ALBERT

Description: Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.

Description: There is a paucity of pharmacokinetic (PK) data about hydroxyurea (HU) in persons with sickle cell anemia (SCA) and none in very young children. HU clearance is predominantly renal. The kidney may be damaged during infancy in SCA, but the first abnormality is usually hyperfiltration which may lead to enhanced HU clearance. Following a 15 mg/kg oral dose of commercially available HU capsules, PKs in 7 adults with SCA and normal renal function have been reported to be a mean±SD half-life (T1/2) of 3.14±0.9 hrs, a maximal concentration (Cmax) of 28.32±11.0 ug/mL, and an area under the curve (AUC) of 81.66±15.5 ug•hr/mL. The Phase II Study of HU in adults concluded that initial 2 hour clearance of HU was not associated with either the maximum tolerated dose or fetal hemoglobin response to treatment. However, the AUC did predict HU toxicity and, in another study, the need for dose adjustment in adults with SCA and renal insufficiency. BABY HUG is an NHLBI-NICHD sponsored Pediatric Phase III Clinical Trial designed to critically assess the efficacy of a novel liquid HU preparation in preventing organ damage in young children with SCA. Forty-five African-American infants, 12 to 18 months (mo) of age (mean 14.7 mo) were recruited without regard to disease severity to the just completed randomized, double-blind, placebo-controlled BABY HUG Feasibility and Safety Pilot Trial. First dose PKs were obtained 0, 1, 2, and 4 hours after oral administration of 20 mg/kg of HU in 22 consecutive patients (mean age 14.5 mo), coincident with measurement of glomerular filtration rate (GFR) by nuclear medicine DTPA clearance. An additional PK sample was obtained between 4 and 8 hours in 15 of the 22 (68%). Samples were frozen at −70°C, shipped, and assayed by high resolution gas chromatography with mass spectrometric detection (limit of detection 0.5 ug/mL). The T1/2 (2.36±0.99 hrs), Cmax (19.81±5.8 ug/mL; 0.26±0.8 uM/L), and AUC (68.82±11.5 ug•hr/mL) were somewhat lower in BABY HUG patients than in the small group of adults with SCA from the literature. There were no apparent relationships between PKs and baseline GFR measured by DTPA or calculated from the Schwartz equation. Younger patients ≤15 mo (n=15) had a trend toward a shorter T1/2 (2.1 vs 2.8 hours; p=0.118) and a lower predicted measurable HU concentration at 8 hours (1.2 vs 2.1 ug/mL; p=0.056) than those 16–18 mo (n=7). There were no age related differences in AUC (67.4 vs 71.8 ug•hr/mL; p=0.421) or Cmax (20.5 vs 18.2 ug/mL; p=0.409). PKs of this novel liquid preparation of HU in young children with SCA may be different than that of adults with the standard capsule formulation. The BABY HUG Trial will continue to refine the PK model by additional evaluations including samples from patients as young as 9 mo of age, the new lower age of eligibility for the open study currently accruing patients.

Description: The identification of bone marrow (BM) niches controlling many aspects of hematopoiesis is of great interest. It is believed that these niches provide the necessary signals to maintain primitive hematopoietic cells including hematopoietic stem cells (HSC). Downstream of HSC are programs for both myelopoiesis and lymphopoiesis, although little is known about the niches that support these processes. Accumulating data implicating osteoblasts (OB) as a component of the HSC niche suggest that these cells are able to regulate HSC properties, and that the HSC are located in direct contact with OB at the bone endosteum. However, the interpretation of these data is controversial, and others have suggested that endothelial cells dictate the structure and function of the HSC niche. In order to determine whether OB are a critical component of hematopoietic niches, we employed Col2.3ΔTK mice, a transgenic mouse strain bearing a fusion gene composed of the rat type I collagen promoter and HSV-thymidine kinase. This construct allows for selective depletion of OB following in vivo gancyclovir (GCV) treatment. Mice received 100 mg/kg GCV for a period of 8, 21, or 28 days. Following 8 days of GCV, OB were depleted from the BM as determined by a lack of staining for osteocalcin. Concommitant with the loss of OB, there was a decrease in total BM cellularity of approximately 50% that included reductions in both myeloid and B lymphoid cells. Among the B cell populations that were measured, pre-pro-B and pro-B cells were reduced to the greatest extent, 64% and 81%, respectively. Despite this marked reduction in B lymphopoiesis, number of Lin− Sca-1+ cKit+ cells (LSK), a population enriched for HSCs, was unchanged immediately following OB depletion. This loss of B cell precursors, occurring before any observable effects on HSC numbers, suggests that OB may directly support the proliferation and maturation of early B cell precursors. Although minimal changes in BM LSK were observed immediately after GCV, BM LSK were reduced after 21 and 28 days of GCV treatment, when their numbers decreased 40% and 88%, respectively. During this same period, however, spleen LSK increased by 165% and 300%, respectively. Assuming that femurs and tibiae constitute 25% of total murine BM, the total number of LSK lost from the BM after 21 and 28 days of GCV is approximately 1.42 and 3.12×10^5 cells, respectively. These values are remarkably consistent with the increase in splenic LSK at the same time points, 1.2 and 3.7×10^5 cells, respectively. These data indicate that loss of OB plays a pivotal role in the partitioning of LSK into and out of the BM. Given the finding that the shift in LSK is most pronounced 2–3 weeks after the loss of OB, these data raise the intriguing possibility that the partitioning of LSK from the BM to the spleen following OB deletion is not the result of increased emigration from the BM but, rather, due to a decreased ability of HSC to home back to the BM following daily limited physiological mobilization. For example, it is plausible that OB supply chemotactic signals that control the ability of HSC to home to the BM. In conclusion, in vivo depletion of OBs causes an immediate reduction in the number of B cell precursors with a delayed shift in LSK from the BM to the periphery, thus demonstrating that OB play a major role in BM HSC trafficking and B lymphopoiesis.

Description: Background: The combination of MPD and amyloidosis is rare, and this patient population's clinical outcome is not well studied. Methods: Pts with a MPD and amyloidosis were identified via an clinical note search engine that searches through the electronic medical records of patients seen at Mayo Clinic Rochester, Jacksonville, and Scottsdale between 1990 and 2016. Terms used included amyloid or amyloidosis, chronic myelogenous leukemia or CML, essential thrombocytopenia or ET, and polycythemia vera or PV. Demographic and clinical data were abstracted from the medical record. Pts with both disorders were analyzed and their mortality rates along with median time to death were calculated. Prevalences at the Mayo Clinic were calculated for the years 2014 and 2015. Results: Twenty-three pts diagnosed with both a MPD and amyloidosis were identified. Thirteen (56.5%) were male, 10 (43%) were female. Eleven (47.8%) were initially diagnosed at the Mayo Clinic. Types of amyloidosis were as follows: Eleven (47%) had immunoglobulin light-chain (AL), four had localized (17%), two (8.7%) had wild-type transthyretin (ATTR), one (4.3%) had mutant ATTR, and five (21.8%) were unknown. Types of MPD were as follows: Seven (30%) had polycythemia vera (PV), seven (30%) had chronic myelogenous leukemia (CML), five (22%) had myelofibrosis, and four (17%) had essential thrombocytosis (ET). Fifteen (65%) were initially diagnosed with a MPD. Median time to last follow-up from second diagnosis was 1.7 years, and median time to death following second diagnosis was 1.4 years. The mortality rate was 87% in the total population. The median time to death for AL and PV was 2.7 years, AL and CML 1.0 years, and AL and ET 1.17 years. Myelofibrosis did not occur with AL. The most common combination was AL and PV, which accounted for five (22%) of the cases. Treatment regimens for the patients with AL were varied. Multiple drugs were combined with dexamethasone including melphalan, velcade, pomalidomide, lenalidomide, doxorubicin, and revlimid. Cyclophosphamide, bortezomib, and dexamethasone (CyBorD) were used in two cases. The mean time to diagnosis of amyloidosis from symptom onset was 10.3 months. Prevalences of AL and a MPD in 2014 and 2015 at the Mayo Clinic were 2.8% and 1.5%, respectively. Conclusions: The mortality rate for the combined diagnoses is high. The most common combination of diagnoses was that of AL and PV, which was associated with a mean time to death of 2.7 years. Disclosures Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.

Description: Introduction: A recent study revealed an antiproliferative and apoptotic effect of propranolol on multiple myeloma (MM) cells. Our previous small matched case-control study showed longer survival in patients with propranolol and other beta-blockers (BB) intake than those without. This larger scale study was conducted to confirm the positive association of BB and MM survival. Methods: We identified 1971 newly diagnosed pts seen at Mayo Clinic between 1995 and 2010. Cardiac medication usage after diagnosis of MM was extracted from patient records and categorized based on BB intake. Cause of death was collected with death due to MM as the primary interest event and death due to cardiac disease or other reasons as competing risk events. The primary outcomes were MM disease-specific survival (DSS) and overall survival (OS). Cumulative incidence functions and Kaplan-Meier method were used to estimate the 5-year cumulative incidence rate (CIR) of MM death and OS rate, respectively. DSS and OS were compared by Gray's test and log-rank test, respectively. Multivarable Cox proportional hazard models were used to estimate the adjusted cause-specific HR (HRCSadj.) and hazard ratio (HRadj.) for DSS and OS, respectively, adjusting for demographics, disease characteristics, diagnosis year, and various chemotherapies. Results: 930 (47.2%) of MM patients had no intake of any cardiac medications; 260 (13.2%) had BB only; 343 (17.4%) used both BB / non-BB cardiac medications; and 438 patients (22.2%) had non-BB cardiac drugs. Five-year CIR of MM death and OS rate were shown in table. Superior MM DSS was observed for BB only users, compared to patients without any cardiac drugs (HRCSadj., .53, 95% confidence interval [CI], .42-.67, padj.

Description: Hyperhaemolysis is a rare, poorly understood complication of red cell transfusion. We report the outcomes of SCT in 2 boys of Middle Eastern origin who developed life-threatening hyperhaemolysis each following their third transfusion for β-TM at the ages of 2.5 and 4 years. The diagnosis of hyperhaemolysis was based on laboratory evidence for haemolysis, post-transfusion haemoglobin (Hb) levels lower than pre-transfusion, positive Coomb’s tests and no allo-antibody able to be identified. Haemolysis was intravascular, extravascular and severe. Precipitous drops in Hb made transfusion unavoidable. The first boy responded to prednisolone, intravenous immunoglobulin (IVIG) and splenectomy. Haemosidderosis and fibrosis were present on liver biopsy, he was therefore regarded as a Class 3 thalassaemia patient and received hydroxyurea, azathioprine, erythropoietin, desferrioxamine, busulfan, cyclophosphamide and fludarabine conditioning prior to an HLA identical sibling SCT. He is alive and well 7 years post BMT with 30% stable donor chimerism and a normal Hb. The second child’s hyperhaemolysis failed to respond to prednisolone, IVIG, rituximab and splenectomy. Provision of the large number of suitably matched red cell units required was problematic. After receiving hydroxyurea, azathioprine, desferrioxamine, busulfan, cyclophosphamide, fludarabine, thiotepa and antithymocyte globulin (ATG) preparation for a CD3/CD19 depleted maternal haplotype peripheral blood SCT, the haemolysis finally stopped. Post-transplant severe veno-occlusive disease and multi-organ failure (MOF) required dialysis and ventilation. The maternal graft was rejected so 28 days after the first transplant he received a 4/6 mismatched unrelated cord blood transplant (UCBT) following further fludarabine and ATG, which fully engrafted. He recovered from MOF and was discharged from hospital 47 days after the UCBT, transfusion independent. On day +86 he contracted Respiratory Syncytial Virus chest infection with acute intravascular haemolysis necessitating transfusions. Fulminant liver failure developed, presumably due to iron toxicity, and death occurred on day +102, having received 112 transfusions in the 12 months since presentation. In conclusion, avoiding red cell transfusion is not always possible in hyperhaemolysis, especially in β-TM. Patients may quickly become classified as Class 3 in terms of predicting BMT outcome. Immune modulation therapy and SCT was effective in 1 case but only temporarily stopped haemolysis in the other, despite full engraftment ultimately being achieved with a mismatched UCBT. SCT should be considered early in cases of hyperhaemolysis in β-TM because it can potentially cure both and result in transfusion independence. Disclosures Off Label Use: Intravenous immunoglobulin and rituximab for treatment of haemolytic anaemia; hydroxyurea, azathioprine, fludarabine, erythropoeitin, busulphan, cyclophosphamide, thiotepa and antithymocyte globulin for use in stem cell transplantation in children with thalassaemia.

Description: Abstract 2195 Introduction: Eltrombopag (epag), a thrombopoietin receptor agonist (TPO-RA), increases platelet counts in patients with chronic immune thrombocytopenia (cITP). TPO-RAs have been associated with varying degrees of increases in bone marrow reticulin (Brynes 2011; Ghanima 2011). Due to lack of pretreatment evaluations, the incidence and clinical significance of these findings have not been established. Inconsistencies in specimen preparation, staining, and analysis across institutions further confound conclusions. The purpose of this 2-year (y) study (NCT01098487) is to assess for bone marrow fibrosis (reticulin and/or collagen) in patients treated with epag for cITP. Baseline and 1y findings are presented. Methods: Bone marrow biopsies are being collected at baseline (before treatment with epag) and at 1 and 2y of treatment. Specimens are centrally processed and stained for reticulin (silver) and collagen (trichrome) and undergo central independent pathology review of cellularity; megakaryocyte, erythroid, and myeloid quantity and appearance; trabecular bone quality; reticulin grade; and presence of collagen (European Consensus scale-MF; Thiele 2005). Results: Baseline and 1y (10–14 months) data are available for 101 patients. Median age is 42y (18–78); 70 patients are female; 50% are Caucasian/European, 22% are East Asian, and 29% are Central South Asian. Median time since ITP diagnosis is 4.2y (0.2–45.7). All patients had received prior ITP therapy, and 8 patients had received prior TPO-RA treatment (epag [7], romiplostim [1]), the last dose ≥6 months before enrollment. At baseline, 91 patients had reticulin grade 0 (MF-0), 10 MF-1, and 0 MF≥2. At 1y, 59 patients had MF-0, 38 MF-1, 3 MF-2, and 1 MF-3 (Figure). Compared with baseline, there was no change at 1y in MF grade in 61 patients, a decrease by 1 grade in 3, an increase by 1 grade in 35, and an increase in 2 or 3 grades in 1 patient each (Table). Three patients had collagen at 1y (1 patient each with MF-1, MF-2, and MF-3). None of the 4 patients with MF≥2 had adverse events or hematologic abnormalities considered related to impaired bone marrow function, and none withdrew due to bone marrow findings. Among the 8 patients with prior TPO-RA treatment, all had baseline reticulin of MF-0 and none had collagen; at 1y, 6 remained MF-0, 1 was MF-1, and 1 MF-3 (collagen demonstrated). Cellularity was normal in 83% and 80% of patients at baseline and 1y, respectively. Other than normalization of erythroid lineage numbers, no changes occurred in marrow cellular composition. In 3 of 4 patients with MF≥2, cellularity was increased at 1y. Trabecular bone thinning was found at baseline in 28 patients (the majority with prior steroid use) and 51 patients at 1y. Discussion: 10% of patients had MF-1 at baseline. After 1y of treatment, no increase or a mild increase in reticulin was observed in 63% and 35% of patients. No patient with MF≥2 (n=4) had clinical signs or symptoms indicative of bone marrow dysfunction and none withdrew from the study. Results were similar to those reported for EXTEND, an eltrombopag extension study (median treatment duration 〉2 years; Brynes 2011). Conclusion: These data suggest that treatment with epag is generally not associated with clinically relevant increases in bone marrow reticulin or collagen. The potential association of TPO-RAs and increased bone marrow reticulin needs further study. Disclosures: Brynes: GlaxoSmithKline: Research Funding. Orazi:GlaxoSmithKline: Research Funding. Wong:Roche: Research Funding; MSD: Research Funding; Johnson & Johnson: Research Funding; Bayer: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biogen-Idec: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; GlaxoSmithKline: Research Funding; Bristol-Myers Squibb: Research Funding. Bakshi:GlaxoSmithKline: Employment, Equity Ownership. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Brainsky:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties.

Description: Background: Recent advances in the therapy of multiple myeloma (MM) have greatly increased the treatment options for this uniformly fatal plasma cell malignancy. It is not clear if introduction of novel therapies and the increased use of high dose therapy (HDT) in the past decade have translated into better outcome for patients (pts) with MM. Methods: We examined the outcome of two cohorts of pts seen at our institution. The first cohort consisted of 387 pts who relapsed after HDT and was examined for potential improvement in survival following relapse after HDT. These pts were divided into two groups; those who relapsed before or after December 31, 2000. The second cohort consisted of 2981 patients with newly diagnosed MM seen between January 1971 and December 2006 and was used to examine the trends in overall survival (OS). Results: Among those relapsing after HDT, there were 245 males (63%); median (range) age at HDT was 57 years (32.8–75.4) and the median time to HDT was 8.1 months (1–90 months) from diagnosis. The median time to relapse was 13.2 months (1.1 months-10.3 years) from HDT. In this cohort, a clear improvement in OS from time of relapse was seen over the past decade, with those relapsing after 2000 having a median OS of 23.9 months (95% CI; 19.8, 27.6) compared to 11.8 months (95% CI; 8.7, 14.9) for the rest (P 〈 0.001) (Figure 1). In a multivariate analysis, the effect of the date of relapse on survival was independent of other prognostic factors such as relapse 5.5 mg/dL. Pts who were treated with one or more of the newer drugs (thalidomide, lenalidomide, bortezomib) had longer survival from relapse (30.9 months (95% CI; 23.6, 38.2) compared to 14.8 months (95% CI; 11.3, 18.4); P 〈 0.001) for others. Among the newly diagnosed MM cohort, the median age at diagnosis was 66 years (20.2 – 97 years), and 1,770 (59.4%) were males. The median follow up for the entire group was 27.4 months (0–29.4 years); and at the time of analysis 558 patients (18.7%) were alive with a median follow up of 32.7 months (0–29.4 years). Pts diagnosed in the last decade had an improved OS (44.8 months) compared to those diagnosed before this period (29.9 months; P 〈 0.001) (Figure 2). The improvement in survival seen in the last decade among newly diagnosed patients was predominantly among those younger than 65 years (60.3 mos vs. 33.3 mos improvement in median survival); compared to those over 65 at diagnosis (26.5 mos vs. 32 mos). Conclusion: In this study, for the first time, we demonstrate definite proof for improved outcome in patients with myeloma, both in the relapsed setting as well as at diagnosis, a change that is likely to continue with increased use of these drugs. Figure Figure Figure Figure

Description: The Pediatric Hydroxyurea Phase 3 Clinical Trial (BABY HUG) was a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial of hydroxyurea in infants (beginning at 9-18 months of age) with sickle cell anemia. An important secondary objective of this study was to compare clinical events between the hydroxyurea and placebo groups. One hundred and ninety-three subjects were randomized to hydroxyurea (20 mg/kg/d) or placebo; there were 374 patient-years of on-study observation. Hydroxyurea was associated with statistically significantly lower rates of initial and recurrent episodes of pain, dactylitis, acute chest syndrome, and hospitalization; even infants who were asymptomatic at enrollment had less dactylitis as well as fewer hospitalizations and transfusions if treated with hydroxyurea. Despite expected mild myelosuppression, hydroxyurea was not associated with an increased risk of bacteremia or serious infection. These data provide important safety and efficacy information for clinicians considering hydroxyurea therapy for very young children with sickle cell anemia. This clinical trial is registered with the National Institutes of Health (NCT00006400, www.clinicaltrials.gov).

Description: Key Points Neutrophils of patients with FHL-5 with Munc18-2/STXBP2 mutations have impaired granule fusion and bacterial killing.