A novel digital PCR-based method to quantify (switched) B cells reveals the extent of allelic involvement in different recombination processes in the IGH locus

Zoutman, Willem H.; Nell, Rogier J.; Versluis, Mieke; Pico, Ingrid; Vu, T. H. Khanh; Verdijk, Robert M.; Burg, Mirjam van der; Langerak, Anton W.; Velden, Pieter A. van der

doi:10.1016/j.molimm.2022.03.003

Cited by 3 publications

(4 citation statements)

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“…Table S8 ). Analysis of immunoglobulin rearrangements using droplet PCR indicated a germline configuration in hematopoietic stem cells, initiation of D H -J H rearrangements in sorted pro-B cells, while V H -(D)J H rearrangements were first observed in pre-B II large cells and class switch recombination occurred exclusively in the most mature B cells, providing an immunogenomic differentiation trajectory 25 which independently confirms our sorting strategy ( Suppl. Figures S4 and S9 ).…”

Section: Resultssupporting

confidence: 79%

“…Stage‐specific gene sets were obtained by multicomparison ANOVA on normalized counts (vst), yielding well discriminative definitions (Figure 5C; http://links.lww.com/HS/A481). Analysis of immunoglobulin rearrangements using droplet PCR indicated a germline configuration in hematopoietic stem cells, initiation of D H ‐J H rearrangements in sorted pro‐B cells, while V H ‐(D)J H rearrangements were first observed in pre‐B II large cells and class switch recombination occurred exclusively in the most mature B cells, providing an immunogenomic differentiation trajectory 25 which independently confirms our sorting strategy (http://links.lww.com/HS/A480). We implemented this newly established model of human B‐lymphopoiesis in ALLCatchR using ssGSEA to define the proximity of each BCP‐ALL sample to all 7 lymphopoiesis stages (Figure 5D; http://links.lww.com/HS/A480).…”

Section: Resultsmentioning

confidence: 99%

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The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age

Beder,

Hansen,

Hartmann

et al. 2023

HemaSphere

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Current classifications (World Health Organization-HAEM5/ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by transcriptome sequencing (RNA-Seq) is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning-based classifier using RNA-Seq gene expression data to allocate BCP-ALL samples to all 21 gene expression-defined molecular subtypes. Trained on n = 1869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n = 1018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High-confidence predictions were achieved in 83.7% of samples with 98.9% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel driver candidates in previously unassigned samples. Additional modules provided predictions of samples blast counts, patient’s sex, and immunophenotype, allowing the imputation in cases where these information are missing. We established a novel RNA-Seq reference of human B-lymphopoiesis using 7 FACS-sorted progenitor stages from healthy bone marrow donors. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory. This identified shared proximity patterns of BCP-ALL subtypes to normal lymphopoiesis stages, extending immunophenotypic classifications with a novel framework for developmental comparisons of BCP-ALL. ALLCatchR enables RNA-Seq routine application for BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocation and novel insights into underlying developmental trajectories.

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Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age

Beder,

Hansen,

Hartmann

et al. 2023

HemaSphere

View full text Add to dashboard Cite

show abstract

“…Stage specific gene sets were obtained by multi-comparison ANOVA on normalized counts (vst), yielding well discriminative definitions ( Figure 4C ; Supplementary Table S6 ). Analysis of immunoglobulin rearrangements using droplet PCR indicated initiation of D H -J H rearrangements in sorted pro-B cells while V H -(D)J H rearrangements were first observed in pre-B II Large cells and class switch recombination occurred exclusively in the most mature B cells, providing an immunogenomic differentiation trajectory 22 which independently confirms our sorting strategy ( Supplementary Figure S8 ). We implemented this newly established model of human B lymphopoiesis in ALLCatchR using ssGSEA to define the proximity of each BCP-ALL sample to all 7 lymphopoiesis stages ( Figure 4D; Supplementary Figure S9 ).…”

Section: Resultssupporting

confidence: 75%

The gene expression classifier ALLCatchR identifies B-precursor ALL subtypes and underlying developmental trajectories across age

Beder

Hansen

Hartmann

et al. 2023

Preprint

View full text Add to dashboard Cite

Current classifications (WHO-HAEM5 / ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes which are defined by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by RNA-Seq is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning based classifier using RNA-Seq expression data to allocate BCP-ALL samples to 21 defined molecular subtypes. Trained on n=1,869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n=1,018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). "High confidence predictions" were achieved in 84.6% of samples with 99.7% accuracy. Only 1.2% of samples remained "unclassified". ALLCatchR outperformed existing tools and identified novel candidates in previously unassigned samples. We established a novel RNA-Seq reference of human B-lymphopoiesis. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory, which identified shared pattenrs of proximity of BCP-ALL subtypes to normal lymphopoiesis stages. ALLCatchR sustains RNA-Seq routine application in BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocations and novel insights into underlying developmental trajectories.

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“…This indicates that a considerable proportion of the DNA present in the vitreous originated from non-malignant cells. It would be interesting to further characterise the cell type of origin of this DNA, for example by applying genetic immune cell measurements or epigenetic profiling [31][32][33][34].…”

Section: Discussionmentioning

confidence: 99%

Vitreous fluid-isolated DNA for the genetic analysis of primary uveal melanoma: a proof-of-concept study

Nell,

Versluis,

Menger

et al. 2024

Preprint

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Background Uveal melanoma is an aggressive ocular malignancy. Early molecular characterisation of primary tumours is crucial to identify those at risk of metastatic dissemination. Although tumour biopsies are being taken, liquid biopsies of ocular fluids may form a less invasive but relatively unexplored alternative. In this study, we aim to evaluate the DNA content of vitreous fluid from eyes with a uveal melanoma to obtain molecular information from the tumour. Methods DNA was isolated from 65 vitreous fluid samples from enucleated eyes with a uveal melanoma and studied using digital PCR. Primary and additional driver mutations (in GNAQ, GNA11, PLCB4, CYSLTR2, BAP1, SF3B1 and EIF1AX) were investigated using accustomed targeted and drop-off assays. The copy numbers of chromosome 3p and 8q were measured using multiplex and single-nucleotide polymorphism-based assays. Our findings were compared to the molecular profile of matched primary tumours and to the clinicopathological tumour characteristics. Results Almost all (63/65) vitreous fluids had measurable levels of DNA, but melanoma-cell derived DNA (containing the primary driver mutation) was detected in 39/65 samples (median proportion 18%, range 0.2%-94%) and was associated with a larger tumour prominence, but not with any of the molecular tumour subtypes. Among the vitreous fluids with melanoma-cell derived DNA, not all samples harboured (analysable) other mutations or had sufficient statistical power to measure copy numbers. Still, additional mutations in BAP1, SF3B1 and EIF1AX were detected in 13/15 samples and chromosome 3p and 8q copy numbers matched the primary tumour in 19/21 and 18/20 samples, respectively. Collectively, a clinically-relevant molecular classification of the primary tumour could be inferred from 27/65 vitreous fluids. Discussion This proof-of-concept study shows that substantial amounts of DNA could be detected in vitreous fluids from uveal melanoma patients, including melanoma-cell derived DNA in 60% of the samples. Prognostically-relevant genetic alterations of the primary tumour could be identified in 42% of the patients. A follow-up study is needed to evaluate our approach in a prospective clinical context.

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A novel digital PCR-based method to quantify (switched) B cells reveals the extent of allelic involvement in different recombination processes in the IGH locus

Cited by 3 publications

References 43 publications

The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age

The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age

The gene expression classifier ALLCatchR identifies B-precursor ALL subtypes and underlying developmental trajectories across age

Vitreous fluid-isolated DNA for the genetic analysis of primary uveal melanoma: a proof-of-concept study

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