BACKGROUND: Multiple myeloma (MM) represents the second most common hematological malignancy characterized by the infiltration of the bone marrow by plasma cells that produce monoclonal immunoglobulin. While the quality and length of life of MM patients have significantly increased, MM remains a hard-to-treat disease; almost all patients relapse. As MM is highly heterogenous, patients relapse at different times. It is currently not possible to predict when relapse will occur; numerous studies investigating the dysregulation of non-coding RNA molecules in cancer suggest that microRNAs could be good markers of relapse. RESULTS: Using small RNA sequencing, we profiled microRNA expression in peripheral blood in three groups of MM patients who relapsed at different intervals. In total, 24 microRNAs were significantly dysregulated among analyzed subgroups. Independent validation by RT-qPCR confirmed changed levels of miR-598-3p in MM patients with different times to relapse. At the same time, differences in the mass spectra between groups were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry. All results were analyzed by machine learning. CONCLUSION: Mass spectrometry coupled with machine learning shows potential as a reliable, rapid, and cost-effective preliminary screening technique to supplement current diagnostics.
- Publikační typ
- časopisecké články MeSH
In chronic lymphocytic leukemia, the reliability of next-generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low-VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low-VAF variants on time-to-second-treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low-VAF TP53 variants had shorter TTST and OS versus wt-TP53 patients, and the relative risk of second-line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low-VAF TP53 variants and wt-TP53 patients, while patients with high-VAF TP53 variants had inferior OS compared to wild type-TP53 cases. Altogether, NGS-based approaches are technically capable of detecting low-VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set-up. Finally, whereas low-VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment.
- Publikační typ
- časopisecké články MeSH
Large-scale next-generation sequencing (NGS) studies revealed extensive genetic heterogeneity, driving a highly variable clinical course of chronic lymphocytic leukaemia (CLL). The evolution of subclonal populations contributes to diverse therapy responses and disease refractoriness. Besides, the dynamics and impact of subpopulations before therapy initiation are not well understood. We examined changes in genomic defects in serial samples of 100 untreated CLL patients, spanning from indolent to aggressive disease. A comprehensive NGS panel LYNX, which provides targeted mutational analysis and genome-wide chromosomal defect assessment, was employed. We observed dynamic changes in the composition and/or proportion of genomic aberrations in most patients (62%). Clonal evolution of gene variants prevailed over the chromosomal alterations. Unsupervised clustering based on aberration dynamics revealed four groups of patients with different clinical behaviour. An adverse cluster was associated with fast progression and early therapy need, characterized by the expansion of TP53 defects, ATM mutations, and 18p- alongside dynamic SF3B1 mutations. Our results show that clonal evolution is active even without therapy pressure and that repeated genetic testing can be clinically relevant during long-term patient monitoring. Moreover, integrative NGS testing contributes to the consolidated evaluation of results and accurate assessment of individual patient prognosis.
BACKGROUND: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. METHODS: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. RESULTS: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).
- MeSH
- buněčné kultury metody MeSH
- chronická lymfatická leukemie * patologie metabolismus MeSH
- gely chemie MeSH
- kolagen * chemie farmakologie MeSH
- lidé MeSH
- polyethylenglykoly * chemie MeSH
- receptory CXCR4 metabolismus MeSH
- sodná sůl karboxymethylcelulosy * chemie farmakologie MeSH
- techniky 3D buněčné kultury metody MeSH
- tkáňové podpůrné struktury * chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Despite significant improvement in the survival of pediatric patients with cancer, treatment outcomes for high-risk, relapsed, and refractory cancers remain unsatisfactory. Moreover, prolonged survival is frequently associated with long-term adverse effects due to intensive multimodal treatments. Accelerating the progress of pediatric oncology requires both therapeutic advances and strategies to mitigate the long-term cytotoxic side effects, potentially through targeting specific molecular drivers of pediatric malignancies. In this report, we present the results of integrative genomic and transcriptomic profiling of 230 patients with malignant solid tumors (the "primary cohort") and 18 patients with recurrent or otherwise difficult-to-treat nonmalignant conditions (the "secondary cohort"). The integrative workflow for the primary cohort enabled the identification of clinically significant single nucleotide variants, small insertions/deletions, and fusion genes, which were found in 55% and 28% of patients, respectively. For 38% of patients, molecularly informed treatment recommendations were made. In the secondary cohort, known or potentially driving alteration was detected in 89% of cases, including a suspected novel causal gene for patients with inclusion body infantile digital fibromatosis. Furthermore, 47% of findings also brought therapeutic implications for subsequent management. Across both cohorts, changes or refinements to the original histopathological diagnoses were achieved in 4% of cases. Our study demonstrates the efficacy of integrating advanced genomic and transcriptomic analyses to identify therapeutic targets, refine diagnoses, and optimize treatment strategies for challenging pediatric and young adult malignancies and underscores the need for broad implementation of precision oncology in clinical settings.
- MeSH
- dítě MeSH
- genomika * metody MeSH
- individualizovaná medicína * metody MeSH
- kohortové studie MeSH
- kojenec MeSH
- lékařská onkologie metody MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- nádory * genetika terapie MeSH
- předškolní dítě MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Inherited thrombocytopenias (ITs) encompass a group of rare disorders characterized by diminished platelet count. Recent advancements have unveiled various forms of IT, with inherited thrombocytopenia 2 (THC2) emerging as a prevalent subtype associated with germline variants in the critical 5' untranslated region of the ANKRD26 gene. This region is crucial in regulating the gene expression of ANKRD26, particularly in megakaryocytes. THC2 is an autosomal dominant disorder presenting as mild-to-moderate thrombocytopenia with minimal symptoms, with an increased risk of myeloproliferative malignancies. In our study of a family with suspected IT, three affected individuals harbored the c.-118C>T ANKRD26 variant, while four healthy members carried the c.-140C>G ANKRD26 variant. We performed a functional analysis by studying platelet-specific ANKRD26 gene expression levels using quantitative real-time polymerase-chain reaction. Functional analysis of the c.-118C>T variant showed a significant increase in ANKRD26 expression in affected individuals, supporting its pathogenicity. On the contrary, carriers of the c.-140C>G variant exhibited normal platelet counts and no significant elevation in the ANKRD26 expression, indicating the likely benign nature of this variant. Our findings provide evidence confirming the pathogenicity of the c.-118C>T ANKRD26 variant in THC2 and suggest the likely benign nature of the c.-140C>G variant.
- MeSH
- 5' nepřekládaná oblast * MeSH
- dospělí MeSH
- genetická predispozice k nemoci MeSH
- lidé středního věku MeSH
- lidé MeSH
- mezibuněčné signální peptidy a proteiny MeSH
- rodokmen * MeSH
- trombocytopenie * genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The CYCS gene is highly evolutionarily conserved, with only a few pathogenic variants that cause thrombocytopenia-4 (THC4). Here, we report a novel CYCS variant NM_018947.6: c.59C>T [NP_061820.1:p.(Thr20Ile)] segregating with thrombocytopenia in three generations of a Czech family. The phenotype of the patients corresponds to THC4 with platelets of normal size and morphology and dominant inheritance. Intriguingly, a gradual decline in platelet counts was observed across generations. CRISPR/Cas9-mediated gene editing was used to introduce the new CYCS gene variant into a megakaryoblast cell line (MEG-01). Subsequently, the adhesion, shape, size, ploidy, viability, mitochondrial respiration, cytochrome c protein (CYCS) expression, cell surface antigen expression and caspase activity were analysed in cells carrying the studied variant. Interestingly, the variant decreases the expression of CYCS while increasing mitochondrial respiration and the expression of CD9 cell surface antigen. Surprisingly, the variant abates caspase activation, contrasting with previously known effects of other CYCS variants. Some reports indicate that caspases may be involved in thrombopoiesis; thus, the observed dysregulation of caspase activity might contribute to thrombocytopenia. The findings significantly enhance our understanding of the molecular mechanisms underlying inherited thrombocytopenia and may have implications for diagnosis, prognosis and future targeted therapies.
- MeSH
- kaspasy * metabolismus genetika MeSH
- lidé MeSH
- rodokmen MeSH
- trombocytopenie * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- chromozomální delece MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- lidské chromozomy, pár 1 genetika MeSH
- mnohočetný myelom * komplikace diagnóza MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
We performed retrospective analysis of relapsed/refractory multiple myeloma (RRMM) patients previously exposed to daratumumab treated with ixazomib, lenalidomide, dexamethasone (IRd) regimen in real clinical practice. Our aim was to evaluate efficacy of IRd in these patients and select a subset of patients that would benefit from this treatment the most. In total, we analyzed 43 daratumumab-exposed RRMM patients treated in our center. Minimal response or better was achieved by 53.5% of patients from the cohort. Median progression free survival (PFS) was 4.56 months (95% CI: 2.56, 8.03) and median overall survival (OS) was 28.92 months (95% CI: 5.4, NR). Duration of response (DOR) was evaluable in 28 patients and reached a median of 21.3 months (95% CI: 6.85, NR). Next, we evaluated hazard ratios (HR) for OS and PFS. There was improved OS in patients that were not-triple refractory or worse (HR = 0.39, 95%Cl (0.14; 1.10), p = .07) and in patients, that had less than three previous lines of treatment (LOT) (HR = 0.13, 95%Cl (0.03; 0.6) p = .003). Similar to OS, there was improved PFS in patients, that were not triple-refractory or worse (HR = 0.52, 95%Cl (0.25; 1.10), p = .08). We concluded, that the best survival benefit for RRMM patients pretreated with daratumumab to IRd regimen was observed in patients that were not triple-refractory and had less than three previous lines of treatment (LOT). The DOR in these patients was 21.3 months (95% CI: 6.85, NR).
- MeSH
- chemorezistence * MeSH
- dexamethason * aplikace a dávkování terapeutické užití MeSH
- dospělí MeSH
- glycin * analogy a deriváty aplikace a dávkování terapeutické užití MeSH
- lenalidomid * aplikace a dávkování terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- mnohočetný myelom * farmakoterapie mortalita MeSH
- monoklonální protilátky * terapeutické užití aplikace a dávkování MeSH
- opakovaná terapie MeSH
- protokoly protinádorové kombinované chemoterapie * terapeutické užití škodlivé účinky MeSH
- recidiva MeSH
- retrospektivní studie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- sloučeniny boru * aplikace a dávkování terapeutické užití MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
