Economic evaluation of plerixafor addition in the mobilization and leukapheresis of hematopoietic stem cells for autologous transplantation: a systematic review.

INTRODUCTION: Although plerixafor in association with granulocyte colony-stimulating factor (G-CSF) can improve mobilization and collection of hematopoietic stem cells (HSC) by leukapheresis, cost may limit its clinical application. The present study systematically reviews economic evaluations of plerixafor plus G-CSF usage compared to G-CSF alone and compares different strategies of plerixafor utilization in multiple myeloma and lymphoma patients eligible for autologous HSC transplantation. AREAS COVERED: Relevant economic evaluations, partial or complete, were searched on PubMed, Embase, LILACS, and Cochrane Central Register of Controlled Trials for a period ending 30 June 2021. This systematic review was reported following the PRISMA Statement. Six economic evaluations were included, considering the use of upfront or just-in-time plerixafor compared to G-CSF alone or other plerixafor strategies. Most comparisons showed both increased cost and health benefits with the addition of plerixafor. Most analyses favored just-in-time plerixafor compared to upfront plerixafor, with a probable preference for broader cutoffs for just-in-time plerixafor initiation. EXPERT OPINION: Plerixafor is a potentially cost-effective technology in the mobilization of HSC in patients with multiple myeloma and lymphomas eligible for autologous HSC transplantation. There is a decreased number of leukapheresis sessions and remobilizations and a higher yield of CD34+ cells.

Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo.

Current understanding suggests that malignant stem and progenitor cells must be reduced or eliminated for prolonged remissions in myeloid neoplasms such as acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Multicolor flow cytometry has been widely used to distinguish stem and myeloid progenitor cells from other populations in normal and malignant bone marrow. In this study, we present a method for assessing drug sensitivity in MDS and AML patient hematopoietic stem and myeloid progenitor cell populations ex vivo using the investigational Nedd8-activating enzyme inhibitor MLN4924 and standard-of-care agent cytarabine as examples. Utilizing a multicolor flow cytometry antibody panel for identification of hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythroid progenitors present in mononuclear cell fractions isolated from bone marrow aspirates, we compare stem and progenitor cell counts after treatment for 24 hours with drug versus diluent. We demonstrate that MLN4924 exerts a cytotoxic effect on MDS and AML stem and progenitor cell populations, whereas cytarabine has more limited effects. Further application of this method for evaluating drug effects on these populations ex vivo and in vivo may inform rational design and selection of therapies in the clinical setting. Stem Cells Translational Medicine 2017;6:840-850.

The Impact of Growth Hormone Therapy on the Apoptosis Assessment in CD34+ Hematopoietic Cells from Children with Growth Hormone Deficiency.

Growth hormone (GH) modulates hematopoietic cell homeostasis and is associated with apoptosis control, but with limited mechanistic insights. Aim of the study was to determine whether GH therapeutic supplementation (GH-TS) could affect apoptosis of CD34+ cells enriched in hematopoietic progenitor cells of GH deficient (GHD) children. CD34+ cells from peripheral blood of 40 GHD children were collected before and in 3rd and 6th month of GH-TS and compared to 60 controls adjusted for bone age, sex, and pubertal development. Next, apoptosis assessment via different molecular techniques was performed. Finally, to comprehensively characterize apoptosis process, global gene expression profile was determined using genome-wide RNA microarray technology. Results showed that GH-TS significantly reduced spontaneous apoptosis in CD34+ cells (p < 0.01) and results obtained using different methods to detect early and late apoptosis in analyzed cells population were consistent. GH-TS was also associated with significant downregulation of several members of TNF-alpha superfamily and other genes associated with apoptosis and stress response. Moreover, the significant overexpression of cyto-protective and cell cycle-associated genes was detected. These findings suggest that recombinant human GH has a direct anti-apoptotic activity in hematopoietic CD34+ cells derived from GHD subjects in course of GH-TS.

Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.

Cytological characterization of murine bone marrow and spleen hematopoietic compartments for improved assessment of toxicity in preclinical gene marking models.

Gene therapy has proven its potential to cure diseases of the hematopoietic system, but potential adverse reactions related to insertional mutagenesis by integrating gene vectors and chromosomal instability in long-lived repopulating cells have emerged as a major limitation. Preclinical gene therapy in murine models is a powerful model for assessment of gene marking efficiency and adverse reactions. However, changes in the hematologic composition after transplantation with retrovirally modified hematopoietic stem cells have not been well investigated in large cohorts of animals by systematic cytological analyses. In the present study, cytological analyses of bone marrow and spleen were performed in a large cohort (n = 58) of C57BL/6J mice over an extended observation period after gene marking. Interestingly, we observed hematological malignancies in four out of 30 animals transplanted with dLNGFR (truncated form of the human p75 low-affinity nerve growth factor receptor) and tCD34 modified stem/progenitor cells. Our data demonstrate that cytological analysis provides important information for diagnosis of hematological disorders and thus should be included in preclinical studies and performed in each investigated animal. Together with histological analysis, flow cytometric analysis, and other analyses, the quality and predictive value of preclinical gene therapy studies will be improved.

Damage control and its costs: BM failure in Fanconi anemia stems from overactive p53/p21.

Despite having well-characterized disease-associated mutations, the mechanisms underlying the progressive bone marrow failure and cancer susceptibility of Fanconi anemia have been unclear. In this issue of Cell Stem Cell, Ceccaldi et al. identify an overactive p53/p21 stress response and cell cycle arrest as an underlying cause that starts during fetal development.

Implementation of peripheral blood CD34 analyses to initiate leukapheresis: marked reduction in resource utilization.

BACKGROUND: Analysis of the peripheral blood (PB) C34 value may determine the optimal time to initiate leukapheresis. STUDY DESIGN AND METHODS: After selecting a threshold PB CD34 value of five CD34 + cells per microL to initiate leukapheresis procedure, a prospective analysis of 50 consecutive patients was initiated to identify the optimal time to initiate leukapheresis and its impact on costs and resource utilization. Clinical decisions were made to commence or to postpone leukapheresis with this PB CD34 threshold number. Based on PB CD34 values for each patient, the number of leukapheresis procedures, postponed or canceled, the number of CD34+ cells per kg, and the total number of cells collected were identified. Costs of mobilization were obtained from the hospital cost accounting system. RESULTS: In 13 months, 50 patients with a hematologic disorder underwent mobilization. There were 34 cancellations or postponements of collections due to a low PB CD34 value in 13 patients. By use of our identified costs per initial collection, this resulted in a savings of 67,660 US dollars. CONCLUSIONS: This prospective study defines how the implementation of the PB CD34 value results in costs savings. A low PB CD34 value canceled or postponed a significant number of leukapheresis procedures, resulting in a substantial cost savings. Use of the PB CD34 value should be the standard of care during mobilization and peripheral blood progenitor cell collection.

The assessment of proliferating cell nuclear antigen immunostaining in myelodysplastic syndromes and its prognostic significance.

Several prognostic factors for patients with myelodysplastic syndromes (MDS) have been identified in previous years. In order to determine prognostic factors characterizing haematopoietic cell kinetics, bone marrow proliferative activity and serum TNF-a levels were measured in 51 cases of MDS. Cell proliferation was evaluated by employing a monoclonal antibody directed against the proliferating cell nuclear antigen (PCNA). The PCNA proliferating index (PCNA PI) and serum TNF-a levels showed significant differences between patients with MDS and normal controls (p<0.0001). PCNA PI and serum TNF-a were significantly higher in the high risk for leukemic transformation FAB subgroups (RAEB, RAEB-t and CMML) in comparison to the low risk group (RA and RARS) (p<0.001). PCNA PI and TNF-a also increased with increasing IPSS score (p<0.05). A positive correlation was noted between TNF-a concentrations and PCNA PI (r:0.36, p<0.008). Univariate analysis using the log-rank test showed that a higher PCNA PI was associated with a significantly shorter survival (p<0.001). We conclude that elevated PCNA PI and TNF-a serum levels are increased in high risk myelodysplastic disease and that a high PCNA PI is predictive of a shorter survival in this group of patients.

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

BACKGROUND AND OBJECTIVES: The percentages of CD34+ cells in the bone marrow of patients with acute myeloid leukemia (AML) vary widely. Especially in the low range (<5% CD34+ cells), the nature (normal or malignant) of the CD34+ cells is uncertain. Since only in a minority of cases are molecular techniques applicable, in this study we explored a multiparameter approach using phenotypic and functional characteristics to discriminate normal CD34+ cells from malignant ones. DESIGN AND METHODS: CD34+ cells from 24 AML patients with <5% CD34+ cells and from 3 patients with >50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants.

Assessment of CD24 expression on bone marrow neutrophilic granulocytes: CD24 is a marker for the myelocytic stage of development.

The understanding of normal surface maturation pattern of granulocytes plays an essential role in identifying abnormal patterns, which may be of diagnostic or pathogenetic significance in some disorders, such as myclodysplastic syndromes. CD24, which is expressed only on granulocytes and not on monocytes, has not been adequately studied. Surface marker studies on eight control marrows indicate that CD24 is expressed on bone marrow granulocytes in a pattern that is similar to CD11b while much different than the more mature markers, CD16b and CD35. Three sorting experiments show that the majority of CD24(-) cells are promyelocytes. We conclude that CD24 is expressed on the neutrophilic granulocytes at the stage of myelocytes, and hence it could potentially be used to study normal and abnormal maturation of granulocytes.

Efficacy and toxicity of a virus-directed enzyme prodrug therapy purging method: preclinical assessment and application to bone marrow samples from neuroblastoma patients.

Autologous stem cell transplantation is used to rescue cancer patients from myelosuppression caused by high-dose chemotherapy. However, autologous grafts often contain tumor cells that can contribute directly to relapse. Current purging methods are useful when fewer than 1% tumor cells contaminate the bone marrow, and patients with tumor burdens of >1% are considered ineligible for chemotherapy that necessitates stem cell rescue. Using neuroblastoma (NB) as a model system, we developed a method that is effective even with tumor burdens of 10-25%. Mixtures of NB-1691 NB cells and CD34(+) hematopoietic cells purged by this method showed no evidence of viable tumor cells as assessed by clonogenic assays or reverse transcription-PCR for the NB cell markers tyrosine hydroxylase and N-MYC. The efficacy and lack of toxicity of the method were verified using in vivo mouse models. Severe combined immunodeficient mice that received purged cell preparations containing 10% NB-1691 cells survived without evidence of disease for the observation period (>1 year), whereas mice that received unpurged cells developed disseminated disease requiring euthanasia 73-96 days after injection of cells. No evidence of toxicity to the mice was detected by numerous laboratory values for bone marrow, liver, and kidney function, and no difference was seen in the ability of purged cell mixtures versus unmanipulated CD34(+) cells to reconstitute the marrow of non-obese diabetic severe combined immunodeficient mice. In a pilot study, marrow was obtained from eight patients who had >/=1% metastatic tumor burden. All eight samples were purged to the level of detection by reverse transcription-PCR (samples from seven patients) or clonogenic potential (sample from one patient), whichever assay was used. The described adenovirus/rabbit carboxylesterase/CPT-11 (irinotecan, 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) virus-directed enzyme prodrug method may be useful for patients whose tumor burdens exceed 1% at the time of stem cell harvest. Assessment of purging efficacy with additional samples from NB patients is ongoing.

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with severe congenital neutropenia.

OBJECTIVE: To investigate further the cellular defect responsible for impaired granulopoiesis in severe congenital neutropenia (SCN), we have evaluated bone marrow (BM) stem cell reserve and function and BM stromal cell myelopoiesis supporting capacity in two patients with SCN. METHODS: BM primitive stem cells and myeloid progenitor cells were assessed using flow cytometry, limiting dilution assay, clonogenic assays, and long-term BM cultures (LTBMC). BM stroma function was assessed by evaluating the ability of irradiated stromal layers from the patients to induce granulocyte-macrophage colony formation (CFU-GM) by normal CD34+ cells. RESULTS: Compared to the normal controls (n = 37), SCN patients displayed a low percentage of CD34+/CD38+ cells (P < 0.05), low CFU-GM colony formation by highly purified CD34+ cells (P < 0.05), low CFU-GM recovery in LTBMC (P < 0.05), and normal primitive stem cells as indicated by the frequency of CD34+/CD38- cells and the number of long-term culture initiating cells. Patient BM stromal layers exhibited normal myelopoiesis supporting capacity as shown by the CFU-GM content of irradiated LTBMC recharged with normal CD34+ cells. In addition, patient LTBMC supernatants displayed 20-fold normal granulocyte colony stimulating factor and 2-fold normal granulocyte-macrophage colony stimulating factor levels. CONCLUSION: These data show that primitive BM stem cells and stromal cells are not affected in SCN patients, while they support further the concept of a primary defect at the myeloid progenitor cell level. To know the differentiation stage at which the underlying defect causes the malfunction will be relevant for further elucidation of its nature at the molecular level.

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with autoimmune cytopenias.

To investigate whether bone marrow (BM) stem cell compartment and/or BM microenvironment are affected by the immune insult in autoimmune cytopenias (AICs), BM stem cell reserve and function and BM stromal function were studied in 15 AIC patients. Stem cells were evaluated by means of flow cytometry, clonogenic progenitor cell assays, long-term BM cultures (LTBMCs), and limiting dilution assay for quantification of long-term-culture initiating cells (LTC-ICs). Stromal cell function was assessed with the use of preformed irradiated LTBMCs from patients and normal controls, recharged with normal CD34(+) cells. AIC patients exhibited a high number of CD34(+), CD34(+)/CD38(+), and CD34(+)/CD38(-) cells; high frequency of granulocyte-macrophage colony forming units in the BM mononuclear cell fraction; high colony recovery in LTBMCs; and normal LTC-IC frequency. Patient BM stromal layers displayed normal hematopoietic-supporting capacity and increased production of granulocyte-colony stimulating factor. Data from this study support the concept that AIC patients with severe, resistant disease might be appropriate candidates for autologous stem cell transplantation.

Hematopoietic growth factors in the pathogenesis and for the treatment of aplastic anemia.

The production and release of hematopoietic growth factors from bone marrow stromas established in vitro from patients with aplastic anemia is normal or increased. Addition of hematopoietic growth factors to aplastic anemia bone marrow cells results in only modest increases in colony growth, with the exception of granulocyte colony-stimulating factor (G-CSF), which corrects their impaired cloning efficiency to normal. Most clinical data on the use of hematopoietic growth factors in aplastic anemia have derived from uncontrolled and small single-arm studies or case reports. Sustained trilineage hematologic responses have not been observed when hematopoietic growth factors have been used alone or in combination. Serious side effects have been reported for most of the hematopoietic growth factors in patients with aplastic anemia, with the exception of G-CSF. There is a major concern that they may further increase the risk of clonal disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Hematopoietic growth factors should not be used alone in newly diagnosed patients as specific treatment for aplastic anemia, and their use in combination with immunosuppressive therapy should be confined to multicenter, prospective randomized studies.

Seeding efficiency of primitive human hematopoietic cells in nonobese diabetic/severe combined immune deficiency mice: implications for stem cell frequency assessment.

Nonobese diabetic/severe combined immune deficiency (NOD/SCID) mouse repopulating cells (SRC) have been proposed to represent a more primitive human stem cell subset than the cobblestone area-forming cell (CAFC) week (wk) 6 or the long-term culture-initiating cell (LTC-IC) wk 5 on the basis of their difference in frequency, phenotype, transfectibility, and multilineage outgrowth potential in immunodeficient recipients. We have assessed the percentage of various progenitor cell populations (colony-forming cell [CFC] and CAFC subsets) contained in unsorted NOD/SCID BM nucleated cells (nc), human umbilical cord blood (UCB) nc, bone marrow (BM) nc, peripheral blood stem cells (PBSC), and CD34(+) selected UCB nc, seeding in the BM and spleen of NOD/SCID mice within 24 hours after transplantation. The seeding efficiency of NOD/SCID BM CAFC wk 5 was median (range) in the spleen 2.9% (0.7% to 4.0%) and in the total BM 8.7% (2.0% to 9.2%). For human unsorted UCB nc, BM nc, PBSC, and CD34(+) UCB cells, the seeding efficiency for CAFC wk 6 in the BM of NOD/SCID mice was 4.4% (3.5% to 6.3%), 0.8% (0.3% to 1.7%), 5.3% (1. 4% to 13.6%), and 4.4% (3.5% to 6.3%), respectively. Using flow cytometry, the percentage CD34(+) UCB cells retrieved from the BM of sublethally or supralethally irradiated NOD/SCID mice was 2.3 (1.4 to 2.8) and 2.5 (1.6 to 2.7), respectively. Because we did not observe any significant differences in the seeding efficiencies of the various stem cell subsets, it may be assumed that the SRC seeding efficiency in NOD/SCID mice is similarly low. Our data indicate that the seeding efficiency of a graft can be of great influence when assessing stem cell frequencies in in vivo repopulation assays.

The assessment of the hematopoietic reservoir after immunosuppressive therapy or bone marrow transplantation in severe aplastic anemia.

We investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P < .00001) both for IS and BMT patients. Their number did not improve with time and was not affected by transplant or treatment-related variables. When IS patients were compared with BMT we found comparable numbers of CFU-GEMM (P = .8) and LTC-IC (P = .9), but lower numbers of BFU-E and CFU-GM (P = .05 and P = .004, respectively), suggestive of a persistent suppressive mechanism. These data indicate that LTC-IC numbers are severely reduced in BMT and IS patients, contradicting the common belief that the former are fully reconstituted as compared with the latter. In addition, the number of mature cells and committed progenitors does not seem to reflect the real size of the hematopoietic reservoir and few stem cells may be sufficient to guarantee normal hematopoiesis long term.

Photochemical purging of autologous bone marrow grafts: assessment of damage to stem cells and the microenvironment in long-term marrow cultures.

Toxicity of merocyanine 540 (MC540) was assessed in long-term (Dexter-type) bone marrow cultures and in a short-term in vitro clonal assay of fibroblast colony-forming cells (CFU-F). Exposure of freshly explanted mouse bone marrow cells to MC540 (15 micrograms/ml) and white light (fluence: 126 kJ/m2) reduced CFU-F by approximately 2 logs but did not abrogate the cells' capacity to establish and maintain long-term bone marrow cultures. Fat cells were rare or absence in adherent layers established with photosensitized bone marrow cells but the cultures' capacity to generate non-adherent cells, granulocyte/macrophage progenitors (CFU-GM), and early erythroid progenitors (BFU-E) was only moderately (28-36%) reduced.

Incidence and role of apoptosis in myelodysplastic syndrome: morphological and ultrastructural assessment.

By application of morphological and ultrastructural methods for identification of apoptosis, we analyzed the incidence of morphologically evident apoptosis in the bone marrow of 30 patients with myelodysplastic syndrome (MDS), and in the bone marrow of 12 healthy individuals. According to FAB classification, out of 30 patients, eight (26.6%) had refractory anemia, three (10%) had refractory anemia with ringed sideroblasts, 14 (46.6%) had refractory anemia with excess of blasts and two (6.8%) had refractory anemia with excess of blasts in transformation. Three patients (10%) had chronic myelomonocytic leukemia. Cells in apoptosis were examined on semithin slides and expressed as the apoptotic index (AI) (percent counted on at least 1000 cells). An overall increase in apoptosis in patients with MDS was found (median AI in patients vs controls, 3.13% vs 1.05%, P < 0.01 by Mann-Whitney U test). Also, negative correlation between AI and white blood cell count was found (linear r= -0.53, or Spearman rank R= -0.52, both P < 0.01). In patients with evident karyotype changes AI was not higher than in patients with normal karyotype. This suggests that discrete alterations in apoptosis are present even in karyotypically 'normal' clones. Our results strongly support the hypothesis that apoptosis has a role in ineffective hematopoiesis and may be a mechanism responsible for the paradox of hypercellular bone marrow and peripheral blood pancytopenia in MDS.

G-CSF post-autologous progenitor cell transplantation: a randomized study of 5, 10, and 16 micrograms/kg/day.

G-CSF is routinely administered after autologous bone marrow or peripheral blood progenitor cell transplantation to enhance neutrophil engraftment. However, many different doses of G-CSF have been described with no clear consensus on the most cost-effective dose. We performed a prospective randomized trial examining the efficacy of three different doses of G-CSF post-autologous transplant (5, 10, or 16 micrograms/kg/day). Fifty-seven consecutive patients with breast cancer (n = 30), non-Hodgkin's lymphoma (n = 16), Hodgkin's disease (n = 6), multiple myeloma (n = 2), acute leukemia (n = 2), and testicular cancer (n = 1) were randomized, with 19 patients enrolled in each of the three treatment groups. All patients underwent a high-dose chemotherapy preparative regimen and received an autologous peripheral blood progenitor cell (PBPC) transplant (without bone marrow), with G-CSF beginning on day 0. There was no difference in time to neutrophil engraftment among the three treatment groups (mean 10.2 to 10.8 days). There is a trend towards earlier platelet engraftment in the patient group receiving 5 microgram/kg/day of G-CSF. The total cost of G-CSF by dose group was $2900, $4400, and $6500 per patient. We conclude that there was no advantage to the use of higher doses of G-CSF after autologous transplantation, and that lower doses are associated with lower costs.