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Background: RNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from different RNA-seq protocols and involves further evaluation of its analytical reliability using patient samples. Here, we applied two commonly used RNA-seq library preparation protocols to samples from acute leukemia patients to understand how poly-A-tailed mRNA selection (PA) and ribo-depletion (RD) based RNA-seq library preparation protocols affect gene fusion detection, variant calling, and gene expression profiling., Results: Overall, the protocols produced similar results with consistent outcomes. Nevertheless, the PA protocol was more efficient in quantifying expression of leukemia marker genes and showed better performance in the expression-based classification of leukemia. Independent qRT-PCR experiments verified that the PA protocol better represented total RNA compared to the RD protocol. In contrast, the RD protocol detected a higher number of non-coding RNA features and had better alignment efficiency. The RD protocol also recovered more known fusion-gene events, although variability was seen in fusion gene predictions., Conclusion: The overall findings provide a framework for the use of RNA-seq in a precision medicine setting with limited number of samples and suggest that selection of the library preparation protocol should be based on the objectives of the analysis.

The article discusses the two case studies investigating genetic heterogeneity between anatomically distinct bone marrow compartments in acute myeloid leukemia (AML). The results from these case studies indicate that genetic heterogeneity exists to a degree between anatomically distinct bone marrow compartments. These studies provide a single snapshoot of subclones in spatially distant sites.

Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-γ response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies. • Molecular subtypes and genetics shape immune landscape in hematological malignancies • Cytotoxic T/NK cell infiltration in MDS-like AML with TP53 mutations and ABC DLBCL • Methylation changes suppress HLA genes in AML and induce cancer antigens in myeloma • Cancer type-specific targets such as VISTA in myeloid and CD70 in lymphoid cancers Dufva et al. define the immune landscape of hematological malignancies by integrating genomic, epigenomic, and transcriptomic data and describe how this influences immune cell infiltration, antigen presentation, anti-tumor immune responses, and patient outcomes. [ABSTRACT FROM AUTHOR]