In Vitro Expanded MSCs From Patients with Myeloprliferative Neoplasms at Late Passages Show Recurrent Cytogenetic Abnormalities
Authors: Achille Valentina , Novara Francesca , Mantelli Melissa , Avanzini Maria Antonietta , Rosti Vittorio , Maccario Rita , Bernardo Maria ester , Locatelli Franco , Zecca Marco , Zuffardi Orsetta , Barosi Giovanni 
Published: October 2019

Abstract
Abstract 4101

Introduction.
Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders that include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Whereas a large body of studies has been performed on hematopoietic cells from patients with MPNs, little is known on bone marrow-derived mesenchymal stem cells (MSCs) of these patients. In particular, beside the demonstration that MPN-derived MSCs do not harbor either the JAK-2 or the MPL mutations, nothing is known about the genetics of ex vivo expanded MSCs from MPN patients. The aim of this study was to obtain data on the genetic characterization of MSCs derived from MPN patients at different in vitro culture passages.

Methods.
MSCs were isolated and expanded ex vivo, according to standard protocols from BM biopsies of 14 patients with MPNs (7 PMF, 6 ET and 1 PV). Four PMF, 3 ET and 1 PV harbored the JAK-2V617F mutation in their granulocytes. Genomic characterization of MSCs was obtained through array-comparative genomic hybridization (array-CGH) both at early and late passages (P). Array-CGH experiments were performed using the 60K platform (41.5 KB overall median probe spacing) according to the manufacturer's protocol (Agilent Technologies). Two well characterized control DNAs (NA10851 and NA 15510 Coriell for males and females, respectively) were used as reference DNAs. Results were compared to those obtained from BM-MSCs of 9 healthy donors (HD). Both patients and HDs gave informed consent for the use of their blood samples.

Results.
Until now, cultures of 5 out of 14 MPN patients reached a senescence phase at different passages, in particular 2 ET at P10 and P6, 2 PMF at P11 and P13, and 1 PV at P13, respectively. The senescence phase was defined as a decrease in MSCs proliferative capacity, finally leading to cell cycle arrest. Array-CGH analysis showed that none of MPN-MSCs expanded in vitro exhibited chromosomal abnormalities at early passages, while MPN-derived MSCs in senescence phase showed several genetic anomalies. MSCs of 2 ET patients (one with JAK-2 wild type and the other with JAK-2V617 positive hematopoiesis) did not exhibit chromosomal abnormalities even in senescence phase; while MSCs of 2 PMF and the PV patients showed genomic alterations at late passages. In particular, one PMF patient (with JAK-2 wild type hematopoiesis) presented: del(7)(pterp22.2) [≂∼f4.4Mb], del(7)(p21.3) [≂∼f1.2Mb], del(7)(p21.3p15.2) [≂∼f14.3Mb], del(7)(p12.3p12.1) [≂∼f5Mb], del(7)(q11.22) [≂∼f 1Mb] and dup(7)(q11.23qter) [≂∼f 83.5Mb]. The second PMF patient (with JAK-2 V617 positive hematopoiesis) presented a duplication of 7q22.1qter region of about 60Mb. MSCs of the PV patient (with JAK-2 V617 positive hematopoiesis) displayed chromosomal abnormalities including: del(1)(pterp34.3) [≂∼f35Mb], del(1)(q42.11q44) [≂∼f25Mb], del(3)(p21.31p11.1) [≂∼f42.5Mb], del(17)(q11.1q11.2) [≂∼f350Kb] and loss of Y chromosome. None of these abnormalities was found in the hematopoietic lineage of the patients and no correlations were found between the JAK-2 genotype of the granulocyte and the genetic abnormalities of the MSCs. Array-CGH analysis in HD did not show genomic alterations either at early or at late passages in MSCs.

Conclusion.
MPN-derived MSCs cultured in vitro show cytogenetic abnormalities at senescence which, in PMF-derived MSCs, seems to recurrently affect chromosome 7q. Interestingly, some genes, previously described to be associated with various human cancers, such as MET, BRAF, and PIK3CG map within the 7q common duplicated region of PMF- MSCs. Although previous reports have suggested that cytogenetic abnormalities can be found in MSCs derived from hematologic malignancies expanded in vitro, the finding of cytogenetic anomalies affecting the identical 7q region in 2 out of 2 patients with PMF suggests that a predisposition to DNA damage of 7q could be a peculiar feature of the disease. Array-CGH assessment of the actually ongoing cultures (5 PMF and 4 ET) will help in addressing this issue. Moreover, further studies are needed to assess whether the genetic abnormalities found in MPN-derived MSCs are acquired at late passages or derive from a small clone originally present in the MSC and expanded through the culture. Our results also raise the issue of whether bone marrow MSCs contain genomic alterations which may contribute to the pathogenesis of MPNs.

Disclosures:
No relevant conflicts of interest to declare.