scholarly journals Targeted deep sequencing of CD34+ cells from peripheral blood can reproduce bone marrow molecular profile in myelodysplastic syndromes

2018 ◽  
Vol 93 (6) ◽  
Author(s):  
Roman Martin ◽  
Pamela Acha ◽  
Christina Ganster ◽  
Laura Palomo ◽  
Sascha Dierks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Deep Sequencing ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Molecular Profile ◽  
Cd34 Cells ◽  
Targeted Deep Sequencing

Targeted Deep Sequencing of Peripheral CD34+ Cells can Reproduce Bone Marrow Molecular Profile in MDS Patients

2017 ◽  
Vol 55 ◽  
pp. S154
Author(s):  
P. Acha ◽  
R. Martin ◽  
L. Palomo ◽  
C. Ganster ◽  
S. Dierks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Deep Sequencing ◽  
Molecular Profile ◽  
Cd34 Cells ◽  
Targeted Deep Sequencing

scholarly journals In MDS Targeted Deep Sequencing of CD34+ Blood Cells but Not Peripheral Blood Mononuclear Cells Give Results Which Are Representative for Bone Marrow

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2383-2383
Author(s):  
Roman Martin ◽  
Laura Palomo ◽  
Christina Ganster ◽  
Pamela Acha ◽  
Sascha Dierks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Population ◽  
Deep Sequencing ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Somatic Mutations ◽  
Malignant Cell ◽  
Cd34 Cells ◽  
Reliable Detection ◽  
Targeted Deep Sequencing

Abstract Myelodysplastic syndromes (MDS) are clonal stem cell diseases of the bone marrow (BM) characterized by a dysfunction of hematopoiesis commonly resulting in cytopenias, dysplasia and an increased risk of acute myeloid leukemia (AML) development. In up to 90% of MDS cases acquired somatic mutations can be identified. Additionally, these aberrations constitute important markers for diagnosis and risk stratification. Currently, the use of BM aspirates is the gold standard for cytogenetic and molecular genetic analysis. However, frequent analyses of peripheral blood (PB) samples are of special interest for monitoring the natural course and therapy response in MDS since this might not be feasible with BM specimens due to ethical and other reasons. The aim of this study was to investigate whether the high sensitivity of targeted deep sequencing (TDS) allows reliable detection of somatic variants also from PB samples and whether the molecular profiles are comparable between these different sources of material. Additionally robustness, feasibility and comparability of the method were verified by inter-laboratory comparisons. This study included 31 patients from two centers, in Barcelona (Spain) and Göttingen (Germany) (12x RCMD, 4x RAEB-2, 4x sAML, 4x CMML-1, 2x MDS-RA, 1x MDS-U, 1x RAEB-1, 1x RCMD-SA, 1x RARS, 1x CCUS). For all patients, genomic DNA was extracted from concurrent mononuclear or total bone marrow cells (BMC), mononuclear peripheral blood cells (PBMC) and immunomagnetically (MACS) enriched circulating CD34+ cells (CD34+). Library preparation was performed using a custom hybridization-probe based panel (Nimblegen SeqCap EZ, Roche) including 83 myeloid-related genes (Barcelona) or the TruSight Myeloid Sequencing-Panel (Illumina) including 53 target genes (Göttingen). Sequencing was performed on MiSeq instruments. Reads were analyzed using local bioinformatic pipelines. Variants were filtered according to read depth (> 100x), population frequency (< 1%), their impact on protein integrity or function and evaluated by visualization on the Integrative Genome Viewer Software. Somatic origin of the variants was confirmed by sequencing of control samples from MACS enriched circulating T-lymphocytes (CD3+). Somatic mutations were discovered in 29 of the 31 analyzed patients. Overall we identified 76 aberrations in 22 genes (12x TET2, 7x SF3B1, 7x SRSF2, 5x EZH2, 5x ASXL1, 5x U2AF1, 5x RUNX1, 4x ZRSR2, 4x TP53, 3x NRAS, 3x STAG2, 2x DNMT3A, 2x IDH1, 2x BCOR, 2x KRAS, 2x PTPN11, 1x PDGFRB, 1x IKZF1, 1x IDH2, 1x ATRX, 1x CSNK1A1, 1x ETV6). Literally all variants were found in BMC (n=74) as well as in circulating CD34+ cells (n=72) and PBMC (n=73). The discordance between the three sample types was random (5x RCMD, 2x sAML, 1x MDS-RA, 1x RARS). However, for five variants the allele frequency (VAF) values, which correlate with the clone size of the malignant cell population, were below 5% in PBMC samples. Therefore these variants were likely to be overlooked in case only PBMC would have been tested. There was no significant difference (p=0.774) between the VAF values measured in BMC (average: 40.0%) and enriched CD34+ cells (average: 41.3%). In contrast VAF values of PBMC (average: 30.1%) deviate significantly from both, BMC (p=0.007) as well as circulating CD34+ cells (p=0.027) (Figure 1). Our findings indicate that TDS enables the adequate detection of somatic mutations from BM, circulating CD34+ cells and for the most part also in PBMC preparations. However, the malignant cell population seems to be less abundant in the PBMC fraction and therefore the detection and especially the quantification of clonal somatic variations is more challenging in this sample type. In the present study we demonstrate that enrichment of circulating CD34+ cells from PB can overcome this problem and provide data that are equivalent to the results obtained from the analysis of BMC, especially for follow up or minimal residual disease analyses. We conclude that enrichment of CD34+ cells from PB constitutes an appropriate alternative for the reliable detection and quantification of somatic aberrations in MDS patients. Usage of circulating CD34+ cells in routine diagnostic next generation sequencing applications would significantly reduce invasive clinical interventions and could therefore improve diagnostics and disease monitoring. Support: PI 11/02010, PI/14/00013, RD12/0036/0044, AR 14/34, R14/03, SGR225 Disclosures No relevant conflicts of interest to declare.

Adhesion receptor expression by CD34+ cells from peripheral blood or bone marrow grafts: Correlation with time to engraftment

2006 ◽  
Vol 34 (5) ◽  
pp. 680-687 ◽  
Author(s):  
Jack Gold ◽  
Helen M. Valinski ◽  
Adrianne N. Hanks ◽  
Karen K. Ballen ◽  
Chung-Cheng Hsieh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Receptor Expression ◽  
Adhesion Receptor ◽  
Cd34 Cells

Myelodysplastic syndromes/myeloproliferative overlap neoplasms

2020 ◽  
pp. 191-203
Author(s):  
Eric Padron ◽  
Tariq I. Mughal ◽  
David Sallman ◽  
Alan F. List
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Stem Cell ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Myeloproliferative Neoplasms ◽  
Bone Marrow Failure ◽  
Disease Outcomes ◽  
Genomic Landscape ◽  
Bone Marrow Dysplasia

The myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are haematologically diverse stem cell malignancies sharing phenotypic features of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) that display a paradoxical bone marrow phenotype hallmarked by myeloid proliferation in the context of bone marrow dysplasia and ineffective haematopoiesis. The unfolding MDS/MPN genomic landscape has revealed numerous mutations in signalling genes, such as CBL, JAK2, NRAS, KRAS, CSF3R, and others involving the spliceosome complex. These observations suggest that comutation of genes involved in dysplasia and bone marrow failure along with those of cytokine receptor signalling may, in part, explain the dual MDS/MPN phenotype. The respective MDS/MPN diseases are identified by the type of myeloid subset which predominates in the peripheral blood. Currently there are no standard treatment recommendations for most patients with MDS/MPN. To optimize efforts to improve the management and disease outcomes, it is essential to identify meaningful clinical and biologic endpoints and standardized response criteria for clinical trials.

scholarly journals Detection of chromosome 20q deletions in bone marrow metaphases but not peripheral blood granulocytes in patients with myeloproliferative disorders or myelodysplastic syndromes

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1561-1570 ◽  
Author(s):  
FA Asimakopoulos ◽  
TL Holloway ◽  
EP Nacheva ◽  
MA Scott ◽  
P Fenaux ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Myeloproliferative Disorders ◽  
Pcr Analysis ◽  
Multipotent Progenitors ◽  
The Common ◽  
Polymerase Chain ◽  
A Minor

Myeloproliferative disorders and myelodysplastic syndromes arise in multipotent progenitors and may be associated with chromosomal deletions that can be detected in peripheral blood granulocytes. We present here seven patients with myeloproliferative disorders or myelodysplastic syndromes in whom a deletion of the long arm of chromosome 20 was detectable by G-banding and/or fluorescence in situ hybridization in most or all bone marrow metaphases. However, in each case, microsatellite polymerase chain reaction (PCR) using 15 primer pairs spanning the common deleted region on 20q showed that the deletion was absent from most peripheral blood granulocytes. The human androgen receptor clonality assay was used to show that the vast majority of peripheral blood granulocytes were clonal in all four female patients. This represents the first demonstration that the 20q deletion can arise as a second event in patients with pre-existing clonal granulopoiesis. Microsatellite PCR analysis of whole bone marrow from two patients was consistent with cytogenetic studies, a result that suggests that cytogenetic analysis was not merely selecting for a minor subclone of cells carrying the deletion. Furthermore, in one patient, the deletion was present in both erythroid and granulocyte/monocyte colonies. This implies that the absence of the deletion in most peripheral blood granulocytes did not reflect lineage restriction of the progenitors carrying the deletion but may instead result from other selective influences such as preferential retention/destruction within the bone marrow of granulocytes carrying the deletion.

scholarly journals Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3754-3761 ◽  
Author(s):  
R Haas ◽  
B Witt ◽  
R Mohle ◽  
H Goldschmidt ◽  
S Hohaus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Lymphoblastic Leukemia ◽  
Peripheral Blood Progenitor Cell ◽  
Myelogenous Leukemia ◽  
High Dose ◽  
Platelet Recovery ◽  
Cd34 Cells

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non- Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty-four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst-forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.

Predictive factors for peripheral-blood progenitor-cell collections using a single large-volume leukapheresis after cyclophosphamide and granulocyte-macrophage colony-stimulating factor mobilization.

1995 ◽  
Vol 13 (3) ◽  
pp. 705-714 ◽  
Author(s):  
J L Passos-Coelho ◽  
H G Braine ◽  
J M Davis ◽  
A M Huelskamp ◽  
K G Schepers ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Large Volume ◽  
Peripheral Blood ◽  
High Dose ◽  
Colony Stimulating Factor ◽  
Cell Content ◽  
Cd34 Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

PURPOSE (1) To study the ability of mobilized peripheral-blood progenitor cells (PBPC) collected in a single large-volume leukapheresis performed on a predetermined date to accelerate engraftment after high-dose cyclophosphamide and thiotepa; (2) to establish the minimum dose of PBPC associated with early engraftment; and (3) to identify parameters predictive of collection of large numbers of PBPC. PATIENTS AND METHODS Twenty-three patients with breast cancer received cyclophosphamide (4 g/m2) and granulocyte-macrophage colony-stimulating factor ([GM-CSF] 5 micrograms/kg/d x 15 days) for PBPC mobilization. A single leukapheresis was performed 15 days after cyclophosphamide administration. Then, patients received high-dose cyclophosphamide and thiotepa followed by reinfusion of PBPC and 4-hydroperoxycyclophosphamide (4HC)-purged bone marrow. PBPC concentration was measured in serial peripheral-blood samples and in the leukapheresis product. Correlation analysis between PBPC dose and engraftment and between leukapheresis yield and patient characteristics was attempted. RESULTS A single leukapheresis processed a median 36 L (range, 24 to 46) blood and collected 5 x 10(6) CD34+ cells/kg (< 0.3 to 24) and 6.2 x 10(5) colony-forming units granulocyte-macrophage (CFU-GM)/kg (< 0.001 to 29). All sixteen patients (70%) reinfused with > or = 2.9 x 10(6) CD34+ cells/kg reached a level of greater than 1,000 leukocytes/microL by day 13 and greater than 50,000 platelets/microL by day 15. All of these patients had a percentage of peripheral-blood CD34+ cells > or = 0.5%, and all but one, a level of greater than 100,000 platelets/microL, on the day of leukapheresis. The bone marrow CD34+ cell percentage at study entry predicted the number of CD34+ cells collected after PBPC mobilization (R2 = .42, P = .002). All patients with > or = 2.5% bone marrow CD34+ cells experienced early engraftment. CONCLUSION Reinfusion of PBPC collected in a single leukapheresis accelerates engraftment in the majority of patients. Pretreatment bone marrow CD34+ cell content determines PBPC mobilization capacity and may help select hematopoietic rescue strategies.

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