According to WHO recommendations, diagnosis of chronic myelomonocytic leukemia (CMML) beforehand requires microscopic examination of peripheral blood to identify dysplasia and/or blasts when ...monocytes are greater or equal to 1.0 × 10
9
/L and 10% of leucocytes. We analyzed parameters derived from Sysmex
TM
XN analyzers to improve the management of microscopic examination for monocytosis. We analyzed results of the complete blood count and the positioning and dispersion parameters of polymorphonuclear neutrophils and monocytes in 61 patients presenting with CMML and 635 control patients presenting with a reactive monocytosis. We used logistic regression and multivariate analysis to define a score for smear review. Three parameters were selected: neutrophil/monocyte ratio, structural neutrophil dispersion (Ne-WX) and monocyte absolute value. We established an equation in which the threshold of 0.160 guided microscopic examination in the search for CMML abnormalities with a sensitivity of 0.967 and a specificity of 0.978 in the learning cohort (696 samples) and 0.923 and 0.936 in the validation cohort (1809 samples) respectively. We created a score for microscopic smear examination of patients presenting with a monocytosis greater or equal to 1.0 × 10
9
/L and 10% of leucocytes, improving efficiency in laboratory routine practice.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
OBJECTIVES
Animal studies have demonstrated the feasibility of tracheal replacement by silicone-stented allogenic aortas (AAs), showing mature cartilage regeneration into the grafts. In clinical ...trials, this graft did not prove stiff enough to allow long-term stent withdrawal. This graft insufficiency could be due to ischaemic phase prior to neoangiogenesis. To solve this issue, we investigated both the efficacy of the rabbit lateral thoracic fascial flap as a vehicle for revascularization of the AA and construction of a tube-shaped graft with transferable vascular pedicle, for more efficient replacement of the trachea.
METHODS
Thirty-four New Zealand rabbits were used. After harvesting of donors 'thoracic aortas', the fresh aortic allografts were transplanted within 1 h, and the others were cryopreserved. Fifteen male and four female rabbits were used as recipients for fresh (n = 9) or cryopreserved (n = 10) aortic allografts that were implanted under the skin of the chest wall, after graft wrap using a pedicled lateral thoracic fascial flap. Animal sacrifice was scheduled at regular intervals up to 61 days. Macroscopic and microscopic examinations and fluorescence in situ hybridization (FISH) were used to study the morphology, revascularization process and viability of the construct.
RESULTS
There was no operative death. Animals showed no graft rejection, despite the absence of immunosuppressive therapy. They all had a satisfactory tubular morphology of their construct. Of the 19 rabbits, 15 were found to have a generally preserved histological structure of the aorta and satisfactory neoangiogenesis. In the last four, a severe wound complication was associated with necrosis of the aortic graft. FISH on three aortic grafts with satisfactory neoangiogenesis showed migration of recipient cells into the aortic graft, decreasing from the adventitial to the luminal side, associated with the persistence of cells from the donor.
CONCLUSIONS
Our results showed that the chimeric construct transformed into a well-vascularized tube-shaped organ with transferable pedicle and some degree of stiffness. Persistence of donor's cells of normal morphology into the aortic graft was suggestive of minimal ischaemia during the initial phase of revascularization. This construct might be investigated in the setting of tracheal replacement in the rabbit model.
Approximately 30% of the patients who fulfil the criteria of Waldenström's macroglobulinemia (WM) are diagnosed while asymptomatic (indolent), and will not require immediate therapy. Conversely, ...patients with a disease-related event will be considered for therapy. The physiopathology of these 2 groups remains unclear, and the mechanisms of progression from indolent to symptomatic WM have yet to be fully understood. Seventeen patients diagnosed with WM were included in this study, 8 asymptomatic WM (A-WM) and 9 symptomatic WM (S-WM). A differential analysis was performed on a first series of 11 patients and identified 48 genes whose expression separated samples from A- to S-WM. This gene signature was then confirmed on a second independent validation set of 6 WM. Within this expression profile,
, a B-cell transcription factor known to be a tumor suppressor gene, was found to be over-expressed in A-MW relatively to S-MW. We specifically over-expressed
in a WM-related cell line and observed a significant reduction of the clonogenic activity. To the best of our knowledge, we report for the first time a specific gene expression signature that differentiates A-WM and S-WM. Within this expression profile, BACH2 was identified as a candidate gene that may help to understand better the behavior of tumor cells in indolent WM.
Mutation of the MYD88 gene has recently been identified in activated B-cell–like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and ...nuclear factor κB (NF-κB) signaling pathways. A whole exome–sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-κB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM.
•The MYD88 locus is altered in 91% of patients with WM.•MYD88 might be new target for therapeutic in WM.
Myelodysplastic syndromes (MDS) are clonal malignant stem cell disorders characterized by inefficient hematopoiesis. The role of the marrow microenvironment in the pathogenesis of the disease has ...been controversial and no study has been performed so far to characterize mesenchymal cells (MC) from MDS patients and to analyse their ability to support hematopoiesis. To this end, we have isolated and characterized MC at diagnostic marrow samples (n=12) and have purified their CD34+CD38- and CD34+CD38+ counterparts (n=7) before using MC as a short- and long-term hematopoietic support. We show that MC can be readily isolated from MDS marrow and exhibit a major expansion potential as well as an intact osteoblastic differentiation ability. They do not harbor the abnormal marker identified by FISH in the hematopoietic cells and they stimulate the growth of autologous clonogenic cells. Conversely, highly purified stem cells and their cytokine-expanded progeny harbor the clonal marker with variable frequencies, and both normal and abnormal long-term culture-initiating cell-derived progeny can be effectively supported by autologous MC. Thus, we demonstrate that MDS marrow is an abundant source of MC appearing both cytogenetically and functionally noninvolved by the malignant process and able to support hematopoiesis, suggesting their possible usefulness in future cell therapy approaches.
Abstract 297
Current models of cancer progression are based on evolution and clonal selection i.e. evolution of tumour cells inducing expansion of cells that acquire genetic lesions over time, ...related to ongoing mechanisms of genomic instability. An initiating event may be followed by gene mutation, copy number alteration (CNA) or copy neutral loss of heterozygosity (CN-LOH) that drive tumor progression and emergence of mechanisms of resistance to drugs. WM is a lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM antibody. The clinical course is characterized either by an indolent or smoldering status or a symptomatic profile that needs chemotherapy to control the progression of tumour cells. The majority of patients (pts)evolved from indolent to symptomatic, and the mechanisms of progression of WM are not fully understood to date. We hypothesized that we could gain insights into clonal evolution underlying disease progression of WM on a paired serial analysis of WM samples using genome wide SNPa, that allow both the detection of LOH and CNA.
BM samples of 19 untreated pts with WM (12 males, mean age: 67 years, 11 symptomatic pts) were analysed. All patients had Genome-Wide Human SNP Array 6.0 (Affymetrix chips) on at least two sequential tumor samples > 6 months apart (42 samples from 19 patients with two to three points). Tumoral DNA was extracted following CD19 B cells selection. Paired samples (tumor/normal T lymphocytes) were used as an intra-individual reference to identify germline polymorphisms. Size, position and location of genes were identified with UCSC Genome Browser HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix). FISH analysis was performed to detect deletion 6q; 13q14, 11q22, TP53, trisomy 4 and 12 chromosomal aberrations using Vysis probes. P53 and MYD88 mutation were analyzed by sanger sequencing.
At initial sampling, SNPa detected a total of 76 CNA genetic aberrations (range 0 – 24 per genome) including 22 gains and 54 losses; 85% of patients had MYD88 L265P mutation. During the follow-up of all indolent WM that remained indolent, we haven’t observed any new genetic aberrations, gain or loss (either CNA or CN-LOH) (n=8, mean follow-up: 63 months, range: 16–107 months). Among the 11 remaining patients (mean follow-up: 44.2 months, range: 7–92 months), a clonal evolution was observed in 6 cases. Three of them were symptomatic and acquired CNA or CN-LOH during clinical evolution: one case with acquisition of several CN-LOH, including one at 3p22 involving MYD88 locus associated to a mutation, one case with an additional deletion 7q31 which was observed in a patient with a chromothripsis at diagnosis, one case with emergence of subclonal del17p. The two remaining patients evolved from smoldering to symptomatic with a major variation in CNA number. Finally, loss of CNAs displayed at diagnosis was observed in one patient after chemotherapy and during indolent follow-up. In 5 cases, no detectable CNA changes were identified between the initial and subsequent sample at relapse.
Our results using high resolution SNP array support the hypothesis that a symptomatic WM disease will favour genetic clonal evolution of tumor cells. In our study, 3 distinct genotypic patterns were observed: (i) absence of genomic variation in stable smoldering WM disease or in symptomatic relapsed patient. (ii) unstable genotype in symptomatic or asymptomatic patients switching to symptomatic suggesting high risk tumors that are less stable and more prone to change with time. (iii) loss of abnormalities suggesting that chemotherapy eradicated the dominant clone or that a genetically distinct relapsed clone has emerged. This study refines our understanding of the dynamic genetic changes in progressive WM. Further confirmation of the role of some candidate genes is underway.
No relevant conflicts of interest to declare.
PDF
