Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis

<p>Abstract</p> <p>Background</p> <p>Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects.</p> <p>Results</p> <p>Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status.</p> <p>Conclusion</p> <p>HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.</p

Utility Of An Algorithm For Use Of Plerixafor In Filgrastim-based Hematopoietic Progenitor Cell Mobilization In Patients With Plasma Cell Myeloma Treated With Carfilzomib-Lenalidomide-Dexamethasone

Abstract Mobilization of hematopoietic progenitor cells (HPC) for subsequent autologous transplantation is difficult in patients with plasma cell myeloma (PCM) due to poor marrow reserve. Targeted HPC yields are generally not achieved in a single apheresis procedure without use of plerixafor as a supplement to standard filgrastim. Strategies to limit use of plerixafor, due to its expense, to cases of poor CD34 mobilization have been developed, but their applicability in patients receiving the novel induction regimen carfilzomib-lenalidomide-dexamethasone (CRD) (Blood 2012;120:1801) has not been described. We prospectively studied the CD34 cell mobilization responses of PCM patients following CRD induction, using a CD34 cell predictive algorithm to determine when plerixafor should be added to the mobilization regimen. Thirty patients, including 23 with PCM and 7 with smoldering PCM, mean age 55 (range 40-72), 47% male, received 4 to 7 cycles of CRD (median, 5 cycles), with the last dose of lenalidomide given at least one week prior to mobilization. Filgrastim 10-16 mcg/kg/day was given as a single evening dose for 5 days, with circulating CD34 count assessed 12 hours after the 4th dose. The pre-apheresis CD34 count after the 5th dose of filgrastim was predicted to be 10% greater than that after the 4th dose; this prediction was validated with an actual pre-apheresis CD34 count obtained the following day. Prior mobilization data derived from healthy HPC apheresis donors was used to generate a regression formula, y=0.45x+0.86, where x=the pre-apheresis circulating CD34 count after the 5th dose of filgrastim, and y=the expected yield of the apheresis procedure, expressed as millions of CD34 cells harvested per liter processed. Targeted yield was ≥ 4 x 106 CD34 cells/kg, with minimum acceptable yield ≥ 2 x 106 CD34 cells/kg. Plerixafor 240 mcg/kg was given with the 5th dose of filgrastim, 8-10 hours prior to apheresis, if the regression equation predicted a CD34 cell yield of < 4 x 106 CD34 cells/kg in a single procedure with a maximum of 30 liters processed. The actual volume processed was based on the stat blood CD34 count drawn immediately prior to apheresis. Procedures were performed on the Cobe Spectra device; continuous intravenous calcium was used to mitigate citrate toxicity. Central lines were required in 67% of subjects. Mean CD34 cell count in the entire group after the 4th dose of filgrastim was 29/uL (range 2-88/uL). Using the regression formula as a guide, 17/30 (57%) of patients received plerixafor. CD34 counts increased 4.2-fold in patients receiving plerixafor, from 15 ± 9/uL (m ± SD) on the day prior to apheresis to 53 ± 30/uL immediately pre-apheresis; CD34 counts did not change in patients who received filgrastim alone (from 48 ± 17/uL to 45 ± 19/uL). Guided by the stat pre-apheresis CD34 count, the volume processed in the first apheresis procedure was the same, 23 ± 7 (range 12-30) liters, with or without plerixafor. CD34 cells were collected with 72 ± 14% efficiency. First-procedure CD34 cell yields were 6.4 ± 2.5 x 106/kg (range 2.5-10.1) with supplemental plerixafor vs 5.8 ± 2.5 x 106/kg (range 1.1-9.3) with filgrastim alone. Only 2/30 patients underwent a second procedure; neither received plerixafor prior to the first procedure, and both received it prior to the second. In one patient, criteria for plerixafor administration were met, but the drug was inadvertently not given prior to the first procedure; in the second patient, an unexpectedly low pre-apheresis CD34 count was traced to inadequate self-administration of the 5th dose of filgrastim. All 30 patients achieved the minimum CD34 collection goal of ≥ 2 x 106 cells/kg and 29/30 did this in one procedure. The higher targeted collection goal of ≥ 4 x 106 CD34 cells/kg was achieved in a single procedure by 76% of patients in both the plerixafor group and the filgrastim-alone group. There was a trend for higher cumulative lenalidomide and carfilzomib doses to be associated with lower CD34 mobilization responses to filgrastim. Induction treatment with CRD does not appear to impair HPC mobilization response to filgrastim in patients with PCM, compared to older regimens. An algorithm that uses the CD34 cell count after 4 doses of filgrastim to predict the following day’s pre-apheresis CD34 count and thus determine whether plerixafor supplementation is needed, was useful in identifying the 40% of CRD-treated myeloma patients who do not need plerixafor. Disclosures: No relevant conflicts of interest to declare

Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis-1

S from 5 subjects, AMD3100 (A)-mobilized CD133+ cells from 4 subjects and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells from 4 subjects. cDNA expression was analyzed using an expression microarray with 17,500 cDNA. Unsupervised hierarchical clustering of Eisen was used to analyze the 11,023 genes that remained after filtering (cDNA expressed in ≥ 80% of samples).<p><b>Copyright information:</b></p><p>Taken from "Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis"</p><p>http://www.translational-medicine.com/content/6/1/39</p><p>Journal of Translational Medicine 2008;6():39-39.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2503968.</p><p></p

A Phase 1 trial of autologous monocytes stimulated ex vivo with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for intra-peritoneal administration in recurrent ovarian cancer

Abstract Background Ovarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells. Methods Patient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response. Discussion We have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local–regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma. Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT0294842

Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare