Treatment‐free remission (TFR) by tyrosine kinase inhibitors (TKI) discontinuation in patients with deep molecular response (DMR) is a paramount goal in the current chronic myeloid leukemia (CML) therapeutic strategy. The best DMR level by real‐time quantitative PCR (RT‐qPCR) for TKI discontinuation is still a matter of debate. To compare the accuracy of digital PCR (dPCR) and RT‐qPCR for BCR‐ABL1 transcript levels detection, 142 CML patients were monitored for a median time of 24 months. Digital PCR detected BCR‐ABL1 transcripts in the RT‐qPCR undetectable cases. The dPCR analysis of the samples, grouped by the MR classes, revealed a significant difference between MR 4.0 and MR 4.5 ( P = 0.0104) or MR 5.0 ( P = 0.0032). The clinical and hematological characteristics of the patients grouped according to DMR classes (MR 4.0 vs MR 4.5‐5.0 ) were superimposable. Conversely, patients with dPCR values <0.468 BCR‐ABL1 copies/µL (as we previously described) showed a longer DMR duration ( P = 0.0220) and mainly belonged to MR 4.5‐5.0 ( P = 0.0442) classes compared to patients with higher dPCR values. Among the 142 patients, 111 (78%) discontinued the TKI treatment; among the 111 patients, 24 (22%) lost the MR 3.0 or MR 4.0 . RT‐qPCR was not able to discriminate patients with higher risk of MR loss after discontinuation ( P = 0.8100). On the contrary, according to dPCR, 12/25 (48%) patients with BCR‐ABL1 values ≥0.468 and 12/86 (14%) patients with BCR‐ABL1 values <0.468 lost DMR in this cohort, respectively ( P = 0.0003). Treatment‐free remission of patients who discontinued TKI with a dPCR <0.468 was significantly higher compared to patients with dPCR ≥ 0.468 (TFR at 2 years 83% vs 52% P = 0.0017, respectively). In conclusion, dPCR resulted in an improved recognition of stable DMR and of candidates to TKI discontinuation.
BackgroundDetecting somatic mutations in whole exome sequencing data of cancer samples has become a popular approach for profiling cancer development, progression and chemotherapy resistance. Several studies have proposed software packages, filters and parametrizations. However, many research groups reported low concordance among different methods. We aimed to develop a pipeline which detects a wide range of single nucleotide mutations with high validation rates. We combined two standard tools – Genome Analysis Toolkit (GATK) and MuTect – to create the GATK-LODN method. As proof of principle, we applied our pipeline to exome sequencing data of hematological (Acute Myeloid and Acute Lymphoblastic Leukemias) and solid (Gastrointestinal Stromal Tumor and Lung Adenocarcinoma) tumors. We performed experiments on simulated data to test the sensitivity and specificity of our pipeline.ResultsThe software MuTect presented the highest validation rate (90 %) for mutation detection, but limited number of somatic mutations detected. The GATK detected a high number of mutations but with low specificity. The GATK-LODN increased the performance of the GATK variant detection (from 5 of 14 to 3 of 4 confirmed variants), while preserving mutations not detected by MuTect. However, GATK-LODN filtered more variants in the hematological samples than in the solid tumors. Experiments in simulated data demonstrated that GATK-LODN increased both specificity and sensitivity of GATK results.ConclusionWe presented a pipeline that detects a wide range of somatic single nucleotide variants, with good validation rates, from exome sequencing data of cancer samples. We also showed the advantage of combining standard algorithms to create the GATK-LODN method, that increased specificity and sensitivity of GATK results. This pipeline can be helpful in discovery studies aimed to profile the somatic mutational landscape of cancer genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1190-7) contains supplementary material, which is available to authorized users.
Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.
Background Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. Methods To understand the molecular bases of aneuploid acute myeloid leukemia (A‐AML), this study examined the genomic profile in 42 A‐AML cases and 35 euploid acute myeloid leukemia (E‐AML) cases. Results A‐AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E‐AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A‐AML, which was associated with a 3‐gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A‐AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E‐AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. Conclusions These findings indicate that aneuploidy‐related and leukemia‐specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets.
Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.
The physio-pathologic interrelationships between endothelium and GvHD have been better elucidated and have led to definition of the entity 'endothelial GvHD' as an essential early phase prior to the clinical presentation of acute GvHD. Using the CellSearch system, we analyzed circulating endothelial cells (CEC) in 90 allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients at the following time-points: T1 (pre-conditioning), T2 (pre-transplant), T3 (engraftment), T4 (onset of GvHD) and T5 (1 week after steroid treatment). Although CEC changes in allo-HSCT represent a dynamic phenomenon influenced by many variables (that is, conditioning, immunosuppressive treatments, engraftment syndrome and infections), we showed that CEC peaks were constantly seen at onset of acute GvHD and invariably returned to pre-transplant values after treatment response. Since we showed that CEC changes during allo-HSCT has rapid kinetics that may be easily missed if blood samples are drawn at pre-fixed time-points, we rather suggest an 'on demand' evaluation of CEC counts right at onset of GvHD clinical symptoms to possibly help differentiate GvHD from other non-endothelial complications. We confirm that CEC changes are a suitable biomarker to monitor endothelial damage in patients undergoing allo-transplantation and hold the potential to become a useful tool to support GvHD diagnosis (ClinicalTrials.gov NCT02064972).
due to the discovery of their role in intra-cellular communications, exosomes, which carry information specific to the cell of origin, have garnered considerable attention in cancer research. Moreover, there is evidence to suggest the possibility of isolating different exosome sub-populations based on target antigens at the cell surface. Philadelphia chromosome-positive (Ph +) chronic myeloid leukemia (cML) is a clonal myeloproliferative neoplasia characterized by the breakpoint cluster region-proto-oncogene 1 tyrosine-protein kinase (BcR-ABL1) fusion-gene, derived from the t (9;22) translocation. Tyrosine kinase inhibitors (TKIs) target BcR-ABL1 protein and induce major or deep molecular responses in the majority of patients. despite the fact that several studies have demonstrated the persistence of leukemic cells in the bone marrow niche, even following treatment, TKIs prolong patient survival time and facilitate treatment-free remission. These characteristics render cML a plausible model for investigating the feasibility of tumor-derived exosome fraction enrichment. In the present study, patients in the chronic phase (cP) of CML were treated with TKIs, and the quantification of the BCR-ABL1 exosomal transcript was performed using digital PcR (dPcR). The possibility of tumor-derived exosomes enrichment was confirmed, and for the first time, to the best of our knowledge, the detection of the BcR-ABL1 transcript highlighted the presence of active leukemic cells in patients with CP-CML. According to these findings, tumor-derived exosomes may be considered a novel tool for the identification of active leukemic cells, and for the assessment of innovative monitoring focused on the biological functions of exosomes in cML.
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