Integration of RNA Sequencing, Whole Exome Sequencing, and Flow Cytometry Into Routine Diagnostic Workup of Pediatric Lymphomas.

The study was conducted to assess the feasibility of integrating state-of-the-art sequencing techniques and flow cytometry into diagnostic workup of pediatric lymphoma. RNA sequencing (RNAseq), whole exome sequencing, and flow cytometry were implemented into routine diagnostic workup of pediatric biopsies with lymphoma in the differential diagnosis. Within 1 year, biopsies from 110 children (122 specimens) were analyzed because of suspected malignant lymphoma. The experience with a standardized workflow combining histology and immunohistochemistry, flow cytometry, and next-generation sequencing technologies is reported. Flow cytometry was performed with fresh tissue in 83% (102/122) of specimens and allowed rapid diagnosis of T-cell and B-cell non-Hodgkin lymphomas. RNAseq was performed in all non-Hodgkin lymphoma biopsies and 42% (19/45) of Hodgkin lymphoma samples. RNAseq detected all but one of the translocations found by fluorescence in situ hybridization and PCR. RNAseq and whole exome sequencing identified additional genetic abnormalities not detected by conventional approaches. Finally, 3 cases are highlighted to exemplify how synergy between different diagnostic techniques and specialists can be achieved. This study demonstrates the feasibility and discusses the added value of integrating modern sequencing techniques and flow cytometry into a workflow for routine diagnostic workup of lymphoma. The inclusion of RNA and DNA sequencing not only supports diagnostics but also will lay the ground for the development of novel research-based treatment strategies for pediatric lymphoma patients.

Prediction of response to immune checkpoint blockade in patients with metastatic colorectal cancer with microsatellite instability.

BACKGROUND: Mismatch repair-deficient (dMMR) tumors displaying microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). However, a proportion of patients with dMMR/MSI mCRC exhibit resistance to ICI. Identification of tools predicting MSI mCRC patient response to ICI is required for the design of future strategies further improving this therapy. PATIENTS AND METHODS: We combined high-throughput DNA and RNA sequencing of tumors from 116 patients with MSI mCRC treated with anti-programmed cell death protein 1 ± anti-cytotoxic T-lymphocyte-associated protein 4 of the NIPICOL phase II trial (C1, NCT03350126, discovery set) and the ImmunoMSI prospective cohort (C2, validation set). The DNA/RNA predictors whose status was significantly associated with ICI status of response in C1 were subsequently validated in C2. Primary endpoint was progression-free survival by immune RECIST (iRECIST) (iPFS). RESULTS: Analyses showed no impact of previously suggested DNA/RNA indicators of resistance to ICI, e.g. MSIsensor score, tumor mutational burden, or specific cellular and molecular tumoral contingents. By contrast, iPFS under ICI was shown in C1 and C2 to depend both on a multiplex MSI signature involving the mutations of 19 microsatellites hazard ratio cohort C2 (HRC2) = 3.63; 95% confidence interval (CI) 1.65-7.99; P = 1.4 × 10-3] and the expression of a set of 182 RNA markers with a non-epithelial transforming growth factor beta (TGFB)-related desmoplastic orientation (HRC2 = 1.75; 95% CI 1.03-2.98; P = 0.035). Both DNA and RNA signatures were independently predictive of iPFS. CONCLUSIONS: iPFS in patients with MSI mCRC can be predicted by simply analyzing the mutational status of DNA microsatellite-containing genes in epithelial tumor cells together with non-epithelial TGFB-related desmoplastic RNA markers.

Polyploidy: its consequences and enabling role in plant diversification and evolution.

BACKGROUND: Most, if not all, green plant (Virdiplantae) species including angiosperms and ferns are polyploids themselves or have ancient polyploid or whole genome duplication signatures in their genomes. Polyploids are not only restricted to our major crop species such as wheat, maize, potato and the brassicas, but also occur frequently in wild species and natural habitats. Polyploidy has thus been viewed as a major driver in evolution, and its influence on genome and chromosome evolution has been at the centre of many investigations. Mechanistic models of the newly structured genomes are being developed that incorporate aspects of sequence evolution or turnover (low-copy genes and regulatory sequences, as well as repetitive DNAs), modification of gene functions, the re-establishment of control of genes with multiple copies, and often meiotic chromosome pairing, recombination and restoration of fertility. SCOPE: World-wide interest in how green plants have evolved under different conditions - whether in small, isolated populations, or globally - suggests that gaining further insight into the contribution of polyploidy to plant speciation and adaptation to environmental changes is greatly needed. Forward-looking research and modelling, based on cytogenetics, expression studies, and genomics or genome sequencing analyses, discussed in this Special Issue of the Annals of Botany, consider how new polyploids behave and the pathways available for genome evolution. They address fundamental questions about the advantages and disadvantages of polyploidy, the consequences for evolution and speciation, and applied questions regarding the spread of polyploids in the environment and challenges in breeding and exploitation of wild relatives through introgression or resynthesis of polyploids. CONCLUSION: Chromosome number, genome size, repetitive DNA sequences, genes and regulatory sequences and their expression evolve following polyploidy - generating diversity and possible novel traits and enabling species diversification. There is the potential for ever more polyploids in natural, managed and disturbed environments under changing climates and new stresses.

Developing a sensitive HBV genotyping assay for HBV DNA suppressed patients using both DNA and RNA sequencing.

Hepatitis B virus (HBV) genotypes impact treatment outcomes and disease progression. The current genotyping methods have limitations in patients with low HBV viral load. In this study, a more sensitive assay has been developed for determining the HBV genotype in HBV DNA suppressed patients. Fifty-five serum samples from 55 chronic hepatitis B patients (HBeAg-, n = 20; HBeAg+, n = 35) across genotypes A to H with long-term nucleos(t)ide analogs (NAs) treatment were collected. All samples had HBV DNA less than 29 IU/mL. Total nucleic acid (viral DNA and RNA) was extracted and a 341 bp amplicon located at HBV S gene overlapping with reverse transcriptase domain of polymerase (pol/RT) was amplified via real time (RT)-nested polymerase chain reaction (PCR) followed by population sequencing. HBV genotype was determined by phylogenetic analysis. The assay successfully amplified HBV S/RT gene from 53 of 55 (96.4%) patient serum samples. Phylogenetic analysis demonstrated that the genotypes of all the 53 PCR positive samples matched the historical genotypes as determined by INNO-LiPA or RT sequence from the corresponding baseline samples. This assay was able to accurately determine HBV genotype irrespective of baseline genotype, HBeAg status, or duration of viral suppression. The ability to determine genotype in virally suppressed patients may facilitate the evaluation of novel treatment agents for HBV in this patient population.