Five Percent Variant Allele Frequency Is a Reliable Reporting Threshold for TP53 Variants Detected by Next Generation Sequencing in Chronic Lymphocytic Leukemia in the Clinical Setting

The clinical significance of small TP53 clones detected with next generation sequencing (NGS) in chronic lymphocytic leukemia is an issue of active debate. According to the official guidelines, treatment decisions should be guided only by variants with variant allele frequency (VAF) >= 10%. We present data on 325 consecutive patients with chronic lymphocytic leukemia analyzed with NGS. In total 47 pathogenic/likely pathogenic (P/LP), TP53 variants were detected in 26 patients (8%). Eleven of these (23%) were in the 5% to 10% VAF range and reported according to our institutional policy. All TP53 variants in the 5% to 10% VAF range were confirmed (100% concordance) with a second NGS panel. Our results where further validated with the performance of Sanger sequencing and digital droplet PCR (ddPCR). In 12 patients with available fluorescence in situ hybridization data and TP53 mutations within 5% to 10% VAF, deletion of chromosome 17p (del(17p)) was detectable in only 1 patient. We propose a robust diagnostic algorithm, which allows the safe detection and reporting of TP53 variants with VAF down to 5% in the clinical setting. Our study provides evidence that NGS is equally potent to detect variants with VAF 5% to 10% compared to those with VAF 10% to 15%, highlighting the urgent need for harmonization of NGS methodologies across diagnostic laboratories

Additional file 1: Table S1. of Loss of DIP2C in RKO cells stimulates changes in DNA methylation and epithelial-mesenchymal transition

- Primers for generation and validation of isogenic DIP2C cell lines and DIP2C overexpressing cells. Primers are indicated by their number in the text. Primers were purchased from Sigma Aldrich. Table S2. - Primers for RT-qPCR. Primer pairs for amplification of the transcript indicated by the respective name. Primers were purchased from Sigma Aldrich. Table S8. - Summary of DNA methylation analysis in DIP2C −/− cells. Table S9. - Genes with change in methylation (beta diff ≥│0.3│) at promoter sites and gene expression (log2 fold change ≥│2│) in DIP2C −/− #1-1 compared to RKO. (PDF 199 kb

Additional file 2: Table S3. of Loss of DIP2C in RKO cells stimulates changes in DNA methylation and epithelial-mesenchymal transition

- Genes with ≥4 fold change in expression in DIP2C knockout cells. Table S4. - Gene set overlap results for differentially expressed genes (≥4-fold change up or down) investigated with GSEA MSigDB Hallmarks gene set. Table S5. - Functional annotation chart for differentially expressed genes (>4-fold change up or down) investigated with the DAVID Functional annotation tool using the GO Biological process (GO_BP_FAT) annotation category. Table S6. - Genes with ≥│0.3│ units change in median promoter DNA methylation in DIP2C knockout cells. Table S7. - Genes with ≥│0.3│ units change in median gene body DNA methylation in DIP2C knockout cells. Table S10. - Gene set overlap results for promoter differentially methylated genes (≥│0.3│ change in methylation level at promoter sites in DIP2C−/− #1-1) investigated with GSEA MSigDB Hallmarks gene set. (XLSX 152 kb

Mechanistic characterization of a copper containing thiosemicarbazone with potent antitumor activity

Background: The thiosemicarbazone CD 02750 (VLX50) was recently reported as a hit compound in a phenotype-based drug screen in primary cultures of patient tumor cells. We synthesized a copper complex of VLX50, denoted VLX60, and characterized its antitumor and mechanistic properties. Materials and Methods: The cytotoxic effects and mechanistic properties of VLX60 were investigated in monolayer cultures of multiple human cell lines, in tumor cells from patients, in a 3-D spheroid cell culture system and in vivo and were compared with those of VLX50. Results: VLX60 showed >= 3-fold higher cytotoxic activity than VLX50 in 2-D cultures and, in contrast to VLX50, retained its activity in the presence of additional iron. VLX60 was effective against non-proliferative spheroids and against tumor xenografts in vivo in a murine model. In contrast to VLX50, gene expression analysis demonstrated that genes associated with oxidative stress were considerably enriched in cells exposed to VLX60 as was induction of reactive oxygen. VLX60 compromised the ubiquitin-proteasome system and was more active in BRAF mutated versus BRAF wild-type colon cancer cells. Conclusions: The cytotoxic effects of the copper thiosemicarbazone VLX60 differ from those of VLX50 and shows interesting features as a potential antitumor drug, notably against BRAF mutated colorectal cancer

Highly similar genomic landscapes in monoclonal B-cell lymphocytosis and ultra-stable chronic lymphocytic leukemia with low frequency of driver mutations

Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we per-formed whole-genome sequencing in 11 individuals with monoclonal B-cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lym phocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia

Familial platelet disorder due to germline exonic deletions in RUNX1 : a diagnostic challenge with distinct alterations of the transcript isoform equilibrium

Germline pathogenic variants in RUNX1 are associated with familial platelet disorder with predisposition to myeloid malignancies (FPD/MM) with intragenic deletions in RUNX1 accounting for almost 7% of all reported variants. We present two new pedigrees with FPD/MM carrying two different germline RUNX1 intragenic deletions. The aforementioned deletions encompass exons 1-2 and 9-10 respectively, with the exon 9-10 deletion being previously unreported. RNA sequencing of patients carrying the exon 9-10 deletion revealed a fusion with LINC00160 resulting in a change in the 3 sequence of RUNX1. Expression analysis of the transcript isoform demonstrated altered RUNX1a/b/c ratios in carriers from both families compared to controls. Our data provide evidence on the impact of intragenic RUNX1 deletions on transcript isoform expression and highlight the importance of routinely performing copy number variant analysis in patients with suspected MM with germline predisposition.Funding Agencies|Lions Cancer Research Foundation, Uppsala</p

Loss of Y and clonal hematopoiesis in blood : two sides of the same coin?

These authors contributed equally: Viktor Ljungström, Jonas Mattisson, Jan P. Dumanski, Panagiotis Baliakas, Lars A. Forsberg</p

Cell-of-origin determined by both gene expression profiling and immunohistochemistry is the strongest predictor of survival in patients with diffuse large B-cell lymphoma

The tumor cells in diffuse large B-cell lymphomas (DLBCL) are considered to originate from germinal center derived B-cells (GCB) or activated B-cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC-based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non-GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression-free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression-free survival differed significantly. Importantly, patients assigned as non-GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome

Clonal haematopoiesis as a risk factor for therapy-related myeloid neoplasms in patients with chronic lymphocytic leukaemia treated with chemo-(immuno)therapy

: Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0-6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1-5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p&nbsp;=&nbsp;0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases