Effect on Patients' Outcomes of a Change to Biosimilar Filgrastim Product in Autologous Stem Cell Mobilization.

BACKGROUND: Following addition of a biosimilar filgrastim product to the formulary, sites in the authors' provincial health authority transitioned from using the originator filgrastim to the biosimilar for autologous stem cell mobilization. OBJECTIVE: To assess the effect on patient outcomes of a universal change to use of the biosimilar filgrastim in stem cell mobilization. METHODS: This retrospective pre-post study included patients undergoing autologous stem cell mobilization at 2 cancer hospitals in Alberta, Canada, between July 1, 2018, and November 30, 2019. Clinical outcomes were investigated for patients treated with a granulocyte colony-stimulating factor (biosimilar or originator product) for mobilization before stem cell transplant, approximately 6 months before and after the defined date of product change. RESULTS: In total, 102 patients were treated with the originator product and 101 patients with the biosimilar. Effectiveness was similar between the originator and biosimilar products, with 98% successful harvest of stem cells in all patients treated. Independent t tests showed no statistically significant differences between patients receiving the originator and those receiving the biosimilar in terms of time from mobilization to collection (difference of means -0.9 days, 95% confidence interval [CI] -2.12 to 0.32), time for neutrophil engraftment (difference of means 0 days, 95% CI -0.36 to 0.36), time for platelet engraftment (difference of means 1 day, 95% CI -0.55 to 2.55), average length of stay (difference of means -0.7 day, 95% CI -2.71 to 1.31), and CD34+ value (difference of means -1 × 106/kg body weight, 95% CI -2.11 to 0.11). A 98% rate of conversion to use of the biosimilar filgrastim was achieved, with an estimated annual drug-cost saving of $67 500. CONCLUSIONS: In this pre-post study, changing to the biosimilar product from the originator maintained clinical effectiveness outcomes while decreasing overall drug expenditures. A well-planned change to the biosimilar product, executed in conjunction with clinician consultation and monitoring of effectiveness outcomes, can ensure appropriate patient therapy while significantly improving the uptake of biosimilars and decreasing expenditures for biologic drugs.

CONTEXTE: À la suite de l'ajout d'un produit filgrastim biosimilaire à la liste des médicaments, les sites relevant de l'autorité sanitaire provinciale des auteurs sont passés de l'utilisation du filgrastim princeps à la version générique pour la mobilisation des cellules souches autologues. OBJECTIF: Évaluer l'effet sur les résultats des patients d'un changement généralisé visant à utiliser le filgrastim générique pour la mobilisation des cellules souches. MÉTHODES: Cette étude rétrospective pré-post comprenait des patients soumis à une mobilisation des cellules souches autologues dans deux hôpitaux de cancérologie en Alberta (Canada) entre le 1er juillet 2018 et le 30 novembre 2019. L'examen des résultats cliniques des patients traités à l'aide d'un facteur stimulant les colonies de granulocytes (G-CSF) (générique ou princeps) pour une mobilisation avant la greffe de cellules souches a eu lieu environ six mois avant et après la date du changement de produit. RÉSULTATS: Au total, 102 patients ont été traités à l'aide du produit princeps et 101 patients à l'aide du générique. Les deux produits présentaient une efficacité similaire, et 98 % de réussite dans la récolte de cellules souches chez tous les patients traités. Des tests t indépendants n'ont montré aucune différence statistique significative entre les patients recevant le princeps et ceux recevant le biosimilaire en termes de temps allant de la mobilisation à la collecte (différence des moyennes −0,9 jour, intervalle de confiance [IC] 95 % −2,12 à 0,32); temps de la prise de la greffe neutrophile (différence des moyennes 0 jour, IC 95 % −0,36 à 0,36); temps de la prise de la greffe des plaquettes (différence des moyennes 1 jour, IC 95 % −0,55 à 2,55); durée moyenne du séjour (différence des moyennes −0,7 jour, IC 95 % −2,71 à 1,31) et valeur CD34+ (différence des moyennes −1 × 106/kg masse corporelle, IC 95 % −2,11 à 0,11). Un taux de conversion de 98 % visant à utiliser le filgrastim générique a été atteint, avec une estimation des économies annuelles sur le coût des médicaments de 67 500 $. CONCLUSIONS: Dans cette étude pré-post, le passage du produit princeps au générique a préservé l'efficacité des résultats cliniques, tout en diminuant les dépenses générales liées au médicament. Un changement bien programmé pour passer au produit générique, mené conjointement avec la consultation d'un clinicien et un contrôle des résultats d'efficacité, peut assurer une thérapie du patient appropriée tout en améliorant grandement la prise de produits génériques et en diminuant les dépenses associées aux médicaments biologiques.

Hematopoietic Stem Cell Mobilization and Homing after Transplantation: The Role of MMP-2, MMP-9, and MT1-MMP.

Hematopoietic stem/progenitor cells (HSPCs) are used in clinical transplantation to restore hematopoietic function. Here we review the role of the soluble matrix metalloproteinases MMP-2 and MMP-9, and membrane type (MT)1-MMP in modulating processes critical to successful transplantation of HSPC, such as mobilization and homing. Growth factors and cytokines which are employed as mobilizing agents upregulate MMP-2 and MMP-9. Recently we demonstrated that MT1-MMP enhances HSPC migration across reconstituted basement membrane, activates proMMP-2, and contributes to a highly proteolytic bone marrow microenvironment that facilitates egress of HSPC. On the other hand, we reported that molecules secreted during HSPC mobilization and collection, such as hyaluronic acid and thrombin, increase MT1-MMP expression in cord blood HSPC and enhance (prime) their homing-related responses. We suggest that modulation of MMP-2, MMP-9, and MT1-MMP expression has potential for development of new therapies for more efficient mobilization, homing, and engraftment of HSPC, which could lead to improved transplantation outcomes.

Membrane Type-1 Matrix Metalloproteinase Expression in Acute Myeloid Leukemia and Its Upregulation by Tumor Necrosis Factor-α.

Membrane type-1 matrix metalloproteinase (MT1-MMP) has been implicated in tumor invasion, as well as trafficking of normal hematopoietic cells, and acts as a physiologic activator of proMMP-2. In this study we examined MT1-MMP expression in primary acute myeloid leukemia (AML) cells. Because tumor necrosis factor (TNF)-α is known to be elevated in AML, we also investigated the effect of TNF-α on MT1-MMP expression. We found (i) MT1-MMP mRNA expression in 41 out of 43 primary AML samples tested; (ii) activation of proMMP-2 in co-cultures of AML cells with normal bone marrow stromal cells; and (iii) inhibition of proMMP-2 activation and trans-Matrigel migration of AML cells by gene silencing using MT1-MMP siRNA. Moreover, recombinant human TNF-α upregulated MT1-MMP expression in AML cells resulting in enhanced proMMP-2 activation and trans-Matrigel migration. Thus, AML cells express MT1-MMP and TNF-α enhances it leading to increased MMP-2 activation and most likely contributing to the invasive phenotype. We suggest that MT1-MMP, together with TNF-α, should be investigated as potential therapeutic targets in AML.

Hyaluronic acid and thrombin upregulate MT1-MMP through PI3K and Rac-1 signaling and prime the homing-related responses of cord blood hematopoietic stem/progenitor cells.

One of the hurdles of cord blood (CB) transplantation is delayed hematopoietic engraftment. Previously, we demonstrated that supernatants isolated from leukapheresis products of granulocyte-colony stimulating factor (G-CSF)-mobilized patients primed the homing of hematopoietic stem/progenitor cells (HSPC) by enhancing their chemotactic responses to stromal cell-derived factor (SDF)-1 and stimulating matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Since membrane type 1 (MT1)-MMP activates proMMP-2 and localizes proteolytic activity at the leading edge of migrating cells, in this study we investigated whether MT1-MMP contributes to the priming of the homing-related responses of CB HSPC. We found that components of supernatants of leukapheresis products such as hyaluronic acid and thrombin (i) increase the secretion of proMMP-9 and transcription and protein synthesis of MT1-MMP in CB CD34(+) cells; (ii) increase the levels of active MMP-2 in co-cultures of CD34(+) cells with endothelial cells; (iii) increase the chemoinvasion across reconstituted basement membrane Matrigel of CD34(+) cells toward a low SDF-1 gradient (20 ng/mL); and (iv) activate mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and Rac-1 signaling pathways. Inhibition of phosphatidylinositol 3-kinase and Rac-1 by their respective inhibitors LY290042 and NSC23766 attenuated MT1-MMP expression in CB CD34(+) cells, leading to reduced proMMP-2 activation and HSPC trans-Matrigel chemoinvasion toward SDF-1. Thus, our data suggest that MT1-MMP plays an important role in the homing-related responses of HSPC, and we propose that pretreatment of CB HSPC with hyaluronic acid or thrombin before transplantation could improve their homing and engraftment.

Complement C1q enhances homing-related responses of hematopoietic stem/progenitor cells.

BACKGROUND: Previously, we reported that the complement cleavage fragments C3a and C5a are important modulators of trafficking of hematopoietic stem/progenitor cells (HSPCs). The aim of this study was to examine a possible role for complement component 1, subcomponent q (C1q) in HSPC migration. STUDY DESIGN AND METHODS: CD34+ HSPCs isolated from cord blood (CB), bone marrow (BM), and granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood (mPB) were evaluated for the expression of C1q and its receptor for phagocytosis (C1qRp) using reverse transcription-polymerase chain reaction, Western blotting, and fluorescence-activated cell sorting. Chemotactic responses and chemoinvasiveness toward stromal cell-derived factor (SDF)-1 and expression of matrix metalloproteinase (MMP)-9 were also examined after C1q stimulation. Moreover, G-CSF- and zymosan-induced mobilization was evaluated in C1q-deficient mice. RESULTS: C1q was expressed in CD34+ cells from mPB, but not from CB or steady-state BM; however, stimulation of the latter with G-CSF induced C1q expression. C1qRp receptor was found on BM, CB, and mPB CD34+ cells and more mature ex vivo expanded myeloid and megakaryocytic precursors. Although C1q itself was not a chemoattractant for HSPCs, it primed/enhanced the chemotactic response of CD34+ cells to a low SDF-1 gradient and their chemoinvasion across the reconstituted basement membrane Matrigel and increased secretion of MMP-9 by these cells. Moreover, in in vivo studies C1q-deficient mice were found to be easy G-CSF mobilizers compared to wild-type mice and normal zymosan mobilizers. CONCLUSION: We demonstrated that C1q primes the responses of CD34+ HSPCs to an SDF-1 gradient, which may enhance their ability to stay within BM niches, suggesting that the C1q/C1qRp axis contributes to HSPC homing/retention in BM.

MT1-MMP association with membrane lipid rafts facilitates G-CSF--induced hematopoietic stem/progenitor cell mobilization.

OBJECTIVE: Soluble matrix metalloproteinases (MMPs) facilitate the egress of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF)-induced mobilization. Because membrane-type (MT)1-MMP, which is localized on the leading edge of migrating cells, activates the latent forms of soluble MMPs, we investigated its role in HSPC mobilization. MATERIALS AND METHODS: We examined the effect of G-CSF on the expression of MT1-MMP and its activities (proMMP-2 activation and migration) in hematopoietic cells. We also investigated the subcellular localization of MT1-MMP and the signaling pathways that regulate its expression and function in hematopoietic cells after exposure to G-CSF. RESULTS: We found that G-CSF increases MT1-MMP transcription and protein synthesis in hematopoietic cells; proMMP-2 activation in cocultures of HSPC with BM fibroblasts; chemoinvasion across reconstituted basement membrane Matrigel toward a stromal cell-derived factor-1 gradient, which is reduced by small interfering RNA silencing of MT1-MMP; and localization of MT1-MMP to membrane lipid rafts through a mechanism that is regulated by the phosphatidylinositol 3-kinase signaling pathway. Disruption of raft formation (by the cholesterol-sequestering agent methyl-beta-cyclodextrin) abrogated phosphatidylinositol 3-kinase phosphorylation and MT1-MMP incorporation into lipid rafts resulting in reduced proMMP-2 activation and HSPC migration. CONCLUSION: G-CSF-induced upregulation of MT1-MMP in hematopoietic cells and its enhanced incorporation into membrane lipid rafts contributes to proMMP-2 activation, which facilitates mobilization of HSPC.

Fifth complement cascade protein (C5) cleavage fragments disrupt the SDF-1/CXCR4 axis: further evidence that innate immunity orchestrates the mobilization of hematopoietic stem/progenitor cells.

OBJECTIVE: Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/(desArg)C5a) in human HSPC mobilization. MATERIALS AND METHODS: C5 cleavage fragments in the plasma were evaluated by enzyme-linked immunosorbent assay using human anti-(desArg)C5a antibody, and expression of the C5a/(desArg)C5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and expression of stromal cell-derived factor-1 (SDF-1)-degrading proteases that perturb retention of HSPC in bone marrow, namely matrix metalloproteinase (MMP)-9, membrane type (MT) 1-MMP, and carboxypeptidase M. RESULTS: We found that plasma levels of (desArg)C5a are significantly higher in patients who are good mobilizers and correlate with CD34(+) cell and white blood cell counts in mobilized peripheral blood. C5 cleavage fragments did not chemoattract myeloid progenitors (colony-forming unit granulocyte-macrophage), but (desArg)C5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34(+) cells, but appeared on more mature myeloid precursors, monocytes, and granulocytes. Moreover, granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells and polymorphonuclear cells had a significantly higher percentage of cells expressing CD88 than nonmobilized peripheral blood. Furthermore, C5a stimulation of granulocytes and monocytes decreased CXCR4 expression and chemotaxis toward an SDF-1 gradient and increased secretion of MMP-9 and expression of MT1-MMP and carboxypeptidase M. CONCLUSION: C5 cleavage fragments not only induce a highly proteolytic microenvironment in human bone marrow, which perturbs retention through the CXCR4/SDF-1 axis, but also strongly chemoattracts granulocytes, promoting their egress into mobilized peripheral blood, which is crucial for subsequent mobilization of HSPC.

The HGF/c-Met axis synergizes with G-CSF in the mobilization of hematopoietic stem/progenitor cells.

As granulocyte-colony-stimulating factor (G-CSF)-induced mobilization of hematopoietic stem/progenitor cells (HSPCs) increases human serum levels of hepatocyte growth factor (HGF), our aim was to investigate the role of HGF and its receptor, c-Met, in the mobilization of HSPC. CD34(+) cells and leukocytes were isolated from the bone marrow (BM) of normal donors and the peripheral blood (PB) of patients mobilized with G-CSF and chemotherapy. Plasma HGF levels were evaluated by ELISA and HGF and c-Met expression by RT-PCR, fluorescence-activated cell sorter (FACS) analysis, and confocal microscopy. Because matrix metalloproteinases (MMPs) facilitate migration across extracellular matrix (ECM) and basement membranes, we also examined expression of MMP-9 and membrane type 1 (MT1)-MMP in hematopoietic cells after HGF stimulation. We found that plasma HGF levels in mobilized (m)PB were higher in patients who are good mobilizers and correlated with their white blood cell (WBC) and CD34(+) cell counts. Moreover, HGF and c-Met expression was significantly higher in mPB CD34(+) cells and leukocytes than in their steady-state BM counterpart cells and was up-regulated by G-CSF. Like G-CSF, HGF increased the secretion of MMP-9 and the expression of MT1-MMP in leukocytes, which was abrogated by the c-Met inhibitor K-252a. This inhibitor also significantly reduced the trans-Matrigel migration of mPB CD34(+) cells toward HGF. Our results suggest that G-CSF-mediated HSPC mobilization occurs in part through the HGF/c-Met axis in HSPC and myeloid cells, eliciting increased production of matrix-degrading enzymes and subsequently facilitating egress of HSPC.

Carboxypeptidase M expressed by human bone marrow cells cleaves the C-terminal lysine of stromal cell-derived factor-1alpha: another player in hematopoietic stem/progenitor cell mobilization?

Carboxypeptidase M (CPM) is a membrane-bound zinc-dependent protease that cleaves C-terminal basic residues, such as arginine or lysine, from peptides/proteins. We examined whether CPM is expressed by hematopoietic and stromal cells and could degrade stromal cell-derived factor (SDF)-1alpha, a potent chemoattractant for hematopoietic stem/progenitor cells (HSPC). We found that (a) CPM transcript is expressed by bone marrow (BM) and mobilized peripheral blood CD34(+) cells, myeloid, erythroid, and megakaryocytic cell progenitors, mononuclear cells (MNC), polymorphonuclear cells (PMN), and stromal cells, including mesenchymal stem cells; and that (b) granulocyte-colony-stimulating factor (G-CSF) significantly increases its expression at the gene and protein levels in MNC and PMN. Moreover, we found that recombinant CPM cleaves full-length SDF-1alpha (1-68) rapidly, removing the C-terminal lysine and yielding des-lys SDF-1alpha (1-67). We demonstrated that such CPM treatment of SDF-1alpha reduced the in vitro chemotaxis of HSPC, which, however, was preserved when the CPM was exposed to the carboxypeptidase inhibitor dl-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. Thus, we present evidence that CPM is expressed by cells occurring in the BM microenvironment and that the mobilizing agent G-CSF strongly upregulates it in MNC and PMN. We suggest that cleavage of the C-terminal lysine residue of SDF-1alpha by CPM leads to attenuated chemotactic responses and could facilitate G-CSF-induced mobilization of HSPC from BM to peripheral blood.

Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient.

We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte-colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)- and bone marrow (BM)-derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.