bluuebunny/crossref_metadata_2025 huggingface dataset:
Acceptability and Validity of Use of a Patient-Reported Cost Diary To Capture Health Resource Utilization and Costs Associated with Deep Venous Thrombosis and Post-Thrombotic Syndrome.
Authors: El-Hadi Wissam , Ducruet Thierry , Johri Mira , Kahn Susan R.
Published: October 2019

OBJECTIVE: To assess the acceptability and validity of a patient-reported cost diary to estimate health resource utilization (HRU) and costs associated with deep vein thrombosis (DVT) and post-thrombotic syndrome (PTS).

METHODS: We developed a cost diary for use in the Venous Thrombosis Outcomes (VETO) Study, a prospective multicenter cohort study of long-term outcomes after DVT. The VETO cost diary is a 28-item patient-reported questionnaire used to capture information pertaining to HRU and direct and indirect costs attributable to DVT and PTS. Data for HRU and direct costs included hospitalizations, physician visits, purchase of prescription and over-the-counter medications, use of transportation and other medical services and purchase or rental of equipment. Data pertaining to indirect costs included time lost from work by patients or their caregivers. Patients were asked to complete the cost diary monthly for 1 year, then for 3 randomly chosen months for the 2nd year. Acceptability of the cost diary was assessed by estimating the percent of time coverage per patient over the study follow-up period. Internal consistency was assessed by comparing data reported by patients in the cost diary to similar data collected in nurse-administered study questionnaires. External validity was assessed by comparing HRU data reported in the cost diaries to patient-specific data obtained from the administrative database of the Province of Quebec Health Insurance Board (RAMQ) for the same time period.

RESULTS: 387 patients with objectively diagnosed DVT participated in the VETO Study, of whom 359 from 7 hospitals in Quebec, Canada contributed to this analysis (∼ 40% developed PTS). Analyses showed that acceptability of the cost diary was high, with more than 82% of patients reporting data for > 90% of the study follow-up period. Internal consistency analyses revealed discrepancies in reporting of hospitalizations in cost diaries compared to nurse-administered questionnaires (144 vs. 97 hospitalizations, respectively, reported in the first year of the study). Analyses of external validity showed that for the first 4-month study period, the mean number of DVT-related prescriptions reported in the cost diary was comparable to that reported in the RAMQ database (2.48 vs. 3.84, respectively), as was the mean number of DVT-related medical visits (6.39 vs. 7.18, respectively).

CONCLUSION: Overall, the VETO cost diary appears to be an acceptable and valid patient-reported instrument that can be used to estimate HRU, direct and indirect costs attributable to DVT and PTS. Further analyses are needed to identify the factors affecting the internal consistency of the cost diary.
Cost of Myelosuppression with the Second Generation Tyrosine Kinase Inhibitors (TKIs) in Imatinib Resistant/Intolerant Chronic Myelogenous Leukemia (CML): An Economic Analysis Using Global Literature.
Authors: Stephens J. , Carpiuc K. , Botteman M. , Feng W.
Published: October 2019

Background: New targeted therapies such as nilotinib and dasatinib show promise as options for patients with imatinib-resistant or -intolerant CML; however, concerns exist for these agents related to myelosuppression. The objective of this study was to compare the short-term cost of myelosuppression, from a global perspective, for chronic and accelerated phase CML patients treated with nilotinib and dasatinib.

Methods: A global systematic literature review of the past 10 years was conducted using MEDLINE, Econlit and EMBASE to examine the potential costs of myelosuppression (specifically anemia, neutropenia and thrombocytopenia) in adult cancer patients, from the perspective of a health-care payer. An economic analysis was then developed using the ranges of cost data reported in literature, as well as the incidence rates of Grade 3/4 anemia, neutropenia and thrombocytopenia from nilotinib phase II clinical study reports and the dasatinib package insert for chronic and accelerated phase CML. All costs/currencies were converted to US dollars and inflated to 2006 using the country-specific medical inflation rates.

Results: The cost of managing myelosuppression varied widely among countries based on patterns of inpatient compared to outpatient treatment, as well as the use of transfusions compared to recombinant growth factors. The reported cost ranges per AE episode were $124–$12,646 for anemia, $300–$27,440 for neutropenia and $239–$12,223 for thrombocytopenia from the following countries: UK, France, Germany, Italy, Spain, Netherlands, Finland, Switzerland, Belgium, Canada and the US. Expected 6-month costs per patient for Grade 3/4 myelosuppression adverse events with nilotinib and dasatinib are shown as ranges below: Chronic Phase Accelerated Phase Nilotinib $238–$5,550 $394–$9,064 Dasatinib $415–$9,929 $1,155–$23,674

Conclusions: Reflecting a broad range of countries, the expected short-term costs of Grade 3/4 myelosuppression with dasatinib may be 1.7 to 2.9 times higher than that associated with nilotinib. When considering the economic implications of different treatment options for imatinib-resistant or -intolerant CML, both the cost of the drug and the cost of managing adverse events should be taken into account. Future observational studies should be conducted to prospectively assess real-world adverse event costs with the second generation TKIs.
Assessment of Epoetin Alfa in Patients with Myelodysplastic Syndrome Utilizing an Electronic Medical Record Database.
Authors: Feinberg Bruce , Gilmore James , Franco Carlos , Gondesen Tom
Published: October 2019

BACKGROUND: There are limited data from electronic medical record studies that provide a real-world view of practice patterns of epoetin alfa (EPO) as supportive therapy in patients with myelodysplastic syndrome (MDS). This retrospective, observational study was conducted to gain a better understanding of the demographics, real-world treatment patterns, and clinical outcomes for patients with MDS receiving EPO.

METHODS: Electronic medical record data from a large oncology/hematology practice in the southeastern United States were collected from January 1, 2005 to September 30, 2006. Inclusion criteria consisted of patients receiving ≥ 2 doses of EPO with a diagnosis of MDS within 30 days prior to or during EPO treatment episode. All other cancer diagnoses were excluded. Baseline characteristics, EPO dosing, change in Hb levels, and response to EPO (attainment of Hb ≥ 11.0 g/dL at any point during the treatment episode from a baseline Hb < 11g.dL) were measured. EPO treatment episode was defined as continuous treatment with EPO and without EPO administration 35 days prior to or after the last administration. Weekly (QW) dosing was defined as ≤ 9 days, every 2 week dosing (Q2W) was defined as 9.1–18.0 days, and every 3 weeks (Q3W) was defined as > 18.1 days.

RESULTS: A total of 166 patients were included in the analysis; 63.9% women, mean age was 76.2 ± 11.3 years. The mean starting dose was 44,518 ± 11,258 units (median = 40,000 units), and the mean administered dose (any dose of EPO) was 45,587 ± 10,351 units (median=40,000 units). The mean cumulative dose (sum of all EPO doses during the treatment episode) was 312,349 ± 453,551 units. Weekly dosing was administered in 38.0% of patients while 34.9% of patients received Q2W dosing and 27.1% received Q3W dosing. Mean Hb at baseline was 10.6 ± 1.1 mg/dL and increased to 11.3 ± 1.7 mg/dL after 12 weeks of EPO therapy. Fifty percent of the patients responded to treatment and the mean time to response was 33.2 days.

DISCUSSION: This study provides insight into real-world treatment patterns and clinical outcomes for patients with MDS treated with EPO. Additional real-world studies may be warranted to validate these findings.
Vitamin D Deficiency Is Associated with Cardiac Iron Loading in Thalassemia Major.
Authors: Wood John C. , Claster Susan , Carson Susan , Rachna Khanna , Hofstra Thomas , Coates Thomas D.
Published: October 2019

Vitamin D deficiency is epidemic in the United States and is associated with decreased calcium absorption and metabolism, leading to bone loss, muscle weakness, and impaired pancreatic function. Many thalassemia major patients, as a result of decreases sunlight exposure and increased metabolic demand, are vitamin D deficient. We hypothesized that vitamin D deficiency might be associated with cardiac siderosis and impaired cardiac function through its modulation of calcium signaling in these patients. Methods: Permission for review of medical records was obtained from the Committee on Clinical Investigation at Children’s Hospital Los Angeles. We compared vitamin D25-0H and D1-25 levels in our thalassemia major patients with cardiac R2* (1/T2*) and left ventricular ejection fraction (LVEF) from the patient’s most recent cardiac MRI. Time difference between the exams was 2.8 ± 3.3 months with a range of 0.2 to 9.4 months. Other parameters recorded included age, gender, ferritin, liver iron (by MRI) and transferrin saturation. Univariate and multivariate regression was performed using JMP 5.1 (SAS, Cary, NC). Results: Twenty four patients had records suitable for review. There were 11 women and 13 men with a mean age of 14.7 ± 7.6 years [1.4 – 25.8]. Population was moderately iron overloaded with ferritin values of 2089 ± 1920 ng/ml [246 – 8230], liver iron 13.7 ± 11.4 mg/g dry wt [2–39.5], cardiac R2* 65 ± 61 Hz [19.8 – 229], and transferrin saturation 84 ± 18% [36%–106%]. Vitamin D25-OH levels were markedly depressed, 17.1 ± 8.5 pg/ml [1–33], with 13/24 values below the lower limit of 20 ng/ml. Surprisingly, vitamin D1-25OH levels were normal or elevated in all patients, 59.9 ± 19.5 pg/ml [32–103] with four patients exceeding the upper limit of normal of 71 pg/ml. There was no correlation between D25-0H and D1-25OH levels. D25-OH levels (but not D1-25OH levels) fell sharply with age (r2 = 0.48) and were negatively associated with liver iron (r2 = 0.20). Figures 1 and 2 demonstrated cardiac R2* and LVEF as functions of D25-OH levels. Cardiac R2* was log-linearly correlated with D25-OH level (r2 = 0.44, p=0.0001; levels below 13 ng/ml were associated with severe cardiac iron loading. Multivariate analysis of D25-OH, D1-25, HIC, ferritin, age, and transferrin saturation demonstrated that D25-0H and ferritin are the sole predictors of abnormal cardiac R2*, accounting for 38% and 5% of the variability respectively. LVEF was also negatively related to D25-OH levels (r2 = 0.35, p = 0.002). In multivariate analysis, vitamin D250H and vitamin D1-25OH levels accounted for 50% of the LVEF variability, independent of cardiac R2*. Conclusion Vitamin D deficiency is common in thalassemia major patients and strongly associated with cardiac iron uptake and ventricular dysfunction.

Figure Figure Figure Figure
Prevalence of T315I, Dasatinib-Specific Resistant Mutations (F317L, V299L, and T315A), and Nilotinib-Specific Resistant Mutations (P-loop and F359) at the Time of Imatinib Resistance in Chronic-Phase Chronic Myeloid Leukemia (CP-CML).
Authors: Deininger Michael W.N. , Mauro Michael J. , Matloub Yousif , Sinha Ritwik , Ploughman Lynn , Liu David , Radich Jerald
Published: October 2019

Mutations in the catalytic domain of ABL kinase (AKD) are a major mechanism of resistance to imatinib. Over 70 mutations in more than 50 amino acid residues have been reported to date. The ‘gatekeeper’ mutation, T315I, which causes complete resistance to all three FDA-approved tyrosine kinase inhibitors (TKIs), was reported to be not uncommon among a heterogeneous set of patients who had failed first-line imatinib therapy. In addition, the F317L, V299L, and T315A mutations were reported to convey a high degree of resistance to dasatinib, and higher frequency mutations within the P-loop (Y253H/F, E255V/K) and F359 mutations were associated with a high degree of resistance to nilotinib. We studied the prevalence of AKD mutations in the START-C phase II trial of dasatinib in patients who have failed imatinib (resistance or intolerance). Baseline mutation data were available for 95 of 99 patients with imatinib intolerance, and 274 of 288 patients with imatinib resistance. Of these patients, 13 (14%) with imatinib intolerance and 136 (50%) with imatinib resistance had AKD mutations. Of the 149 patients with mutations, only 3 (2%) had the T315I mutation. A total of 57 (38%) subjects had mutations in the P-loop (between 248–256): 13 patients with Y253H/F (9%), and 6 patients with E255V/K (4%). Four subjects (3%) had the F317L mutation, and 8 (5%) had F359 mutations. No subjects with V299L or T315A mutations were detected at baseline. The rates of complete cytogenetic response (CCyR) were 52% in patients with any mutation, 69% in patients with Y253H/F, 40% among those with E255V/K, 0% for T315I mutations, 0% for F317L mutations, and 50% for F359 mutations. Patients without mutations achieved a 55% rate of CCyR. These results confirm that select P-loop and F359 mutations are sensitive to dasatinib, while F317L and T315I mutations are resistant to dasatinib treatment. However, the overall incidence of these dasatinib-resistant mutants is low. In contrast, nilotinibresistant mutations in the P-loop (Y253H/F, E255V/K) or at F359 are more common, representing 15% and 5% of all patients with mutations, respectively. Therefore, the likelihood of harboring a nilotinib-resistant mutation at the time of imatinib resistance appears higher than the likelihood of harboring a dasatinib-resistant mutation, and suggests mutation testing may become instrumental for choosing between the various second-line TKI inhibitors to optimize outcomes.
Allelic Variation within the NKG2DL COMPLEX Influences Event Free Survival Following Stem Cell Transplantation.
Authors: Antoun Ayman , Briggs David , Jobson Shirley , Cook Mark , Moss Paul
Published: October 2019

NKG2D, an important activating receptor on NK cells, is capable of transmitting costimulatory signals by certain subsets of T cells. Viruses and tumours have evolved strategies to evade the NKG2D-mediated immune recognition thus illustrating the importance of NKG2D in mediating anti-tumour and anti-viral responses. Although NKG2D is a highly conserved molecule, it interacts with several structurally diverse ligands that are upregulated by cellular stress. The currently identified Ligands (NKG2DL) are the MHC class I-chain related proteins A and B (MICA and -B) and the ULBP/RAET1 families. MICA and MICB are structurally similar to MHC class I proteins, but share only 27% identity and do not associate with β2-microglobulin or bind peptides. MICA and MICB are very polymorphic with more than 50 MICA and 13 MICB alleles so far reported. Some of their alleles have been shown to be associated with auto-immune diseases such as Behcet’s disease and psoriasis vulgaris. Furthermore, there are currently six identified ULBPs and RAET genes, which are related to MHC class I in their α1 and α 2 domains, but lack the α3 domain and are located in a linked cluster on the long arm of chromosome 6. These proteins are closely related and polymorphic; however, the functional significance of this polymorphism remains unclear. We developed a novel polymerase chain reaction sequence specific primer (PCR-SSP) method for typing NKG2DL using genomic DNA and newly designed primers. First, we established the population genetics of 29 SNPs of NKG2DL, both in the promotor and coding regions in a control population of 223 Healthy controls and confirmed the presence of 24 of these SNPs. For our clinical studies we have limited the analysis to 10 NKG2DL SNPs with allele frequencies of at least 10%, which were used in subsequent analysis of association with clinical outcome of allogeneic stem cell transplantation. The allele and haplotype frequencies were determined in 211 patient and donor pairs who had undergone sibling stem cell transplantation (SSCT) for a range of diseases. Interestingly, a strong association between event free survival (EFS) and a specific SNP within this group of ligands was observed, which resulted in greater than a 25% difference in Event Free Survival at 6 years between its two allelic forms, (p=0.001). Initial analysis of the effect of matching of the SNPs between donor and recipient does not indicate any clear association with SSCT outcome. This is the first study of genetic variation within NKG2D ligands and effect on stem cell transplantation outcome and the findings of the significant associations of their SNPs with EFS indicates a critical role for the NKG2D-ligand interactions with transplant outcome.
Outcome of Patients with IgD and IgM Multiple Myeloma Undergoing Autologous Hematopoietic Stem Cell Transplants: A Retrospective CIBMTR Study.
Authors: Reece Donna E. , Vesole David H. , Shrestha Smriti , Dispenzieri Angela , Milone Gustavo , Peréz Waleska S. , Zhang Mei-Jie , Hari Parameswaran
Published: October 2019

Multiple Myeloma (MM) is the most common indication for autologous hematopoietic stem cell transplantation (auto HCT). However, little information is available regarding the outcome of patients (pts) with the rarer immunoglobulin subtypes IgD and IgM, which represent 2% and 0.5%, respectively, of all MM cases. In addition, IgD MM has been reported previously to have a poorer prognosis, at least after conventional therapy. The CIBMTR conducted a retrospective analysis of MM pts transplanted between 1995–2005 to describe the characteristics and results of auto HCT in IgD (n=36) and IgM (n=11) among 3578 MM patients with auto HCT during this period. Median follow-up of survivors was 41 (range 2–130) months for pts with IgD and 58 (range 5–101) months for those with IgM MM. Among pts with IgD subtype, median age was 52 years (yrs), 67% were male, 36% had a creatinine >2 mg/L, 61% had Durie-Salmon stage III disease at diagnosis and 33% had Kappa Serum Light chain; the corresponding values for IgM pts were 58 yrs, 36%, 73% and 55% respectively. Prior to auto HCT, 75% of IgD pts were chemosensitive and 25% had received >2 lines of chemotherapy, while all IgM pts were chemosensitive and none had received more than 2 lines of prior therapy. Median time from diagnosis to auto HCT was 9 months in both subtypes. The most common conditioning regimen was singleagent melphalan, and all but 1 pt with IgD disease were grafted with blood stem cells. The small sample size precluded multivariate analysis for potential prognostic factors for outcome. Below table summarizes the post-auto HCT results in these pts contrasted with a reference pool of IgG and IgA MM receiving auto HCT in the same time period.

Outcomes, probability (95% CI) IgD IgM IgG/IgA (n=36) (n=11) (n=1475) 100-day mortality, % 0 9 ( 0 – 32) 7 (5 – 8) Non MM deaths, % @ 1 yr 0 9 (0 – 32) 5 (4 – 7) @ 3 yrs 3 (0 – 13) 21 (2 – 51) 16 (14 – 18) Relapse/ Progression, % @ 1 yr 21 (9 – 37) 20 (2 – 49) 18 (16 – 20) @ 3 yrs 59 (41 – 76) 32 (8 – 64) 36 (34 – 39) Progression-free survival, % @ 1 yr 79 (63 – 91) 71 (41 – 93) 77 (74 – 79) @ 3 yrs 38 (21 – 56) 47 (17 – 78) 47 (44 – 50) Overall survival, % @ 1 yr 87 (74 – 97) 91 (68 – 100) 79 (77 – 85) @ 3 yrs 69 (51 – 84) 68 (36 – 93) 53 (50 – 57)

No striking differences are apparent in the post auto HCT outcomes of patients with IgD and IgM MM. These results are also consistent with published outcomes of pts with IgD/ IgM MM (Wechaleker et al Ann Hematol. 2005 Feb; 84(2):115–7; Maisner et al Bone Marrow Transplant. 2008 Jan; 41(1):51–4).
Dosing Patterns and Outcomes in African American, Asian, and Hispanic Patients with Heparin-Induced Thrombocytopenia Treated with Argatroban.
Authors: Hursting Marcie J , Jang Ik-Kyung
Published: October 2019

Nonwhites, as compared with whites, have increased risk for thromboembolic complications in heparin-induced thrombocytopenia (HIT) (Lewis et al, Chest 2006). To better characterize the risk in nonwhite patients, we retrospectively evaluated dosing patterns and clinical outcomes in African American, Asian, and Hispanic patients administered argatroban therapy for clinically diagnosed HIT. Patients were from previously reported, multicenter, prospective studies of argatroban therapy (2 mcg/kg/min, adjusted to achieve aPTTs 1.5–3 times baseline) in HIT (defined as an otherwise unexplained platelet count <100 x109/L, or 50% reduction in platelet count, following heparin therapy). For African American (n=52), Asian (n=13), and Hispanic (n=14) patient groups, baseline characteristics, therapeutic argatroban dose and duration, aPTTs during therapy, and clinical outcomes (death, including death due to thrombosis; amputation, including amputation secondary to ischemic complications of HIT; new thrombosis; and major bleeding within a 37-day period) were summarized and compared. Among groups, no differences were detected in demographic or baseline features, excepting weight (median values: 62, 77 and 85 kg, respectively, for Asians, African Americans, and Hispanics). Median baseline platelet counts were 45–71 x109/L, and approximately 50% of patients in each group had HIT-related thrombosis before argatroban was initiated. Each group received a similar duration of argatroban therapy (4.0–5.5 days), yet Asians needed a lesser dose (1.0 mcg/kg/ min, versus 1.9 mcg/kg/min in other groups) to achieve comparable aPTTs (61–69 s). New thrombosis occurred most often in African Americans (21%, versus 7–8% in other groups) and when baseline HIT-related thrombosis was present (9/13 events, 69%). Amputation occurred most often in Hispanics (21%, versus 0–12% in other groups) and when diabetes was present (6/9 events, 67%). Mortality was 21–31%, and mortality due to thrombosis was 0–7%. Asians had the lowest frequency of any thrombotic-related outcome (composite of death due to thrombosis, amputation secondary to ischemic complications of HIT, or new thrombosis: 8%, versus 27–29% in other groups). Two patients, each African American, experienced major bleeding (gastrointestinal), for an overall major bleeding rate of 2.5% and by-group rates of 0–4%. Although limited by small group sizes, our study is among the first to characterize outcomes in minority patients administered nonheparin therapy for HIT. Our findings suggest that, despite achieving comparable levels of anticoagulation with argatroban, African Americans and Hispanics are higher risk patients than Asians for poor outcomes in HIT and also that in minority patients with adverse outcomes, baseline HIT-related thrombosis or diabetes is often present.
Successful Harvesting of Peripheral Hematopoietic Stem Cells after Induction Treatment with Bortezomib, Adriamycin, Dexamethasone (PAD) in Patients with Newly Diagnosed Multiple Myeloma (MM)
Authors: Goldschmidt Hartmut , Lokhorst Henk M. , Bertsch Uta , van der Holt Bronno , Jarari Laila el , Salwender Hans-Jürgen , Zweegman Sonja , Vellenga Edo , Schubert Jörg , Blau Igor W. , Jie Asiong , Beverloo Berna , Hose Dirk , van de Velde Helgi , Schaafsma Martyn Ronald , Kersten Marie Jose , Delforge Michel , de Weerdt Okke , Van der Griend Rene , Wijermans Pierre , Martin Hans , Scheid Christof , Hänel Mathias , Schmidt-Wolf Ingo G.H. ,   Pieter Sonneveld for the HOVON GMMG group
Published: October 2019

The HOVON-65/GMMG-HD4 trial is a prospective, randomized phase III trial to evaluate the efficacy of bortezomib prior to high-dose melphalan (200 mg/m2, HDM) on response and progression-free survival (PFS) in patients with newly diagnosed MM stage II or III according to Salmon & Durie (SD). Until May 2008 in total 833 patients aged 18–65 years were included in the trial. Patients were randomized to receive three cycles of VAD (arm A; vincristin 0,4mg, days 1–4, adriamycin 9 mg/m2, days 1–4, dexamethasone 40 mg, days 1–4, 9–12, and 17–20) or PAD (arm B; bortezomib 1.3 mg/m2, days 1,4,8,11, adriamycin 9 mg/m2, days 1–4, dexamethasone 40 mg, days 1–4, 9–12, and 17–20). Hematopoietic stem cells were mobilized in patients using the CAD regimen (cyclophosphamide 1000 mg/m2 iv day 1, adriamycin 15mg/m2, days 1–4, dexamethasone 40mg, days 1–4) and G-CSF. After stem cell harvesting all patients received one or two cycles of HDM with autologous stem cell transplantation followed by maintenance therapy with thalidomide 50 mg daily (arm A) and bortezomib 1.3 mg/m2 once every 2 weeks (arm B), respectively.

As of August 2008 stem cell harvesting data from the first consecutively enrolled 150 patients (75 per arm) were analyzed. The data of the initial 300 patients (150 per arm) will be available by November 2008. Both treatment arms did not differ in age, SD stage of disease, ISS stage, and FISH abnormalities. 132 patients (88%) were treated with CAD plus G-CSF. Dosing and type of G-CSF treatment were comparable in both arms.

In all patients stem cell collection was successful and at least two autografts could be harvested (minimal number of stem cells permitted per autograft was 2.0 ×106 CD 34+ cells per kg BW).

Induction treatment Days until first leukapheresis Median (range) Number of leukaphereses Median (range) Number of harvested CD34+ stem cells (× 106) per kg BW Median (range) PAD 110 (96–149) 1 (1–4) 10.5 (4.1–37.6) VAD 111 (95–156) 1 (1–5) 9.3 (4.0–37.0)

In 64% of the patients in arm A (VAD) and 79% of the patients in arm B (PAD) only one leukapheresis was sufficient for stem cell harvesting. In all patients hematopoietic reconstitution was achieved after HDM followed by autografts harvested after PAD or VAD. We conclude that stem cell harvesting after PAD is very well feasible.
The Role of C5a in the Mobilization of Hematopoietic Stem/Progenitor Cells
Authors: Jalili Ali , Shirvaikar Neeta , Korol Chris , Janowska-Wieczorek Anna
Published: October 2019

The complement system, a vital component of the immune system, has been shown to play a role in hematopoietic stem/progenitor cell (HSPC) trafficking. C3a is known to be important in the retention of HSPC in the bone marrow (BM) as C3a-deficient mice are good mobilizers, and C5a is important in the mobilization of HSPC because C5a-deficient mice are poor mobilizers (Stem Cells2007; 25: 3093). Further, granulocyte-colony stimulating factor (G-CSF) activates the complement system via the classical pathway, down-regulates stromal-derived factor (SDF)-1 in BM stromal cells and decreases expression of its receptor, CXCR4, in myeloid cells. In this work investigated the mechanism of C5a involvement in HSPC mobilization. Using RT-PCR and FACS we examined the expression of the C5a receptor (CD88) on mobilized peripheral blood (PB) and steady-state PB HSPC and mature white blood cells, and in vitro-expanded myeloid, erythroid, and megakaryocytic progenitor cells. We found that

CD88, like the G-CSF receptor, is not expressed on BM, PB or cord blood HSPC (CD34+ cells); during CD34+ cell differentiation the expression of CD88 increases in myelocytic and megakaryocytic progenitors; and the percentage of monocytes and polymorphonuclear (PMN) cells expressing CD88 is significantly higher in mobilized than in steady-state PB.

Examing the function of C5a (using flow cytometry) we found that, unlike C3a, C5a decreases CXCR4 expression in a dose-dependent manner in monocytes and PMN, but not in lymphocytes; and this effect was not seen when anti-C5a antibody was added. Interestingly, we found that G-CSF down-regulation of CXCR4 expression on PMN was partially restored by anti-C5a antibody, suggesting that the mobilizing effects of G-CSF are at least in part due to the action of C5a. Moreover, chemotaxis of PMN towards SDF-1 (chemotaxis assay) increased when these cells were stimulated with C3a but decreased with both C5a and G-CSF, reflecting the CXCR4 expression status of these cells after stimulation. We also examined the effect of C5a on matrix metalloproteinase (MMP) secretion in BM leukocytes and found that, like G-CSF, C5a increased MMP-9 and MMP-2 secretion into media (zymography). Since the SDF-1/CXCR4 axis plays an integral role in the retention of HSPC in the BM, we conclude that C5a promotes mobilization by disrupting this axis, as well as increasing MMP-9 and MMP-2 secretion by BM leukocytes, thereby allowing egress of HSPC into the PB.
Development of a Limiting Dilution Assay and MHC-Matched Transplantation Model to Detect Hematopoietic Stem Cell Activity in Zebrafish
Authors: de Jong Jill L. O. , Burns Caroline E. , Tinmaung Aye , Pugach Emily , Mayhall Elizabeth , Smith Alexandra C.H. , Feldman Henry A. , Zhou Yi , Zon Leonard I.
Published: October 2019

The zebrafish is a powerful model for the discovery of novel genetic mechanisms through large scale forward and reverse screening methodologies, and has been used prominently for the study of hematopoiesis. Methodologies capable of assessing hematopoietic stem cell (HSC) activity, however, are largely undeveloped. Here, we created a long-term limit dilution hematopoietic reconstitution assay in adult zebrafish, defined zebrafish haplotypes at the proposed core Major Histocompatibility Complex (MHC) locus, and tested their functional significance by performing the first matched and unmatched transplants. We identified a sublethal radiation dose of 25Gy which was optimal for hematopoietic reconstitution while minimizing mortality presumably due to radiation damage of other organs. Our method detects multi-lineage repopulation in primary and secondary recipients. Using Poisson statistics, our limit dilution analyses suggest that at least 1 in 65,000 nucleated marrow cells in zebrafish contain repopulating activity, consistent with mammalian marrow HSC frequency. Finally, we characterized the genes in the proposed MHC core locus on chromosome 19 for one family used for sibling marrow transplantations. We were able to identify the four parental MHC haplotypes by sequencing PCR products amplified for specific MHC Class I genes: mhc1uea, mhc1ufa, mhc1uda, mhc1uba, mhc1uca, loc751750, and mhc1uxa2. F1 generation siblings were subsequently typed, and we demonstrated that matching the donor and recipient MHC haplotypes at this chromosome 19 locus dramatically increases engraftment and percentage of donor chimerism in recipients compared to MHC-mismatched donors and recipients. At 15 weeks post-transplant, MHC-matched recipients showed engraftment in 4 of 5 surviving fish with mean donor chimerism of 59.15% +/− 25.1% for myeloid cells and 8.26% +/− 6.2% for lymphoid cells. Those animals receiving MHC-mismatched donor marrow had only 3.92% +/− 2.1% myeloid donor chimerism and 0.97% +/− 0.5% lymphoid donor chimerism. These data represent the first assay allowing long term HSCs to be distinguished from other hematopoietic progenitor fates and provides the first functional test of MHC genes between zebrafish haplotypes. This method opens the door to MHC-matched long-term transplantation experiments in zebrafish that have previously not been possible, including competitive transplantation experiments with zebrafish mutants already identified in prior genetic screens, and long-term tumor transplantation assays. By harnessing the unique genetic and screening advantages of the zebrafish model, such experiments may provide critical insight into mammalian transplantation biology.
In Vivo Imaging to Monitor Trafficking and Engraftment of Human CD34+ Hematopoietic Stem and Progenitor Cells in Rhesus Monkeys
Authors: Tarantal Alice F. , Lee C. Chang I. , Batchelder Cynthia A. , Sutcliffe Julie L. , Cherry Simon R.
Published: October 2019

Significant advancements have been made in the use of noninvasive imaging techniques which includes dramatic improvements in resolution and sensitivity, and imaging systems designed specifically for animals. The non-invasive nature of these techniques allows longitudinal assessments over time, and the evaluation of protocols that could be applied in a human clinical setting. Imaging is also of great utility in situations when it is unclear where to focus the analyses, such as the challenges presented by systemic administration of transplanted cells, which can traffic to different anatomical sites. Our prior studies have shown that we can monitor gene expression in fetal and infant monkeys using reporter genes, microPET, and optical imaging. We have also shown that human mobilized peripheral blood stem cells (hPBSC) from adult male donors and male cord blood CD34+ hematopoietic stem cells (HSC) can readily engraft in fetal rhesus monkeys and persist over time. Other recent findings suggest that growth factor expansion of cord blood CD34+ HSC in vitro prior to transplant substantially increases the level of engraftment, with evidence of human hematopoietic cells in the rhesus host at levels significantly greater than the approximate 2% we previously reported. Our studies have also focused on new ways to use optical imaging to monitor transplanted hPBSC, cord blood HSC, and CD34+ precursors differentiated from human embryonic stem cells (hESC) that express firefly luciferase and a drug resistance gene, and the development of PET techniques for short-term tracking of transplanted cells. Transduced cells (1×104–20×106 cells/fetus) were transplanted intraperitoneally into fetal monkeys in the late first trimester under ultrasound guidance (N=27), then animals were monitored sonographically during gestation, and delivered by cesarean-section at term. At 1 week postnatal age and monthly thereafter animals were imaged for firefly luciferase expression (bioluminescence) following the intravenous injection of 100 mg/kg D-luciferin, and after the collection of blood and bone marrow for analysis. While evidence of the human Y chromosome (hSRY, hTSPY) was found in peripheral blood mononuclear cells and CD34+ immunoselected cells from marrow by qRT-PCR, the range of copies detected over time did not reflect the imaging findings. Foci of engrafted human cells were found to persist for ≥1 year, to date, in the rhesus host and in anatomical areas not previously identified (e.g., ribs, sternum, liver). In addition, 64Cu-PTSM radiolabeled hPBSC from the same donors were injected postnatally and cell trafficking monitored without evidence of toxicity or adverse effects. These in vivo imaging studies have provided unique insights into the fate of CD34+ cells post-transplantation, and indicate the importance of these techniques for cell transplant protocols.
A Report from the International Collaborative Gaucher Group (ICGG) Gaucher Registry
Authors: Weinreb Neal J. , Dahl Stephan vom
Published: October 2019

Objective: To report the latest data on patients with Gaucher disease (GD) enrolled in the ICGG Gaucher Registry.

Methods: Data from all patients enrolled in the ICGG Gaucher Registry from 1991 through 2007 were analyzed.

Results: As of December 31, 2007, 4,936 GD patients were enrolled in the ICGG Gaucher Registry by 772 physicians in 60 countries. The countries with the largest numbers of patients were the United States (36%), Israel (14%) and Brazil (10%). The majority of patients were diagnosed with GD between 4 and 30 years (mean age, 19 years). The most common genotypes were N370S/N370S (31%), N370S/L444P (16%), N370S/Rare Allele (13%), and N370S/unknown (11%). The most frequent genotype for patients with neuronopathic GD was L444P/L444P (68%). At diagnosis, anemia was reported in 37% of patients and moderate to severe thrombocytopenia in 60%. Splenomegaly was reported in 86% of patients (>5 multiples of normal [MN]) and hepatomegaly in 65% (liver volume >1.25 MN). Bone pain was present in 34% of patients and radiologic bone disease was reported in 83%. Long-term treatment with imiglucerase resulted in improved haematological parameters, decreased visceral involvement, decreased bone pain and abolition of bone crises.

Conclusions: For GD and other rare “ultra-orphan” diseases, a large longitudinal international disease registry provides the best means to investigate the natural history of the disease and the long-term effects of therapy. The strength of the ICGG Gaucher Registry data is the inclusion of a large, worldwide patient population with long periods of follow-up data, allowing for studies not otherwise possible.
The Influence of Spatially Distinct Bone Marrow Niches on HSCs
Authors: Liu Sali , Tozer Tigue , Rosa Dilani , Cunningham Cynthia , Tseng Alan , Rashidi Narges , Adams Gregor B.
Published: October 2019

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow (BM), which remains the site of hematopoiesis throughout adulthood. In the BM the HSCs are located at the endosteal surface, where cells of the osteoblastic lineage comprise a key component of the stem cell niche. While hematopoiesis occurs in many bones, the process of bone formation can actually be split into those bones that develop through endochondral ossification (long bones) and those that form through membranous ossification (flat bones). We examined the role played by the microenvironment in these two distinct bones and whether these microenvironments have differing effects on the HSCs. In vitro analysis of the BM stromal cells isolated from long bones and flat bones has demonstrated that calvaria derived stromal layers can support cobblestone area-forming cells 10-fold greater than stromal layers derived from femurs and tibia. Real-time PCR analysis of gene expression has demonstrated that flat bone stromal cells have 5-fold greater expression of N-cadherin than long bone, while other cadherins such as VE-cadherin show no difference. Correlating with this, we found that calvarial derived HSCs demonstrated increased expression of N-cadherin and also increased expression of other genes associated with cadherin signaling, such as cyclinD1. However, no difference in the cell cycle status of the HSCs derived from long bone and flat bone was noted. Functional assays are being performed in order to assess the function of these distinct BM marrow niches in vivo. It is anticipated that we will be able to begin to define the molecular cues the govern HSC physiology in different locations within the mammalian skeleton and thus provide an understanding not only into the continual migration of HSCs between different HSC niches but also the regression of hematopoiesis that occurs from the appendicular skeleton to the axial skeleton during the adult human lifespan.
Identification and Isolation of the Hematopoietic Stem Cell Niche Initiating Cell Population
Authors: Chan Charles , Chen Ching-Cheng , Kraft Daniel L. , Luppen Cynthia , Kim Jae-Beom , DeBoer Anthony , Wei Kevin Wei , Weissman Irving L.
Published: October 2019

Introduction: Identification and understanding of the cells and processes that can generate, sustain and influence the HSC niche and hematopoiesis are critical for the development of a more comprehensive knowledge of normal hematopoiesis, stem cell homing, trafficking, differentiation and hematopoietic pathology. Growth and renewal in many tissues are initiated by stem cells, supported by the microenvironment (niche) in which they reside. While recent work has begun to describe functional interactions between stem cells and their niches, little is known about the formation of stem cell niches.

Methods & Results: We established a functional, in vivo assay (via implantation of cells under the renal capsule) to isolate the determinants of hematopoietic stem cell (HSC) niche formation and activity. Using this novel assay, we show that a population of progenitor cells (CD45−Tie2-aV+CD105+Thy1.1−; CD105+Thy1−) sorted from 15.5 dpc fetal limbs and transplanted under the adult mouse renal capsule recruit host-derived vasculatures in a VEGF dependent manner, produce donor-derived ectopic bones through endochondral ossification, and generate a marrow cavity populated by host-derived long term reconstituting HSC (LT-HSC). In contrast, CD45−Tie2-aV+CD105+Thy1a+ (CD105+Thy1+) progenitors form bone that does not contain a marrow cavity. While analyzing these and other sorted populations, we did not observe any instances where niche was present without bone, suggesting that skeletal progenitors are necessary for initiating an HSC niche but osteoblasts alone cannot initiate and support niche activity. Suppression of factors important for HSC maintenance, such as steel factor (SLF), in progenitor populations prior to transplant did not alter their ability to initiate and support an HSC niche. On the other hand, suppression of factors involved in endochondral ossification, such as osterix and VEGF, inhibited niche generation. Furthermore, CD105+Thy1− progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification form only bone without marrow in our assay.

Conclusions: In addition to identifying the limb-derived skeletal progenitor capable of endochondral ossification involved and the basic mechanisms of HSC niche initiation, our study provides a functional framework by which future studies on HSC-niche interactions at the cellular level can be carried out.
High-Dose Methotrexate and Early Intensification of Therapy Do Not Improve 3 Year EFS in Children and Adolescents with Disseminated Lymphoblastic Lymphoma. Results of the Randomized Arms of COG A5971
Authors: Abromowitch Minnie , Termuhlen Amanda , Chang Myron , Perkins Sherrie L. , Gross Thomas , Weinstein Howard J. , Finlay Jonathan
Published: October 2019

The treatment of pediatric lymphoblastic lymphoma (LL) has developed in parallel with treatment strategies for childhood acute lymphoblastic leukemia using a BFM backbone. The excellent results of the NHL/BFM 90 trial prompted us to design this randomized factorial study to determine whether a regimen without high dose methotrexate (HDMTX) the CCG BFM will result in the same outcome as NHL/BFM-90 and whether intensification with anthracycline and cyclophosphamide would further improve disease free survival.

From June 2000 to October 2005, 257 patients with Murphy’s Stage III and IV (excluding CNS disease) LL were randomized to one of the four regimens. All regimens used the BFM/NHL95 backbone. The CCG BFM regimen had intrathecal (IT) methotrexate throughout interim maintenance and maintenance without IV methotrexate. The NHL BFM utilized I.V. Methotrexate 5 Gms/m2 and intrathecal MTX every 2 weeks for four doses during interim maintenance without further intrathecal MTX during maintenance. One of each backbone regimens was further intensified with anthracycline and cyclophosphamide early in induction and delayed intensification.

The median age was 10.3 years, 195 (76%) were males; 43 (17%) had >5% bone marrow involvement. Twelve patients with CNS disease were not randomized and received intensification and HD HDMTX with delayed CNS radiation (data not reported here).

Major toxicities have been related to bone marrow suppression with 4 toxic deaths, 3 due to sepsis and 1 from cerebral hemorrhage. The frequency of grade III/IV neutropenia (alone, with fever or with infection), anemia, and thrombocytopenia were higher in the intensified arms during induction. Three of the four toxic deaths occurred on the intensified arms. The three years EFS of the HDMTX vs. none is 84.5% ± .3.5% vs. 82.7± 3.8 (ρ= 0.93) and the intensification vs. none is 83.4% ±3.7 vs 83.0% ± 3.6 (ρ= 0.66). Therefore, there was no significant difference between treatment arms.

These results suggest that neither HDMTX nor early intensification improves EFS in LL. Future direction should focus on identifying biological factors early in therapy so alternative therapies may be investigated.
5-Azacytidine, Valproic Acid and ALL-Trans Retinoic Acid in INT-2/High Risk Myelodysplastic Syndromes: Results of the GIMEMA MDS0205 Multicenter Trial
Authors: Voso Maria Teresa , Santini Valeria , Finelli Carlo , Borin Lorenza , Angelucci Emanuele , Fioritoni Giuseppe , Neri Benedetta , Cortellezi Agostino , Villani Oreste , Buccisano Francesco , Martinelli Giovanni , Petti Maria Concetta , Zini Gina , Piciocchi Alfonso , Fazi Paola , Alimena Giuliana , Gobbi Marco , Liso Vincenzo , Leone Giuseppe
Published: October 2019

Epigenetic changes have been shown to play a role and to cooperate with genetic alterations in the pathogenesis of myelodysplastic syndromes (MDS). The potential reversibility of DNA and chromatin modifications makes chromatin remodeling enzymes attractive targets for therapeutic intervention in this disease. We conducted a phase II study on the combination of the DNMT inhibitor 5-azacitidine (5-AZA), the histone deacetylase inhibitor valproic acid (VPA), and all-trans retinoic acid (ATRA) in patients with intermediate-2/high-risk myelodysplastic syndromes. Bone marrow morphology was centrally reviewed before enrolment. VPA was given at 600–1500 mg daily to reach a final plasma concentration above 50 microg/ml, then 5-AZA was added at a standard dose of 75 mg/sqm daily, subcutaneously, 7 days for 8 cycles. In case of minor response, stable disease or failure after 4 cycles, ATRA was added at 30 mg/sqm orally daily, on days 8–27 for 4 cycles. Treatment was continued in responding patients until response persisted. The protocol included 62 patients (43 males, 19 females, median age 67 years, range 53–83 yrs). Diagnosis was RAEB for 37 patients (60.7%), RAEB-t for 21 (32.8%), and CMML for 4 patients (6.5%). The IPSS was int-2 (1.5) for 46 patients and High (≥2) for 16 patients. A valproic acid concentration between 45 and 55 microg/ml was reached in a median of 7 days (range 4–28 days). Three patients died before start of treatment, while 58.7% of patients (95% C.I.: 51.3–67.1) were alive at 12 months. Out of 27 patients who completed 8 treatment cycles, 8 patients (29.6%) obtained complete and partial remission, 3 patients (11.1%) major hematological improvement while 10 patients (37.4%) showed a stable disease. Transformation into AML or progression occurred in 20 patients. RBC transfusion needs decreased significantly from a median of 3 units (range 0–16) before start of treatment to 0 (range 0–7) after 8 cycles. Neurological toxicity occurred in 6 patients. Our data show that the 5-AZA/VPA/ATRA combination is safe and feasible in poor prognosis MDS patients.