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.

The significance of positron emission tomography/computed tomography (PET/CT) in chronic lymphocytic leukemia (CLL) has not yet been systematically studied. This prospective study was aimed at assessing the benefit of PET/CT in patients with newly diagnosed or relapsed CLL and Richter transformation (RT). PET/CT examination was performed in 23 patients with newly diagnosed disease, 13 with relapsed disease and eight with suspected or histopathologically confirmed RT. In all patients, the maximum standardized uptake value (SUV(max)) was calculated. The median SUV(max) was 3.4 (range: 1.5-6.3) and 3.1 (range: 1.2-5.9) in newly diagnosed and relapsed patients, respectively. The median SUV(max) of patients with suspected or confirmed RT reached 16.5 (range: 7.2-25.3), a value different from that of the previous groups (p < 0.001). 2-[18F]fluoro- 2-deoxy-D-glucose ((18)F-FDG) PET/CT revealed inflammatory lesions in seven patients (16%) and synchronous tumors in two newly diagnosed patients. (18)F-FDG PET/CT may be a beneficial imaging method when used in individuals with CLL and suspected RT.

Inhibitors of B-cell receptor signaling, ibrutinib and idelalisib, and BCL-2 antagonist, venetoclax, have become the mainstay of treatment for chronic lymphocytic leukemia (CLL). Despite significant efficacy in most CLL patients, some patients develop resistance to these agents and progress on these drugs. We provide a state-of-the-art overview of the acquired resistance to novel agents. In 80% of patients with ibrutinib failure, acquired mutations in BTK and PLCG2 genes were detected. No distinct unifying resistance-associated mutations or deregulated signaling pathways have been reported in idelalisib failure. Acquired mutations in the BCL2 gene were detected in patients who had failed on venetoclax. In most cases, patients who have progressed on ibrutinib and venetoclax experience resistance-associated mutations, often present at low allelic frequencies. Resistance-associated mutations tend to occur between the second and fourth years of treatment and may already be detected several months before clinical relapse. We also discuss the development of next-generation agents for CLL patients who have acquired resistant mutations to current inhibitors.

No abstract

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-1β 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.

Our study has confirmed the safety of individually determined administration of G-CSF. The optimal timing of G-CSF application after ASCT in patients with good-quality grafts is shortly before expected spontaneous engraftment.

BackgroundAbsolute lymphocyte count (ALC) and absolute monocyte count (AMC) have been documented as independent predictors of survival in patients with newly diagnosed Diffuse Large B-cell Lymphoma (DLBCL). Analysis of the prognostic impact of ALC and AMC in the context of International Prognostic Index (IPI) and other significant variables in elderly population treated in the R-CHOP regime has not been carried out yet.Methodology/Principal FindingsIn this retrospective study, a cohort of 443 newly diagnosed DLBCL patients with age ≥60 was analyzed. All patients were treated with the R-CHOP therapy. An extensive statistical analysis was performed to identify risk factors of 3-year overall survival (OS). In multivariate analysis, only three predictors proved significant: Eastern Cooperative Oncology Group performance status (ECOG), age and bulky disease presence. These predictors were dichotomized (ECOG ≥1, age ≥70, bulk ≥7.5) to create a novel four-level score. This score predicted 3-year OS of 94.0%, 77.4%, 62.7% and 35.4% in the low-, low-intermediate, high-intermediate and high-risk groups, respectively (P<0.001). Further, a three-level score was tested which stratifies the population better (3-year OS: 91.9%, 67.2%, 36.2% in the low, intermediate and high-risk groups, respectively) but is more difficult to interpret. Both the 3- and 4-level scores were compared to standard scoring systems and, in our population, were shown to be superior in terms of patients risk stratification with respect to 3-year OS prediction. The results were successfully validated on an independent cohort of 162 patients of similar group characteristics.ConclusionsThe prognostic role of baseline ALC, AMC or their ratio (LMR) was not confirmed in the multivariate context in elderly population with DLBCL treated with R-CHOP. The newly proposed age-specific index stratifies the elderly population into risk groups more precisely than the conventional IPI and its existing variants.

Array-based comparative genomic hybridization (arrayCGH) studies in chronic lymphocytic leukemia (CLL) have revealed novel recurrent chromosomal imbalances, such as a gain of chromosome 2p. However, a detailed cytogenetic analysis of the 2p gain region has not been elucidated. Here, we present cytogenetic and molecular cytogenetic analysis of 16 such cases selected from a group of 200 patients with CLL based on CGH and/or arrayCGH data. We revealed significant heterogeneity of the region of gain on 2p in CLL, including a new recurrent aberration: the dicentric chromosome, dic(2;18). In our cases, the region of gain involved three genes (MYCN, REL, and ALK) and was associated with an unmutated IgVH status in 14 out of 16 cases. We consider this aberration clinically important in CLL and suggest that an examination of the gene(s) located in region of gain should be included in the routine fluorescence in situ hybridization screening method used for patients with CLL.