Jolien M. Bueno-de-Mesquita

Microarray-Based Determination of Estrogen Receptor, Progesterone Receptor, and HER2 Receptor Status in Breast Cancer

Purpose: The level of estrogen receptor (ER), progesterone receptor (PR), and HER2 aids in the determination of prognosis and treatment of breast cancer. Immunohistochemistry is currently the predominant method for assessment, but differences in methods and interpretation can substantially affect the accuracy, resulting in misclassification. Here, we investigated the association of microarray-based mRNA expression levels compared with immunohistochemistry. Experimental Design: Microarray mRNA quantification of ER, PR, and HER2 was done by the developed TargetPrint test and compared with immunohistochemical assessment for breast tumors from 636 patients. Immunohistochemistry was done in a central laboratory and in an independent reference laboratory according to American Society of Clinical Oncology/College of American Pathologists guidelines for 100 cases. For HER2 immunohistochemistry 2+ cases, additional chromogenic in situ hybridization (CISH) was used to determine the final status. Results: ER concordance between microarray and central immunohistochemistry was 93 [95 confidence interval (95 CI), 91-95]. Only 4 of immunohistochemistry-positive samples were classified negative using microarray, whereas 18 of immunohistochemistry-negative samples showed a positive microarray ER status. Concordance for PR was 83 (95 CI, 80-86) and 96 of all samples showed an identical classification of HER2 status by microarray and immunohistochemistry/CISH (95 CI, 94-98). Nine percent of immunohistochemistry HER2-positive samples showed a negative microarray classification. Detailed review of 11 cases with discordant classifications by American Society of Clinical Oncology/College of American Pathologists and central immunohistochemistry indicated that microarray assessment was likely to add additional information in 5 cases. Conclusion: Microarray-based readout of ER, PR, and HER2 shows a high concordance with immunohistochemistry/CISH and provides an additional, objective, and quantitative assessment of tumor receptor status in breast cancer. (Clin Cancer Res 2009;15(22):700311)

Technology Insight: tuning into the genetic orchestra using microarrays--limitations of DNA microarrays in clinical practice.

Scientific advances in the field of genetics and gene-expression profiling have revolutionized the concept of patient-tailored treatment. Analysis of differential gene-expression patterns across thousands of biological samples in a single experiment (as opposed to hundreds to thousands of experiments measuring the expression of one gene at a time), and extrapolation of these data to answer clinically pertinent questions such as those relating to tumor metastatic potential, can help define the best therapeutic regimens for particular patient subgroups. The use of microarrays provides a powerful technology, allowing in-depth analysis of gene-expression profiles. Currently, microarray technology is in a transition phase whereby scientific information is beginning to guide clinical practice decisions. Before microarrays qualify as a useful clinical tool, however, they must demonstrate reliability and reproducibility. The high-throughput nature of microarray experiments imposes numerous limitations, which apply to simple issues such as sample acquisition and data mining, to more controversial issues that relate to the methods of biostatistical analysis required to analyze the enormous quantities of data obtained. Methods for validating proposed gene-expression profiles and those for improving trial designs represent some of the recommendations that have been suggested. This Review focuses on the limitations of microarray analysis that are continuously being recognized, and discusses how these limitations are being addressed.

Methodology of constructive technology assessment in health care

OBJECTIVES Technologies in health care are evolving quickly, with new findings in the area of biotechnological and genetic research being published regularly. A health technology assessment (HTA) is often used to answer the question of whether the new technology should be implemented into clinical practice. International evidence confirms that the results of HTA research sometimes have limited impact on practical implementation and on coverage decisions; the study design is commonly based on the paradigm of stability of both the technology and the environment, which is often not the case. Constructive technology assessment (CTA) was first described in the 1980s. In addition to the traditional HTA elements, this approach also takes into account the technology dynamics by emphasizing sociodynamic processes. With a CTA approach, comprehensive assessment can be combined with an intentional influence in a favorable direction to improve quality. METHODS In this study, the methodological aspects mainly concerning the diagnostic use of CTA are explained. The methodology will be illustrated using the controlled introduction of a new technology, called microarray analysis, into the clinical practice of breast cancer treatment as a case study. Attention is paid to the operationalization of the phases of development and implementation and the research methods most appropriate for CTA. CONCLUSIONS In addition to HTA, CTA can be used as a complementary approach, especially in technologies that are introduced in an early stage of development in a controlled way.

The impact of inter-observer variation in pathological assessment of node-negative breast cancer on clinical risk assessment and patient selection for adjuvant systemic treatment

BACKGROUND It is well known that there is considerable inter-observer variability in assessment of the pathological parameters that are used to select node-negative breast cancer patients for adjuvant systemic treatment. There are only limited data available as to in how many patients this leads to differences in treatment decisions. METHODS Clinical and pathological data of 694 patients <61 years with primary unilateral T1-4N0M0 breast cancer were analysed. Grade, estrogen receptor (ER) status and human epidermal growth factor receptor 2 (HER2) status were first assessed locally; subsequent central re-evaluation of these parameters was carried out. Clinicopathological low or high risk was assessed using national Dutch guidelines and the Adjuvant! Online (www.adjuvantonline.com). RESULTS The local pathological examination was discordant with central review for grade, ER and HER2 in 28% (kappa 0.56; grade 2 tumours 35% discordant), 5% (kappa 0.85) and 4% (kappa 0.81) of patients, respectively. If clinical risk were assessed based on Dutch guidelines or Adjuvant! Online, respectively, 15% (one of seven patients; kappa 0.70) or 8% (kappa 0.83) of patients would have been assigned to a different clinical risk group. CONCLUSION Inter-observer variation in pathological examination of breast carcinomas results in significant differences in grade, ER status, HER2 status, clinicopathological risk and subsequently in adjuvant systemic treatment advice.

Prospective cost-effectiveness analysis of genomic profiling in breast cancer

BACKGROUND The cost-effectiveness of the 70-gene signature (70-GS) (MammaPrint®) has earlier been estimated using retrospective validation data. Based on the prospective 5-year survival data of the microarRAy-prognoSTics-in-breast-cancER (RASTER) study, the aim here was to evaluate the cost-effectiveness reflecting the actual use in clinical practice, including reality-based compliance rates. METHODS Costs and outcomes (quality-adjusted-life-years (QALYs)) were calculated in node-negative (N-) patients included in the RASTER study (n=427). Sensitivity and specificity of the 70-gene and Adjuvant! Online (AO) were based on 5-year distant-disease-free survival (DDFS). Subgroup analyses were performed for two groups for whom benefit of the 70-gene had earlier been reported: (1) ductal, oestrogen receptor-positive (ER+), tumour diameter 10-30 mm, grade II, age 40-70; (2) ductal, oestrogen receptor-positive, tumour diameter 5-30 mm, grade II/III and age 40-70. RESULTS Based on 5-year survival data, the cost-effectiveness of the 70-gene signature versus AO was prospectively confirmed. The total health care costs per patient were €26,786 for the 70-gene and €29,187 for AO. The quality adjusted life years yielded 12.49 and 11.88, respectively. The subgroups retrieved slightly higher life gains and higher costs, but all resulted finally in a favourable position for the 70-gene signature. CONCLUSIONS The use of the 70-gene signature, as judged appropriate by doctors and patients and supported by a low risk 70-gene signature as an oncological safe choice, was also found to be cost-effective.

Additional value and potential use of the 70-gene prognosis signature in node-negative breast cancer in daily clinical practice

BACKGROUND The 70-gene prognosis signature has strong prognostic value in node-negative breast cancer, independent of established prognostic factors. It is unclear whether all node-negative patients should receive a signature result. We therefore evaluated its additional prognostic information to a combination of established prognostic guidelines. METHODS We evaluated 701 patients from three previously described series in whom a signature result was available. Clinical risk was on the basis of Adjuvant! Online (AO), St Gallen guidelines (St G) and Nottingham Prognostic Index (NPI). Overall survival (OS) analyses were carried out in patients treated at the Netherlands Cancer Institute (Amsterdam) who did not receive adjuvant systemic treatment (AST). RESULTS Only 6% (10 of 156) of estrogen receptor (ER)-negative tumours had a good prognosis signature. The signature was not useful for ER-positive tumours and concordant high AO, high St G and/or high NPI clinical risks (N = 139). The 10-year OS estimate for good signature tumours with these characteristics was <80% and AST would therefore be appropriate irrespective of the signature result. In contrast, for patients with a concordant low AO, low St G and/or low NPI risk and in discordant clinical risk patients, the signature identified low-risk patients in whom AST could be safely withheld (10-year OS > 90%). CONCLUSION The 70-gene prognosis signature provides additional prognostic information especially in ER-positive lymph node-negative breast cancer patients with a predominant low or discordant clinical risk on the basis of AO, St G and/or NPI.

Identification of a low risk subgroup in Her2-positive breast cancer by the 70-gene prognosis signature.

Abstract #4171 Introduction:
 Her2/neu-overexpression is observed in 15-20% of invasive breast cancers and considered a negative prognostic factor. Most Her2-positive patients are classified as high risk by current treatment guidelines and thus allocated to adjuvant trastuzumab and chemotherapy. However, 74% of patients remained distant recurrence-free at 3 years without trastuzumab in the HERA-trial (HERceptin Adjuvant). In the present study the 70-gene prognosis signature (MammaPrint™), validated as an independent prognostic indicator for patients with up to three positive lymph nodes, was used to identify a subgroup of patients with low risk and favorable outcome.
 Methods:
 One-hundred-sixty-nine patients with Her2-positive breast cancer were selected from a pooled dataset of 1280 patients with known Her2-status. Patients had a unilateral T1-3, N0-1 tumor and were treated with either breast-conserving therapy or mastectomy. Samples were analyzed and classified by the 70-gene signature as good or poor prognosis by Agendia Laboratories.
 Results:
 After a median follow-up of 65 months (range 4-303) 49 (29%) distant recurrences and 41 (24%) breast cancer-specific deaths occurred. The 70-gene signature classified 27 (16%) patients as good prognosis, with a 10-year distant disease-free survival (DDFS) of 89% (25/27), compared to 142 (84%) patients classified as poor prognosis, who had a DDFS of 64%. The estimated hazard ratios (HR) for genomic risk were 4.9 (95%CI 1.2-20.1,p=0.029) and 4.4 (95%CI 1.1-18.4,p=0.040) for DDFS and breast cancer-specific survival at 10 years, respectively. In multivariate analysis, adjusted for known prognostic factors and adjuvant therapy, only the 70-gene signature and tumor size were independent predictors for 10-year-DDFS with HRs of 5.4 (95%CI 1.3-23.4,p=0.024) and 1.05 (95%CI 1.02-1.08,p=0.001), respectively. The good prognosis group had positive hormone-receptors in 85%, and fewer patients received adjuvant therapy: 16 patients (59%) received no adjuvant treatment, 6 (22%) received adjuvant chemotherapy, 8 (30%) hormonal therapy and 1 (4%) received trastuzumab. For the subgroup of 90 patients who were not treated with adjuvant chemotherapy or trastuzumab, the HR for low versus high 70-gene signature for 10-year DDFS was 4.75 (1.13-19.92,p=0.033).
 Discussion:
 The 70-gene signature is a strong independent prognostic indicator that can identify a subgroup with good clinical outcome in Her2-positive early breast cancer even in the absence of adjuvant chemotherapy and trastuzumab. This subgroup will be further studied in the ongoing MINDACT-trial (MIcroarray for Node-negative and 1-3 positive node Disease may Avoid ChemoTherapy) and beyond. The MINDACT-trial will prospectively evaluate if it is acceptable to withhold chemotherapy and/or trastuzumab in Her2-positive, genomic low risk patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4171.

Microarray-based determination of ER, PR and HER2 receptor status: validation and comparison with IHC assessments.

Abstract #3007 Background
 In breast cancer patients the level of expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 is predictive for prognosis and/or treatment response. However, differences in assessment methods and interpretation can substantially affect the accuracy and reproducibility of the results. Previously, we have determined the association between immunohistochemistry (IHC) and mRNA levels for ER, PR and HER2, and have confirmed the accuracy of microarray readout on >400 samples. In the current study we describe the use of this microarray based readout on prospectively collected samples. We compared these readouts with multiple IHC and fluorescent in situ hybridization (FISH) assessments generated in various hospitals and a CLIA-certified reference laboratory and developed a microarray based test called TargetPrint™.
 Methods
 Gene expression data for ER, PR and HER2 were obtained by analysis of 100 breast carcinomas that have been collected prospectively within the RASTER study. Samples were stratified as receptor positive or negative using thresholds for ER, PR and HER2 mRNA levels. IHC assessment was performed (1) according to local standards of the hospital from where the sample originated, (2) by the central laboratory of the Netherlands Cancer Institute, and (3) at an independent reference laboratory using FDA-approved procedures and ASCO/CAP guidelines. A tumor was classified positive for ER and PR when ≥10% of tumor cells showed positive staining. HER2 IHC status was scored as 0, 1+, 2+ or 3+; a score of 3+ was considered positive. In case of 2+ samples, a FISH was performed to assess final HER2 amplification status. The cohort used in this study was pre-selected to include about two-third ER and PR positive samples and one-third HER2 positive samples.
 Results
 Multiple microarray readouts were highly reproducible (Pearson correlation 0.991) and resulted in 67, 61 and 39 percent positive samples for ER, PR and HER2, respectively. Comparison of microarray results with IHC (including FISH for HER2) performed at the three centers indicated highly similar results for receptor readout with a concordance of 92, 93 and 92% for ER; 84, 81 and 86% for PR; and 93, 95 and 94% for HER2. Overall misclassification rates between microarray and IHC readout were low for ER (0.08) and HER2 (0.06) and quite low for PR (0.14), and were comparable to the misclassification rates between the three IHC methods.
 Conclusion
 A microarray-based assessment of ER, PR and HER2 in relation to mRNA levels gives results comparable to multiple IHC methods and FISH and provides an objective and more quantitative assessment of tumor receptor status than IHC alone. Using TargetPrint™ for microarray readouts for hormone and HER2 receptor in addition to standard IHC will improve molecular characterization of breast cancer tissue. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3007.

Abstract P6-06-13: Optimized prediction of clinical outcome by the PREDICT plus tool and the 70-gene signature in early stage node-negative breast cancer

Background Established breast cancer guidelines and online tools use clinico-pathological factors including age, tumor size and grade to evaluate the risk of recurrence and select patients who are eligible to receive adjuvant chemotherapy (ACT). One of the online tools, PREDICT plus, was recently updated and is the first tool to include HER2 status and method of detection in risk assessment. Another tool to better guide AST decisions is the 70-gene signature. The 70-gene signature is a gene-expression classifier that was developed and extensively validated to predict the risk of distant recurrence in breast cancer. In clinical practice, an ad-hoc combination of clinico-pathological guidelines and gene-expression classifiers are used. The aim of this study is to evaluate the combination of the PREDICT plus tool and the 70-gene signature to optimize adjuvant systemic treatment decisions. Methods For 427 patients participating in the RASTER study (cT1-3N0M0) a 70-gene signature result was available. PREDICT risk estimates at 5 (P5) and 10 (P10) years after diagnosis were calculated using the following variables: age, method of detection, tumor size, tumor grade, number of positive nodes, estrogen receptor, and HER2 status. Patients were considered high risk if their survival probability was less than 95% at 5 years and/or 90% at 10 years. Five-year distant-recurrence-free-interval (DRFI) and distant-disease-free-survival (DDFS) probabilities were evaluated between subgroups based on the 70-gene signature and PREDICT plus. Results Median follow-up was 61.6 months. Patients with a low risk 70-gene signature (n = 219) had a 5-year DRFI of 97.0% (CI: 94.7-99.4) compared to 91.7% (CI: 87.9-95.7) for the 70-gene signature high risk patients (n = 208). The 5-year DRFI for patients with a P5 low risk (n = 228) is 96.8% (CI: 94.2-99.4) compared to 91.7% (CI: 87.9-95.7) for P5 high risk (n = 199). The 5-year DRFI for patients with a P10 low risk (n = 168) is 98.0% (CI: 95.7-100) compared to 92.1% (CI: 88.7-95.6) for P10 high risk (n = 259). ACT data, DRFI and DDFS probabilities in all subgroups are shown in table 1. Among the patients who had a low risk according to PREDICT at 5 years, but a high risk at 10 years (n = 60), the 5-year DRFI was 100% when their tumor was tested as low risk based on the 70-gene signature (13% received CT) compared to 84% in case of a high risk result (55% received CT)(p = 0.03). Conclusion Combining PREDICT plus with the 70-gene signature may help to identify early stage node-negative breast cancer patients for whom limited adjuvant systemic treatment might be appropriate and for whom overtreatment can be avoided. Especially in case of a low risk assessment by PREDICT at 5 years but a high risk at 10 years, the 70-gene signature may aid to select those patients at a high risk of recurrence who will benefit most from ACT. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-13.

Benefit of the 70-gene profile for widely used guidelines: an answer to increased selection for adjuvant chemotherapy in breast cancer (Poster)

Risk assessment for breast cancer patients differs substantially among treatment guidelines. The NCCN, St.Gallen, Adjuvant!Online, and Dutch 2008 guidelines are less restrictive in comparison to the 2004 Dutch guidelines and Nottingham Prognostic Index, when selecting patients for adjuvant systemic treatment. The Dutch Institute for Healthcare Improvement (CBO) has introduced slight changes in the concept 2008 guidelines. Adjuvant systemic treatment is only advised when the absolute 10-years survival benefit is 5% or more. The new recommendations for adjuvant systemic treatment are based on survival tables used in the Adjuvant! software. The changes affect only node-negative patients aged >35 years: since 2008 patients with G2/G3 tumors >1cm or every tumor >2cm are advised to undergo adjuvant systemic therapy, while in 2004 this was the case for tumors G3>1cm, G2>2cm or every tumor >3cm. Patients and Methods: Risk was assessed for 427 lymph-node negative (LNN) patients in the prospective RASTER-study (Bueno-de-Mesquita, 2007) and 151 LNN patients in the NEJM- series (van de Vijver, 2002). Clinical risk was calculated using the 2004 and 2008 Dutch guidelines. Genetic risk was assigned according to the result of the Amsterdam 70-gene signature. Survival analyses were done according to the univariate Kaplan-Meier-method. Results: Instead of 57% in 2004, in 2008 only 24% of patients were assigned to clinical low risk in the RASTER study, and 141 patients (33%) changed from low to high risk (p<0.001). The rate of discordant findings between clinical assessment and 70- gene signature increased from 30% in 2004 to 41% in 2008 (p<0.001, table 1). Similar results were obtained for the 151 patients of the NEJM-series (30% and 35% discordance for the 2004 and 2008 CBO guidelines, respectively). At 10 years follow-up, differences between the high and low risk categories according to the 2004, 2008 guidelines and the 70-gene profile, were best predicted by the latter: distant-disease-free survival (DDFS) log-rank p=0.002, p=0.11 and p<0.001 respectively. Discussion: As adjuvant systemic treatments become more effective, guidelines become less restrictive, resulting in more patients being selected for adjuvant therapy. Since newer treatment guidelines do not better predict survival, the magnitude of the 70- gene profiles benefit, which is defined as proportion of patients in whom over- and undertreatment can be avoided, depends on present guidelines for risk assessment and shows a remarkable increase.