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Molecular subtyping is essential to infer tumor aggressiveness and predict prognosis. In practice, tumor profiling requires in‐depth knowledge of bioinformatics tools involved in the processing and analysis of the generated data. Additionally, data incompatibility (e.g., microarray versus RNA sequencing data) and technical and uncharacterized biological variance between training and test data can pose challenges in classifying individual samples. In this article, we provide a roadmap for implementing bioinformatics frameworks for molecular profiling of human cancers in a clinical diagnostic setting. We describe a framework for integrating several methods for quality control, normalization, batch correction, classification and reporting, and develop a use case of the framework in breast cancer.

Abstract Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. Here, the authors present a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques.

Abstract Axillary lymph node status is an important prognostic factor for breast cancer patients and sentinel lymph node biopsy (SLNB) is a less invasive surgical proxy. We examined if consecutively derived molecular subtypes from primary breast cancers provide additional predictive value for SLNB status. 1556 patients with a breast cancer > 10 mm underwent primary surgical procedure including SLNB and tumor specimens were assigned with a transcriptomics-based molecular subtype. 1020 patients had a negative sentinel node (SN) and 536 a positive. A significant association between tumor size and SN status (p

Breast cancer is a highly heterogeneous disease that can be classified into multiple subtypes based on the tumor transcriptome. Most of the subtyping schemes used in clinics today are derived from analyses of microarray data from thousands of different tumors together with clinical data for the patients from which the tumors were isolated. However, RNA sequencing (RNA‐Seq) is gradually replacing microarrays as the preferred transcriptomics platform, and although transcript abundances measured by the two different technologies are largely compatible, subtyping methods developed for probe‐based microarray data are incompatible with RNA‐Seq as input data. Here, we present an RNA‐Seq data processing pipeline, which relies on the mapping of sequencing reads to the probe set target sequences instead of the human reference genome, thereby enabling probe‐based subtyping of breast cancer tumor tissue using sequencing‐based transcriptomics. By analyzing 66 breast cancer tumors for which gene expression was measured using both microarrays and RNA‐Seq, we show that RNA‐Seq data can be directly compared to microarray data using our pipeline. Additionally, we demonstrate that the established subtyping method CITBCMST (Guedj et al., ), which relies on a 375 probe set‐signature to classify samples into the six subtypes basL, lumA, lumB, lumC, mApo, and normL, can be applied without further modifications. This pipeline enables a seamless transition to sequencing‐based transcriptomics for future clinical purposes.