A broad variety of new biomarkers with potential prognostic or predictive value are discussed in the literature, but only a few have proven to represent an evidence-based gain for daily use.
The tumor-associated fibrinolytic factors urokinase-type plasminogen activator (uPA) and its inhibitor type 1 (PAI-1) are important promoters of tumor invasion and metastasis. The uPA/PAI-1 assay is based on a standardized enzyme-linked immunosorbent assay (ELISA) to measure the protein levels in fresh tumor samples. Single clinical trials as well a recently published metaanalysis with > 8,000 patients confirmed the prognostic value of uPA/PAI-1 [23
]. Node-negative patients with low uPA/PAI-1 levels have a very low risk of recurrence, and in the final analysis of the multicentric prospective Chemo N0 trail a 10-year survival rate of nearly 90% without any adjuvant treatment was observed [24
]. Besides retrospective analyses showing a clear benefit for adjuvant chemotherapy in patients with high versus low uPA/PAI levels, the prospectively designed international multicentric NNBC-3 study will address the question of treatment optimization for node-negative patients with a taxane/anthracycline-based chemotherapy regimen with regard to uPA/PAI expression. Based on the well established standardization and the solid scientific evidence, uPA/PAI-1 detection is recommended by the AGO Breast Committee (AGO LOE la, GR A, +) as well as by the American Society of Clinical Oncology (ASCO) guidelines, especially for the group of G2 node-negative patient, as a tool for treatment decisions [3
Proliferation has been recognized for a long time as an important prognosticator in breast cancer. Ki-67 represents a strong proliferation marker and has been gaining more interest over the last years. Patients with highly proliferating tumors exhibit an increased benefit from adjuvant therapy in comparison to patients with low or intermediate proliferation activity [25
]. In retrospective analyses, higher values of Ki-67 were associated with adverse prognostic factors such as Her2/neu expression, higher grading, or LVI, and with worse disease-free survival with a hazard ratio (HR) of 1.8 in comparison to tumors with low Ki-67 expression [25
]. Since prospective validation is still missing, a general recommendation for Ki-67 determination cannot be given. However, this factor represents an additional potential prognostic marker in selected cases and is gaining more importance due to its role in defining the different intrinsic subtypes of breast cancer [3
Over the last years, molecular characterization of breast cancer has gained more and more importance [26
]. The profound analysis of gene expression profiles has led to the definition of 5 different molecular intrinsic subtypes of breast cancer: ER-positive/luminal A and B, basal-like, ErbB2-positive, and normal breast, which are associated with differences in clinical outcome. The intrinsic subtypes as distinct entities were found to have a significant impact on recurrence-free survival in untreated patients and remained significant in multivariate analysis incorporating standard prognostic factors such as ER status, histological grade, tumor size, and lymph node status. The basal-like subtype – which is mostly G3-differentiated, expressing C5/6 cytokeratins, and negative for ER, PR, and Her2/neu – is characterized by an unfavorable prognosis and represents a potential predictor for specific adjuvant systemic treatment, which is currently under investigation [28
]. According to the St. Gallen Consensus 2011, the intrinsic subtypes can be defined by immunohisto-chemistry, in analogy to the classification recommended by Cheang et al. [29
] using ER, PR, Her2/neu, and Ki-67, respectively: i) Luminal A = ER- and/or PR-positive, Her2/neu-neg-ative, Ki-67 low (< 14%); ii) Luminal B (Her2/neu-negative) = ER- and/or PR-positive, Her2/neu-negative, Ki-67 high (< 14%); iii) Luminal B (Her2/neu-positive) = ER- and/or PR-positive, Her2/neu overexpression/amplification, Ki-67 low or high; iv) Her2/neu-positive (not luminal) = ER- and PR-negative, Her2/neu overexpression/amplification; v) Basal-like, triple-negative (ductal) = ER- and PR-negative, Her2/ neu-negative.
The significant discrepancies between the clinical and molecular classification become apparent for the so-called triple-negative breast cancers (ER-, PR-, Her2/neu-negative) which are defined immunohistochemically. This subgroup is not identical to the molecularly defined basal-like subtype and underlines the fact that ER and Her2/neu status are not accurate surrogates for the true intrinsic subtype status. As a consequence, the optimal classification system for breast cancer subtypes to guide therapeutic decision-making has to yet be defined. Nevertheless, the triple-negative as well as the basal-like subtypes are characterized by an adverse course of disease and the need for adjuvant chemotherapy, even though the optimal regimens (platinum-based chemotherapy, PARP inhibitors) are the current focus of ongoing clinical trials [30
The detection of disseminating tumor cells (DTCs) is an additional field of growing interest in breast cancer. The risk of distant recurrence is biologically based on minimal residual disease (MRD) which can be detected by the identification of DTCs in the bone marrow or circulating tumor cells (CTCs) in the blood. The relevance of DTCs as an independent prognostic parameter was demonstrated in several studies and in 1 metaanalysis [31
]. However, so far, no routine detection method has been established since the therapeutic consequences are unclear. The detection of CTCs in peripheral blood is technically difficult, but a standardized FDA-approved assay is currently available. CTCs represent a validated prognostic marker in the metastasized situation and correlate with significantly shorter survival [32
]. In the adjuvant situation, several studies were able to identify a prognostic value for CTCs [33
]. However, since there is no proof of a clinical benefit resulting from therapeutic measures undertaken in response to the existence of CTCs, the detection of MRD cannot currently be recommended for routine use [3
Multigene analyses offer the possibility of simultaneous investigation of multiple tumor-relevant pathways with the goal of identifying prognostic and predictive gene expression signatures [34
]. Different test platforms are used to detect gene profiles. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and cDNA microarray are complex methods which also require complex mathematical algorithms to avoid false associations. The feasibility of these approaches was demonstrated in several retrospective studies, but the required prospective data are missing so far.
The Oncotype DX™ (Genomic Health Inc., Redwood City, CA, USA) assay is based on qRT-PCR to avoid the problem of fresh frozen tumor tissue, and uses readily available paraffin-embedded tumor blocks. A set of 250 genes was primarily analyzed in different study populations including the NSABP B-20 trial. The combination of gene expression could be linked to clinical outcome, and by applying a continuous variable algorithm, 3 risk categories could be defined – low, intermediate, and high-risk – for developing distant métastases at 10 years. Finally, a 21-gene set (16 cancer-related, 5 reference genes) could be identified for the optimal discrimination of the 3 risk categories, and this set was validated in the NSABP B-14 trial in a population of ER-positive, node-negative patients who underwent endocrine therapy with tamoxifen [35
]. The most dominant genes in this set can be grouped to proliferation (e.g. Ki-67), Her2/neu, and ER-related genes. A recurrence score is calculated with low score (< 18), intermediate score (> 18 and < 30), and high score (> 30). In the validation population of the NSABP B-14 trial, these score translated into a risk for distant recurrence of 7% (low), 14% (intermediate), and 31% (high), respectively [35
]. The molecular classification seems to better predict clinical outcome in comparison to conventional prognostic markers. A recently published retrospective study supported these findings and was able to show that up to 50% of patients who were classified with classical parameters to be at high risk could be downgraded to low risk by the utilization of Oncotype DX [37
]. Besides the validation data for tamoxifen treatment, retrospective analyses (including NSABP B-20) also revealed that node-negative patients had no benefit from an additional chemotherapy in the case of a low recurrence score [36
]. However, until now, the AGO guidelines have not recommended the usage of this assay since prospective data are missing [3
]. Several large prospectively designed multi-center trials (TAILORx, Plan B) are ongoing to evaluate Oncotype DX for treatment decision, especially with regard to chemotherapy.
A different approach for multigene analyses is the utilization of fresh frozen tissue by applying cDNA arrays. The most advanced and analyzed assay is the MammaPrint™ (Tromms-dorff GmbH & Co. KG, Alsdorf, Germany) using a 70-gene signature. Starting from 25,000 genes, the Amsterdam group analyzed the clinical outcome (endpoint: distant-free survival) of 78 patients < 55 years of age with node-negative tumors less than 5 cm which were ER-positive or -negative. The retrospectively chosen validation cohort proofed the 70-gene signature to be a potent discriminator for a good versus bad prognosis with a 10-year survival of 85 vs. 51%, respectively [15
]. A further multicentre validation study of 300 untreated primary breast cancer patients exposed the potential prognostic significance of the assay by showing a hazard ratio of 4.6 vs. 2.1 for patients with a bad prognosis in comparison to a good prognosis signature [38
]. Even if these data for multigene signatures are promising, the clinical value is so far not proven since no prospective data exist. The MammaPrint assay is currently the subject of a large prospective international trial (MINDACT of the BIG study group) to validate its prognostic power. Therefore, the current AGO guidelines do not recommend the clinical utilization of this test outside of clinical trials [3
In the daily clinical setting, the established classical prognostic and predictive biomarkers still represent the backbone for treatment decisions with the highest clinical value. Some innovative markers, like uPA/PA-1, also represent solid and highly validated tools to guide treatment decisions, especially in certain subgroups of patients with early breast cancer, even if long-term data are mostly unavailable. It is of big importance to note that the innovative multigene approaches potentially will open a new chapter in breast cancer therapy but currently fail to reach the required validation level for routine clinical use. Besides all the discussed factors, additional risk modifiers such as lifestyle (obesity, alcohol consumption) have proven influence on disease outcome in breast cancer patients and should as a consequence be considered and verbalized in the communication with the patient.