ALBERT

Description: We previously suggested that a reinfused threshold dose of CD34+38− cells = 5x104/kg b.w. better predict both short- and long-term engraftment after PBSCT than total CD34+ cells assessment, and should thus avoid unecessary postransplant (Tx) G-CSF administration. Therefore, we have further conducted a prospective study comparing postTx data from cancer patients undergoing autologous PBSCT and were administered or not G-CSF depending on the amount of CD34+38− cells reinfused. 48 patients (mean age 49y) were transplanted with, on average 2.5x104 CD34+38− cells/kg b.w. (range 1–49) and were consequently administered G-CSF 5 μg/kg daily from d5 to ANC = 109/1 (Group-I). 46 patients (mean age 50y) received an average of 20.5x104 CD34+38− cells/kg (range 5.5–162) without postTx G-CSF (Group-II). These 2 groups were compared and paired two by two with 2 groups of "historical" patients referred as controls : 11 patients (mean age 44y) had received, on average, 2.5x104 CD34+38− cells/kg b.w. (range 1.1–4.8) without G-CSF (Group-III); 29 patients (mean age 51y) received an average of 15.2x5x104 CD34+38− cells/kg b.w. (range 5.5–60) systematically associated with G-CSF for protocolar reasons (Group-IV). PostTx trilineage hematopoietic engraftment (up to 2 years), clinical and economical parameters were systematically recorded for each group of patients and statistically compared. PostTx ANC recovery occurred sooner, was faster and reached higher levels in the G-CSF groups (II and IV) compared to the others; platelets recovery kinetics was significantly faster in Group-III compared to the others; reticulocytic recovery was not statistically different whichever the group. When age, sex, disease, TBI did not significantly influence trilineage engraftment, a multiparametric study showed strong positive impacts of total CD34+ cells reinfused on ANC kinetics and of CD38− subset amounts on platelet kinetics, which was on the contrary slowered by G-CSF administration. Group-I patients received more transfusions, stayed longer hospitalized and costed more than those of the 3 other groups. Regarding long-term hematopoiesis, platelets and hemoglobin levels were globally higher in Group-III compared to the other groups, but still more dramatically compared to Group-IV from 1 to 9 months, which might be explained by differences in BM CD34+ and 38− subset differentiation. In conclusion, if postTx G-CSF certainly accelerates ANC recovery, it seems to be to the detriment of short- an d median-term platelets and hemoglobin recovery, even in case of reinjection of CD34+38− cell doses = 5x104/kg b.w., which appears thus to be significantly discriminant for G-CSF administration decision.

Description: Introduction Primary myelofibrosis (PMF) is accompanied by an increase in the bloodstream circulation of some adult progenitor cells. Extramedullary hematopoiesis observed in this setting might remind some features related to foetal hematopoiesis. Material and methods We looked for evidence in favour of this hypothesis in blood samples of a small cohort of untreated patients with PMF (4 pre-fibrotic (PF) and 4 fibrotic (F), defined according to the WHO and Thiele's histopathology score (Blood, 2011)). Patient baseline characteristics are shown below. We performed a) flow-cytometric analysis for cell subsets related to VSEL, PEC, MPC, HPC; b) RT-PCR for embryonic transcriptional factors NANOG, OCT4, SOX2, LIN28 from MNC fraction (positive control hES, negative control CPRE2 c) in-vitro development of embryonic stem like cells (ESlC) under specific culture conditions. In addition we looked for SRSF2 mutations in order to better characterize PMF stages. Results As expected we detected high numbers of circulating CD34+ cells (HPC) (mean 233083±307148/ml (range 4600-783000), with similar numbers in PF- (231125±289553/ml) and F-PMF (235040 ±369156/ml). We were able to detect small numbers of the following cell subsets related to VSEL (size 2-4m) (Fig 1) Lin-/CD45-/CD34+ (mean 124±239/ml), Lin-/CD45-/CD133+ (mean 1178±971/ml), Lin-/CD45-/CXCR4+ (mean 1572±1622/ml). Lin-/CD45-CD34+AC133+CXCR4+ cells were detected in 6 of 8 patients (mean 186±375/ml) with F-PMF patients showing higher numbers (279±416/ml) than PF-PMF (63±71/ml). NANOG and OCT4 expression was detected by RT-PCR in all the patients tested. Mean OCT4 expression was about 50% the level of hES, but F-PMF showed higher levels. NANOG expression was similar to that of hES, whereas Sox2 and Lin28 were not expressed in most patients. We failed to observe the in-vitro development of ESlC in the 2 tested patients. PEC (Lin-/CD45-CD34+AC133+KDR+) were detected in all the PF-PMF (185±332/ml) and in 1 of 4 F-PMF (mean 9±18/ml). MPC (Lin-/CD45-CD90+CD105+) were detected in higher numbers in PF-PMF (mean 413±528/ml) than in F-PMF (mean 157±216/ml). We were not able to detect mutations in the hot spot of SRSF2 (codons 93,94,95). Conclusions Small numbers of cell subsets displaying morphologic and immunophenotypic features of VSELs were detected in PMF patients. However, we are not able to define these as fully specific VSELs according to previous works that defined them (Kucia, Leukemia 2006). Interestingly Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in higher numbers in F-PMF, supporting in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the foetal period. This recruitment also involves HPC. Moreover although all patients expressed OCT4 and NANOG, OCT4 expression was higher in F-PMF. As expected PEC circulate in higher numbers in PF-PMF compared to F-PMF. Interestingly both F-PMF and PF-PMF were associated to the circulation of significant numbers of MPC but higher numbers observed in PF-MFP might be interpreted either as a necessary recruitment to establish extra-medullary stroma or due to the exit from bone marrow of highly proliferative MPC. Whether all these different circulating progenitor cells, are clonally or not clonally related to the PMF pathogenesis or to unspecific mobilisation secondary to bone marrow microenvironment injury cannot be determined from these preliminary results. Disclosures: No relevant conflicts of interest to declare.

Description: Several groups, mainly from Germany and Japan, have recently conducted different phase-I clinical studies in which autologous bone marrow (BM) mononuclear cells (MNCs) were reinjected either directly in the ischemic area or intra-coronary in patients with severe post-infarct cardiac failure, resulting in a significant improvement of myocardium viability and/or reperfusion. However, besides “true” HSCs, BM-MNCs represent a mixture of mesenchymal progenitor cells, angioblasts, and maybe other progenitor cells, which does not allow to identify the type(s) of cells potentially responsible for improvement. Moreover, the obstructed coronary artery was always repermeabilized, which biases the evaluation of posttransplant myocardial reperfusion. We have personally chosen an other original approach using mobilized and purified circulating CD34+ cells. We and others have indeed demonstrated that mobilized CD34+cells were in fact subdivided into various subsets: of course, the most important (~75%) is the truly hematopoietic subset (CD34+/133+), of which a CD38− part is probably close to the very primitive HSC. But other smaller subsets are immunophenotypically characterized either as mature (CD34+/VEGFR-2+) or immature (CD34+/133+/VEGFR-2+) endothelial progenitor cells - thus potentially capable of neoangiogenesis -, or as muscle progenitors (Desmin+) and even more as cardiomyocytes (Troponin-T+). In a phase-I trial benefiting of the approval of the regional ethical committee, patients suffering of post-infarct cardiac failure are selected according to the following criteria: left ventricular ejection-fraction (LVEF) =35%; distinct area of left ventricular-wall akinesis determined by PetScan; candidates for coronary artery by-pass grafting (CABG), but without any repermeabilization of the coronary artery involved in the infarction; age =70 y. After a 6-days mobilization by G-CSF, circulating CD34+ cells are collected, then purified by immunomagnetism and immediately reinjected at d+7 during CABG, all around and within the infarcted area. The first evaluable patients well tolerated cell mobilization - and collection phases, as well as operative and post-operative periods. Three patients have presently a follow-up = 1 y post-transplantation. Two show a striking gain in LVEF (14 and 20% respectively) with an important improvement in myocardium viability, reperfusion and contractility, and finally in exercise capacity (from class IV to class I in New-York Association functional class). Although very encouraging, these results have to be confirmed in further patients.

Description: Introduction. Primary myelofibrosis (PMF) can be associated to increased bloodstream traffic of some adult progenitor cells. The extramedullary hematopoiesis developed in this setting might remind some features of the fetal hematopoiesis. Material and methods. Some evidence of this hypothesis was searched by flow-cytometry and molecular analysis of blood samples from 12 untreated patients with PMF, 6 pre-fibrotic (PF) and 6 fibrotic (F). The PF or F phase was defined according to the WHO and Thiele histopathology scores. Results. As expected we detected high numbers of CD34+ cells (258x103±402x103/ml; range 1.2-1218x103/ml) as well as small mean numbers of Lin-/CD45-CD34+AC133+CXCR4+ progenitor cells (130±275/ml; range 0-882/ml) that are related to VSELs (very-small embryonic-like stem cells), which have been described as putative pluripotent adult stem cells. When we looked at some embryonic molecular markers by RT-PCR, NANOG and OCT4 expression was detected in the MNC fraction of all the patients tested (hES and CPRE2 cell lines were the positive and negative controls). OCT4 expression was half the level of hES being stronger in F-PMF than in PF-PMF. In all the tested patients NANOG expression was similar to that of hES, whereas SOX2 and LIN28 were not expressed in most patients. Regarding other circulating progenitor cells we observed that PF-PMF had higher mean values of progenitor endothelial cells (PEC) (130±271/ml; range 0-683) than F-PMF (7±16/ml; range 0-37). Mesenchymal progenitor cells (MPC) had also higher mean values in PF-PMF (289±452/ml; range 0-1165) than in F-PMF (111±199/ml; range 0-458). Conversely, Lin-/CD45-CD34+AC133+CXCR4+ cell subset detection was higher in F-PMF (185±381/ml; range 0-882) than in PF-PMF (38±60/ml; range 0-140). Patients V617F-JAK2pos presented lower levels of PEC and MPC (16±19/ml and 43±61/ml respectively) than those V617F-JAK2neg (173±340/ml and 515±470/ml respectively). Similar numbers of Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in both V617F-JAK2pos and V617F-JAK2neg patients (133±331/ml and 123±115/ml respectively). Patients with elevated IPSS (〉3) showed higher levels of PEC, MPC and Lin-/CD45-CD34+AC133+CXCR4+ cells (respectively 180±335/ml, 334±560/ml and 256±422/ml) than those with low IPSS (