Standardization of sequencing coverage depth in NGS: Recommendation for detection of clonal and subclonal mutations in cancer diagnostics

The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30 mutated reads for a targeted NGS mutation analysis of >= 3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.Web of Science9art. no. 85

Serum protein fingerprinting by PEA immunoassay coupled with a pattern-recognition algorithms distinguishes MGUS and multiple myeloma

Serum protein fingerprints associated with MGUS and MM and their changes in MM after autologous stem cell transplantation (MM-ASCT, day 100) remain unexplored. Using highly-sensitive Proximity Extension ImmunoAssay on 92 cancer biomarkers (Proseek Multiplex, Olink), enhanced serum levels of Adrenomedullin (ADM, P-corr=.0004), Growth differentiation factor 15 (GDF15, P-corr=.003), and soluble Major histocompatibility complex class I-related chain A (sMICA, P-corr=.023), all prosurvival and chemoprotective factors for myeloma cells, were detected in MM comparing to MGUS. Comparison of MGUS and healthy subjects revealed elevation of angiogenic and antia-poptotic midkine (P-corr=.0007) and downregulation of Transforming growth factor beta 1 (TGFB1, P-corr=.005) in MGUS. Importantly, altered serum pattern was associated with MM-ASCT compared to paired MM at the diagnosis as well as to healthy controls, namely by upregulated B-Cell Activating Factor (sBAFF) (P-corr<.006) and sustained elevation of other pro-tumorigenic factors. In conclusion, the serum fingerprints of MM and MM-ASCT were characteristic by elevated levels of prosurvival and chemoprotective factors for myeloma cells.Web of Science841694216940

Neutrophils in chronic lymphocytic leukemia are permanently activated and have functional defects

A growing body of studies highlights involvement of neutrophils in cancer development and progression. Our aim was to assess the phenotypic and functional properties of circulating neutrophils from patients with chronic lymphocytic leukemia (CLL). The percentage of CD54+ and CD64+ neutrophils as well as CD54 expression on these cells were higher in CLL patients than in age-matched healthy controls. Neutrophils from CLL produced more reactive oxygen species (ROS) compared to controls in both resting and activated conditions. Lipopolysaccharide-induced production of IL-1 beta and TNF-a as well as reduced TLR2 expression in neutrophils from CLL than in neutrophils from controls suggesting their tolerant state. Finally, phenotypic alterations of neutrophils, particularly elevation of CD64 and CD54 markers, correlated with disease activity and treatment, and low percentage of neutrophils. Taken together, the alterations in percentage and functional characteristics of neutrophils reflect the clinical course of CLL. Our data provide first evidence that neutrophils in CLL are permanently primed and have functional defects.Web of Science849849018488

High CXCR3 on leukemic cells distinguishes IgHV(mut) from IgHV(unmut) in chronic lymphocytic leukemia: Evidence from CD5(high) and CD5(low) clones

Despite the shared pattern of surface antigens, neoplastic cells in chronic lymphocytic leukemia (CLL) are highly heterogeneous in CD5 expression, a marker linked to a proliferative pool of neoplastic cells. To further characterize CD5(high) and CD5(low) neoplastic cells, we assessed the chemokine receptors (CCR5, CCR7, CCR10, CXCR3, CXCR4, CXCR5) and adhesion molecules (CD54, CD62L, CD49d) on the CD5(high)and CD5(low) subpopulations, defined by CD5/CD19 coexpression, in peripheral blood of CLL patients (n=60) subgrouped according to the IgHV mutational status (IgHV(mut),n=24;IgHV(unmut),n=36). CD5(high) subpopulation showed a high percentage of CXCR3 (P<0.001), CCR10 (P=0.001), and CD62L (P=0.031) and high levels of CXCR5 (P=0.005), CCR7 (P=0.013) compared to CD5(low) cells expressing high CXCR4 (P<0.001). Comparing IgHV(mut) and IgHV(unmut)patients, high levels of CXCR3 on CD5(high) and CD5(low) subpopulations were detected in theIgHV(mut) patients, with better discrimination in CD5(low) subpopulation. Levels of CXCR3 on CD5(low) subpopulation were associated with time to the next treatment, thus further confirming its prognostic value. Taken together, our analysis revealed higher CXCR3 expression on both CD5(high) and CD5(low) neoplastic cells inIgHV(mut) with a better prognosis compared to IgHV(unmut) patients. Contribution of CXCR3 to CLL pathophysiology and its suitability for prognostication and therapeutic exploitation deserves future investigations.Web of Science2020art. no. 708426

Whole-genome optical mapping of bone-marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities

Extramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n=4) and intramedullary MM (n=7). Large intrachromosomal rearrangements (>5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp-50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk IGH/IGK/IGL, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes.Web of Science111art. no. 1467

Deciphering the complex circulating immune cell microenvironment in chronic lymphocytic leukaemia using patient similarity networks

The tissue microenvironment in chronic lymphocytic leukaemia (CLL) plays a key role in the pathogenesis of CLL, but the complex blood microenvironment in CLL has not yet been fully characterised. Therefore, immunophenotyping of circulating immune cells in 244 CLL patients and 52 healthy controls was performed using flow cytometry and analysed by multivariate Patient Similarity Networks (PSNs). Our study revealed high inter-individual heterogeneity in the distribution and activation of bystander immune cells in CLL, depending on the bulk of the CLL cells. High CLL counts were associated with low activation on circulating monocytes and T cells and vice versa. The highest activation of immune cells, particularly of intermediate and non-classical monocytes, was evident in patients treated with novel agents. PSNs revealed a low activation of immune cells in CLL progression, irrespective of IgHV status, Binet stage and TP53 disruption. Patients with high intermediate monocytes (>5.4%) with low activation were 2.5 times more likely (95% confidence interval 1.421-4.403, P=0.002) to had shorter time-to-treatment than those with low monocyte counts. Our study demonstrated the association between the activation of circulating immune cells and the bulk of CLL cells. The highest activation of bystander immune cells was detected in patients with slow disease course and in those treated with novel agents. The subset of intermediate monocytes showed predictive value for time-to-treatment in CLL.Web of Science111art. no. 32