Chromothripsis Is a Recurrent Genomic Abnormality in High-Risk Myelodysplastic Syndromes.

To explore novel genetic abnormalities occurring in myelodysplastic syndromes (MDS) through an integrative study combining array-based comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) in a series of MDS and MDS/myeloproliferative neoplasms (MPN) patients. 301 patients diagnosed with MDS (n = 240) or MDS/MPN (n = 61) were studied at the time of diagnosis. A genome-wide analysis of DNA copy number abnormalities was performed. In addition, a mutational analysis of DNMT3A, TET2, RUNX1, TP53 and BCOR genes was performed by NGS in selected cases. 285 abnormalities were identified in 71 patients (23.6%). Three high-risk MDS cases (1.2%) displayed chromothripsis involving exclusively chromosome 13 and affecting some cancer genes: FLT3, BRCA2 and RB1. All three cases carried TP53 mutations as revealed by NGS. Moreover, in the whole series, the integrative analysis of aCGH and NGS enabled the identification of cryptic recurrent deletions in 2p23.3 (DNMT3A; n = 2.8%), 4q24 (TET2; n = 10%) 17p13 (TP53; n = 8.5%), 21q22 (RUNX1; n = 7%), and Xp11.4 (BCOR; n = 2.8%), while mutations in the non-deleted allele where found only in DNMT3A (n = 1), TET2 (n = 3), and TP53 (n = 4). These cryptic abnormalities were detected mainly in patients with normal (45%) or non-informative (15%) karyotype by conventional cytogenetics, except for those with TP53 deletion and mutation (15%), which had a complex karyotype. In addition to well-known copy number defects, the presence of chromothripsis involving chromosome 13 was a novel recurrent change in high-risk MDS patients. Array CGH analysis revealed the presence of cryptic abnormalities in genomic regions where MDS-related genes, such as TET2, DNMT3A, RUNX1 and BCOR, are located.

Profound blockade of T cell activation requires concomitant inhibition of different class I PI3K isoforms.

PI3K inhibitors have emerged as potential therapeutic tools for a variety of diseases, and thus, a vast array of compounds with specificity for different PI3K isoforms is being developed. Gaining knowledge about the contribution of the different isoforms to PI3K function will allow selecting the most appropriate inhibitor for each pathology. In this study, we have addressed the effect of PI3K inhibitors with specificity for different class I PI3K isoforms on primary human T cell activation. In particular, we have analyzed proliferation, expression of activation and differentiation markers, apoptosis induction, cytokine secretion and Akt phosphorylation in T cells stimulated in vitro with anti-CD3 and anti-CD28 monoclonal antibodies and cultured with either one of these compounds: p110ß-specific inhibitor TGX-221, p110δ-specific inhibitor IC-87114, p110γ inhibitor AS-242525 or pan-class I PI3K inhibitor BKM120. Inhibition of any of the isoforms led to an impairment of T cell activation, mainly of cytokine secretion and granzyme B expression. However, only complete blockade of class I PI3K activity with the pan-class I inhibitor effectively abrogated T cell proliferation. These results indicate that these three p110 isoforms (ß, δ and γ) take part in T cell activation, but all of them are dispensable for T cell proliferation.

Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics.

Multiple myeloma is a hematological malignancy in which clonal plasma cells proliferate and accumulate within the bone marrow. The presence of osteolytic lesions due to increased osteoclast (OC) activity and suppressed osteoblast (OB) function is characteristic of the disease. The bone marrow mesenchymal stromal cells (MSCs) play a critical role in multiple myeloma pathophysiology, greatly promoting the growth, survival, drug resistance and migration of myeloma cells. Here, we specifically discuss on the relative contribution of MSCs to the pathophysiology of osteolytic lesions in light of the current knowledge of the biology of myeloma bone disease (MBD), together with the reported genomic, functional and gene expression differences between MSCs derived from myeloma patients (pMSCs) and their healthy counterparts (dMSCs). Being MSCs the progenitors of OBs, pMSCs primarily contribute to the pathogenesis of MBD because of their reduced osteogenic potential consequence of multiple OB inhibitory factors and direct interactions with myeloma cells in the bone marrow. Importantly, pMSCs also readily contribute to MBD by promoting OC formation and activity at various levels (i.e., increasing RANKL to OPG expression, augmenting secretion of activin A, uncoupling ephrinB2-EphB4 signaling, and through augmented production of Wnt5a), thus further contributing to OB/OC uncoupling in osteolytic lesions. In this review, we also look over main signaling pathways involved in the osteogenic differentiation of MSCs and/or OB activity, highlighting amenable therapeutic targets; in parallel, the reported activity of bone-anabolic agents (at preclinical or clinical stage) targeting those signaling pathways is commented.

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment.

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.

Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.

OBJECTIVE: Optimization of the mesenchymal stem cells (MSC) isolation and expansion method. MATERIALS AND METHODS: Mononuclear cells (MNC) from bone marrow aspirates were obtained by both density gradient centrifugation (standard method) and gravity sedimentation. Cells were cultured in standard conditions (10% fetal calf serum and normal oxygen tension [21% O(2)]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O(2) concentration was compared to a lower (5%) concentration (hypoxia) until the fourth cell passage. Time of expansion, number of cells obtained, morphology, cell surface markers, and differentiation potential were evaluated. RESULTS: MSC obtained by any of the different culture conditions expressed comparable immunophenotype and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. When the number of MSC obtained at fourth passage was analyzed, the highest cell numbers were obtained with gravity sedimentation isolation and PL-supplemented culture and the expansion time was the shortest when cells were cultured under hypoxic conditions. CONCLUSION: MSC isolation by MNC gravity sedimentation together with culture medium supplementation with 5% of PL in a hypoxic atmosphere (5% O(2)) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols.

Immunophenotypic analysis of myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: In contrast with hematologic malignancies in which the value of immunophenotypic studies is well established, information on the immunophenotypic characteristics of myelodysplastic syndromes (MDS) is scanty. The main goal of the present study was to explore the immunophenotypic differences between patients with MDS and normal individuals, including changes in distribution of cell lineages as well as phenotypic aberrations and blockades in cell maturation pathways. DESIGN AND METHODS: In MDS the proportion of bone marrow CD34+ cells was higher than in normal patients but the most immature progenitors (CD34+CD38-) were less represented. By contrast the proportion of myelomonocytic CD34+ cells was greater than in normal individuals, translating into an increased myeloid/non-myeloid CD34+ hematopoietic progenitor cell ratio. RESULTS: This suggests that in MDS, the majority of CD34+ cells are already committed to the myeloid lineage. Upon analyzing the granulo-monocytic differentiation pathway, MDS patients showed an increased proportion of monocytic cells with a decreased percentage of cells of neutrophil lineage, leading to a lower neutrophil/monocytic cell ratio. Maturational arrests in the monocytic but not in the neutrophil differentiation pathway were observed. In refractory anemia with excess blasts in transformation (RAEB-t) such blockades mainly occurred during the earliest stages of differentiation but in the other MDS subtypes they occurred in later stages. INTERPRETATION AND CONCLUSIONS: Phenotypic aberrations occurred in 90% of patients and a high proportion of cases showed >=2 aberrations. In summary, our results show that, in addition to an abnormal distribution of the bone marrow cell compartment, MDS patients frequently show aberrant phenotypes and maturational arrests. Some of these features may help in cases in which the diagnosis of MDS is questionable.