Leonie Delahaye

Converting a breast cancer microarray signature into a high-throughput diagnostic test

BackgroundA 70-gene tumor expression profile was established as a powerful predictor of disease outcome in young breast cancer patients. This profile, however, was generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing of many samples on a routine basis.ResultsTo facilitate its use in a diagnostic setting, the 70-gene prognosis profile was translated into a customized microarray (MammaPrint) containing a reduced set of 1,900 probes suitable for high throughput processing. RNA of 162 patient samples from two previous studies was subjected to hybridization to this custom array to validate the prognostic value. Classification results obtained from the original analysis were then compared to those generated using the algorithms based on the custom microarray and showed an extremely high correlation of prognosis prediction between the original data and those generated using the custom mini-array (p < 0.0001).ConclusionIn this report we demonstrate for the first time that microarray technology can be used as a reliable diagnostic tool. The data clearly demonstrate the reproducibility and robustness of the small custom-made microarray. The array is therefore an excellent tool to predict outcome of disease in breast cancer patients.

Gene-Expression and Immunohistochemical Study of Specific T-Cell Subsets and Accessory Cell Types in the Transformation and Prognosis of Follicular Lymphoma

PURPOSE Despite the generally favorable clinical course in follicular lymphoma (FL), a minority of patients have a poor prognosis-with death within 3 years of diagnosis-most often due to transformation to aggressive disease. PATIENTS AND METHODS In this study, we analyzed the potential of predicting early transformation on the basis of gene expression and immunologic parameters in FL biopsy samples taken at diagnosis. RESULTS At the gene-expression level, FL is a highly uniform disease at the time of diagnosis, precluding the detection of sufficiently validated prognostic gene-expression profiles suitable for a clinical setting. Combinations of differentially expressed genes indicate that immunologic mechanisms play a differential role in the risk of early transformation. Using immunohistochemistry for specific cell populations, the spatial distribution to neoplastic follicles and the activation of CD4-positive T-helper cells (P = .002) and specifically T-helper 1 (P = .004) were shown to be highly discriminatory to predict early transformation. A role for functional modulation of follicular dendritic cells could also be supported (P = .04). Other cell populations, including CD68-positive macrophages and regulatory T cells, were not differentially present. CONCLUSION These results support the identification of FL as an immunologically functional disease in which an interaction of the tumor cells and the functional composition of the microenvironment determines the clinical behavior.

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.

A gene expression profile for detection of sufficient tumour cells in breast tumour tissue: microarray diagnosis eligibility.

BackgroundMicroarray diagnostics of tumour samples is based on measurement of prognostic and/or predictive gene expression profiles. Typically, diagnostic profiles have been developed using bulk tumour samples with a sufficient amount of tumour cells (usually >50%). Consequentially, a diagnostic results depends on the minimal percentage of tumour cells within a sample. Currently, tumour cell percentage is assessed by conventional histopathological review. However, even for experienced pathologists, such scoring remains subjective and time consuming and can lead to ambiguous results.MethodsIn this study we investigated whether we could use transcriptional activity of a specific set of genes instead of histopathological review to identify samples with sufficient tumour cell content. Genome-wide gene expression measurements were used to develop a transcriptional gene profile that could accurately assess a samples tumour cell percentage.ResultsSupervised analysis across 165 breast tumour samples resulted in the identification of a set of 13 genes which expression correlated with presence of tumour cells. The developed gene profile showed a high performance (AUC 0.92) for identification of samples that are suitable for microarray diagnostics. Validation on 238 additional breast tumour samples indicated a robust performance for correct classification with an overall accuracy of 91 percent and a kappa score of 0.63 (95%CI 0.47–0.73).ConclusionThe developed 13-gene profile provides an objective tool for assessment whether a breast cancer sample contains sufficient tumour cells for microarray diagnostics. It will improve the efficiency and throughput for diagnostic gene expression profiling as it no longer requires histopathological analysis for initial tumour percentage scoring. Such profile will also be very use useful for assessment of tumour cell percentage in biopsies where conventional histopathology is difficult, such as fine needle aspirates.

A Computational Workflow Translates a 58-Gene Signature to a Formalin-Fixed, Paraffin-Embedded Sample-Based Companion Diagnostic for Personalized Treatment of the BRAF-Mutation-Like Subtype of Colorectal Cancers

Colorectal cancer patients with the BRAF(p.V600E) mutation have poor prognosis in metastatic setting. Personalized treatment options and companion diagnostics are needed to better treat these patients. Previously, we developed a 58-gene signature to characterize the distinct gene expression pattern of BRAF-mutation-like subtype (accuracy 91.1%). Further experiments repurposed drug Vinorelbine as specifically lethal to this BRAF-mutation-like subtype. The aim of this study is to translate this 58-gene signature from a research setting to a robust companion diagnostic that can use formalin-fixed, paraffin-embedded (FFPE) samples to select patients with the BRAF-mutation-like subtype. BRAF mutation and gene expression data of 302 FFPE samples were measured (mutants = 57, wild-type = 245). The performance of the 58-gene signature in FFPE samples showed a high sensitivity of 89.5%. In the identified BRAF-mutation-like subtype group, 50% of tumours were known BRAF mutants, and 50% were BRAF wild-type. The stability of the 58-gene signature in FFPE samples was evaluated by two control samples over 40 independent experiments. The standard deviations (SD) were within the predefined criteria (control 1: SD = 0.091, SD/Range = 3.0%; control 2: SD = 0.169, SD/Range = 5.5%). The fresh frozen version and translated FFPE version of this 58-gene signature were compared using 170 paired fresh frozen and FFPE samples and the result showed high consistency (agreement = 99.3%). In conclusion, we translated this 58-gene signature to a robust companion diagnostic that can use FFPE samples.

Abstract 5409: Assessment of the MammaPrint 70-gene profile using RNA sequencing technology

Introduction: Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Recently, MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, was successfully translated to FFPE tissue showing to be substantially equivalent to fresh tissue. In recent years, RNA-sequencing (RNA-Seq) became the standard method for transcriptome analysis, because of its low background signal and its ability of quantifying a large dynamic range of expression levels. Here we report a preliminary analysis of the FFPE MammaPrint 70-gene profile using RNA-Seq technology and the comparison with the MammaPrint® microarray diagnostic test in a series of FFPE samples. Methods: RNA-Seq was carried out using a strand-specific RNA library preparation followed by target enrichment of the coding region of the human transcriptome without relying on the presence of poly-A tail. RNA sequencing libraries were prepared starting from a minimal amount of 20 ng of total RNA based on the DV200 metric assessment. The library pools were single-end sequenced on the Illumina HiSeq 2500 instrument at the length of 65bp. The resulting sequences were mapped to the human reference genome (build 38) using TopHat v2.1. Tophat was guided by using a transcriptome index from Ensembl (version 77). The HTSeq-count tool was used to generate the total number of uniquely mapped reads for each gene. Gene expressions were normalized with Count Per Million (CPM) normalization and log2 transformed afterwards. Microarray data of the sample were available for analysis comparison. Results: On average, we obtained 22 million reads assigned to gene per sample (min=15M, max=28M). The number of reads assigned to genes vary from 61% to 70% of the total number of reads. Between 80% and 90% of the reads assigned to genes mapped to protein coding genes which is comparable to fresh frozen material. The 70-gene signature was successfully mapped to the RNA-Seq genes. A median raw read-count of 384 was observed for the 70-gene profile among the samples. Importantly, we observed a high concordance (R2 Pearson correlation=0.97) between the MammaPrint index calculated using the RNA-Seq data and the correspondent Microarray MammaPrint index. Additionally, the BluePrint profile, a microarray diagnostic test for breast cancer molecular subtyping, was successfully translated to the RNA-Seq platform. As with the MammaPrint profile, BluePrint showed high concordance between the two technologies with high correlation values for each of the subtypes (Luminal R2 Pearson correlation=0.98, Basal R2 Pearson correlation=0.97, HER2 R2 Pearson correlation=0.77). Conclusions: Next Generation RNA-sequencing is a feasible technology to assess diagnostic signatures, such as the 70 gene MammaPrint and BluePrint profiles. Citation Format: Lorenza Mittempergher, Jacob B. Spangler, Mireille H. Snel, Leonie J. Delahaye, Iris de Rink, Sun Tian, Annuska M. Glas, Rene Bernards. Assessment of the MammaPrint 70-gene profile using RNA sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5409. doi:10.1158/1538-7445.AM2017-5409