Bichlien Nguyen

MammaPrint Molecular Diagnostics on Formalin-Fixed, Paraffin-Embedded Tissue

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

Central review of discordant samples for microarray-based ER, PR, and HER2 and local IHC/FISH assessment worldwide from 827 patients.

11 Background: Differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and is 98% concordant with centrally assessed ER as presented by Viale et al, SABCS 2011. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. METHODS Fresh tumor samples (core needle biopsies or surgical) were collected for 831 patients diagnosed with breast cancer stage I to IV (Feb 2008 - Jan 2011) from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts with TargetPrint. Discordant cases were centrally reviewed for IHC/FISH assessment. RESULTS Of the 831 samples, IHC assessment was unknown for 4 ER/ PR samples; HER2 was unknown for 12 samples. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 83% for PR and 94% for HER2. In this study, 3% of all IHC ER positive samples were classified negative by microarray, and 11% of IHC PR positive samples were classified negative by microarray. For HER2, 4% of IHC/FISH HER2 positive samples were classified negative by microarray and 2% of IHC/FISH HER2 negative samples were classified positive by microarray. Most notably, all available 5 ER IHC negative/TargetPrint positive samples turned out to be positive with central re-assessment. HER2 IHC2+ samples with discordant classifications for TargetPrint and local assessment are currently being reviewed for FISH/SISH assessment. CONCLUSIONS Microarray based readout of ER, PR and HER2 status using TargetPrint is fairly comparable to local IHC and FISH analysis in 827 analyzed samples in various hospitals worldwide. However, re-assessment of discordant cases-especially IHC ER-/TargetPrint ER+ cases- confirms TargetPrint to be a useful high quality second opinion for local IHC/FISH assessment.

Abstract OT3-4-03: Prospective neo-adjuvant registry trial linking MammaPrint, subtyping and treatment response: Neo-adjuvant Breast Registry – Symphony Trial (NBRST)

Background MammaPrint is performed on a diagnostic multi-gene array featuring >4,500 genes. This platform enables additional gene expression profiles to be analyzed simultaneously on one tumor specimen. BluePrint, an eighty gene Molecular Subtyping Profile, discriminates between three distinctive subtypes; Basal-type, Luminal-type, and ERBB2 (HER2)-type. Studies have shown marked differences in response to neo-adjuvant treatment in groups stratified by MammaPrint and BluePrint. Trial design A prospective observational, case-only study linking MammaPrint, BluePrint, TargetPrint, TheraPrint (together referred to as the Symphony suite) and possible additional profiles of interest to neoadjuvant treatment response and Distant Metastases Free Survival (DMFS) and Relapse Free Survival (RFS). 20-30 institutions in the US will be invited to contribute clinical patient data from enrolled patients after a MammaPrint, TargetPrint, BluePrint and TheraPrint (Symphony suite) has been successfully performed and the patient has started neo-adjuvant therapy. Treatment is at the discretion of the physician, adhering to NCCN approved regimens or a recognized alternative. Eligibility criteria Women with histologically proven breast cancer, who have started or are scheduled to start neo-adjuvant chemotherapy therapy or neo-adjuvant hormone therapy, after successful Symphony suite assessment Age 18–90 Written informed consent No excisional biopsy or axillary dissection No confirmed distant metastatic disease No prior therapy for the treatment of breast cancer Scope The scope of this registry study is to measure chemosensitivity as defined by pCR (primary endpoint), or endocrine sensitivity as defined by partial response, (a primary endpoint for neo-adjuvant endocrine therapy and a secondary endpoint for neoadjuvant chemotherapy), metastasis-free survival and relapse-free survival (secondary endpoints) in molecular subgroups, determined by the MammaPrint and BluePrint; as well as correlation to Targetprint and Theraprint read outs in addition to investigating novel response profiles. Statistical methods The response rate and corresponding confidence intervals will be presented as a proportion of all patients enrolled. The confidence intervals will be calculated using the normal approximation to the binomial distribution. Comparison of response rates between different molecular subgroups will be conducted using Pearson Chi-square test. Correlation of chemosensitivity and endocrine sensitivity (as defined by pCR) to TheraPrint will be determined using Pearson correlation and linear fit models. Kaplan-Meier curves for RFS and DMFS will be calculated for different molecular subgroups. Present accrual and target accrual The target accrual is to enroll approximately 500 patients in 4 years. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT2-03-02.

P3-04-06: Comparison of MammaPrint, BluePrint, and TargetPrint with Clinical Parameters in Patients with Breast Cancer: Findings from a Prospective United States Cohort.

47 Background: MammaPrint (MP) is a powerful predictor of disease outcome in early stage breast cancer. In addition, TargetPrint (TP), a microarray-based test that measures the mRNA expression level of ER, PR and HER2 and an 80 gene expression Molecular Subtyping profile BluePrint (BP) were developed. In the present study, MP, BP and TP were measured in a prospective U.S. breast cancer patient cohort. METHODS MP results were evaluated in fresh tumor samples from 127 breast cancer patients (T1-4N0-2; median age 62 [39-97 yr]) collected by core needle biopsy or from a surgical specimen between July 2008 and January 2011. We compared treatment advice as recommended by NCCN guidelines and classification according to MP. In addition, we compared IHC/FISH ER, PR and HER2 assessments with TP. The MP and BP results were used to subtype the patients into molecular subgroups. RESULTS For the group of patients (n=59) for which NCCN recommends the use of a multi-gene signature for determining chemotherapy treatment recommendations, 42 patients were classified as High Risk and 17 as Low Risk by MP. Comparison of TP with IHC/FISH indicated a concordance of 98% for ER, 94% for PR, and 98% for HER2. For a subgroup of 53 patients combined MP and BP results were available; 18 patients were Luminal-type/MP Low Risk, 27 patients were Luminal-type/MP High risk, 1 patient was Her2-type/MP Low Risk, 1 patient was Her2-type/MP High Risk and 6 patients were Basal-type/MP High Risk. CONCLUSIONS Adding the multi-gene signature MammaPrint, as well as BluePrint and TargetPrint provides additional information for treatment guidance. By combining MammaPrint with the BluePrint molecular subtyping profile, specific groups of patients can be recognized that are at high risk of recurrence and that would possibly benefit from specific treatment. This study shows that TargetPrint provides high quality second opinion for local IHC/FISH assessment.