficiency at the DNA level in the germ line of newly diagnosed breast cancer patients and has been the most widely employed technique for the study of tamoxifen response [78–80]. This assay has been approved by the FDA, and recently the Laboratory Corporation of America announced a partnership with Medco Health Solutions Inc. (Franklin Lakes, NJ), designed to offer CYP2D6 testing on a large scale. Several smaller laboratories have announced the availability of home brew assays for this biomarker. It is too early to know whether this approach toward personalizing the selection of hormonal therapy will become widely used.
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SUMMARY AND CONCLUSIONS
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The commercialized multigene predictors for breast cancer are summarized in Table 3
. The assays are grouped according to their major clinical functions: classification, grading, prognosis alone, and prediction of response to therapy. As seen in Table 3 and in the text, the two tests that have achieved the most advanced commercial success are oncotype DXTM and MammaPrint®. Virtually all the tests considered in this review, regardless of their final assay platform, used transcriptional profiling for the discovery of their test's gene, mRNA, or protein biomarkers. As the process of commercialization continues in this extremely competitive landscape, concerns continue to arise over the scientific validity, true clinical utility, and ultimate cost–benefit ratios for these significantly expensive tests [79]. Rapid adoption of these assays before they have been proven in their clinical effectiveness is a serious concern among both breast cancer oncologists and health care economists. Experts in the fields of both oncology and biostatistics continue to raise concerns as to whether the independently published significant statistical contributions of these new assays will hold up over time as more patients are tested. In addition, it has been noted that these new tests can easily be misused, including employing the test in the wrong clinical setting and ending up with misleading reassurance about test-driven decisions [80]. For example, gene-expression predictors that use extracted mRNA from a resection specimen can give an inaccurate high risk result if the brisk proliferative response of benign tissue associated with the healing of a previous biopsy wound site is included in the overall specimen assessment. Although the prospective clinical trials that are currently underway, such as the TAILORx and the MINDACT trial, could provide significant answers about the clinical value of these multigene predictors, many questions will remain unanswered for several years.
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AUTHOR CONTRIBUTIONS
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Conception/design: Jeffrey S. Ross, Christos Hatzis, W. Fraser Symmans, Lajos Pusztai, Gabriel N. Hortobágyi
Administrative support: Jeffrey S. Ross
Collection/assembly of data: Jeffrey S. Ross, Christos Hatzis, W. Fraser Symmans, Lajos Pusztai, Gabriel N. Hortobágyi
Data analysis and interpretation: Jeffrey S. Ross, Christos Hatzis, W. Fraser Symmans, Lajos Pusztai, Gabriel N. Hortobágyi
Manuscript writing: Jeffrey S. Ross, Christos Hatzis, W. Fraser Symmans, Lajos Pusztai, Gabriel N. Hortobágyi
Final approval of manuscript: Jeffrey S. Ross, Christos Hatzis, W. Fraser Symmans, Lajos Pusztai, Gabriel N. Hortobágyi
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Learning Objectives
Abstract
