ALBERT

Description: Submyeloblative conditioning regimens for bone marrow transplantation (BMT) are desirable to reduce host toxicity in many settings. We previously demonstrated that hosts conditioned with 160 cGy or with two sequential doses of the antimetabolite 5-fluorouracil (5-FU) displayed ~50% and ~35% donor chimerism 6 months after transplantation of 20 x 106 fresh congenic bone marrow (BM) donor cells, respectively. Donor chimerism in 160 cGy- or 5-FU-conditioned mice was proportional to the reduction in host long-term repopulating ability (LTRA) caused by the conditioning regimen (~80–90% vs. ~55%, respectively). In this study, we sought to determine if donor marrow homing efficiency also correlated with long-term engraftment in these models of submyeloablative conditioning. We hypothesized that sequential doses of the cell cycle-specific agent 5-FU (5-FU x 2) may damage the marrow microenvironment less than low-dose radiation, resulting in more efficient homing in 5-FU x 2-conditioned mice. C57Bl6/J (Bl/6) hosts were conditioned with either 5-FU x 2 administered on days -5 and -1 (150 mg/kg IP) or with 160 cGy given the day of transplantation (day 0). Bl/6 (CD 45.2+) hosts were transplanted with 20 x 106 PKH-26 stained Bl/6 or with Boy J (CD 45.1+) whole BM cells via tail vein injection. The percentage of homed cells 20 hours after BMT in 5-FU x 2-conditioned mice was 3.53 % ± 3.1% (n = 13 from 3 independent experiments), while the percentage of homed cells in the 160 cGy group was 8.67 % ± 2.1% (n = 12; p = 0.0375). The fraction of donor cells which homed to the spleen or remained in the peripheral blood after 20 hours was similar between conditioning regimens. This difference in homing efficiency led us to consider other possible explanations as to why BM cells home less efficiently in 5-FU x 2-conditioned hosts than in low-dose irradiated mice. We hypothesized that 5-FU x 2 may have a more injurious effect on the marrow microenvironment than previously thought. Examination of marrow stromal cell potential using a colony-forming unit-fibroblast (CFU-F) assay revealed that marrow from 5-FU x 2-treated mice (n = 3) contained 1.1 ± 0.4 colonies/marrow femur equivalent (FE), whereas marrow from 160 cGy-treated mice (n = 3) contained 33.8 ± 13.4 colonies/FE. In comparison, marrow from untreated mice (n = 3) averaged 95.6 ± 27.5 colonies/FE. These data suggest that homing efficiency correlates with donor chimerism in these two models of submyeloablative conditioning, and establish at least two potential mechanisms (a lesser reduction of host LTRA, and decreased donor cell homing efficiency) for the lower level of long-term donor chimerism observed in 5-FU x 2- compared to 160 cGy-conditioned hosts. Furthermore, 5-FU x 2 conditioning appears to disrupt the marrow microenvironment to a greater degree than low-dose radiation. Current efforts are aimed at exploring the molecular interactions of homed donor cells with conditioned marrow.

Description: Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.

Description: The failure to devise a satisfactory means of adjusting all sedimentation rates to a standard cell volume, together with a critical analysis of the results of other investigators, casts serious doubt on the validity of any such correction charts. Two formulae are presented by means of which the sedimentation rate of a given sample of blood can be predicted on the basis of the plasma/cell ratio and plasma proteins concentrations. One formula utilizes the results of saltingout analysis of plasma proteins, the other those of electrophoretic analysis of serum proteins. The most influential factors in determininsg sedimentation rate are plasma/cell ratio, fibrinogen, alpha-2 globulin, and gamma globulin. The results suggest that the effective concentrations of the plasma proteins are most adequately expressed as concentrations per unit of cell volume. Addition of purified protein fractions (fibrinogen, gamma globulin, and albumin) to the blood in vitro confirm the findings from the statistical studies and suggest a direct cause and effect relationship between the concentration of these proteins and the rate of erythrocyte sedimentation. Determination of the erythrocyte sedimentation rate cannot give, per se, any clue as to the level of any one of the responsible factors. An appreciation of the multiple factors involved is essential for an adequate interpretation of the clinical significance of a sedimentation rate determination.

Description: Seventy-seven cytomegalovirus (CMV)-seronegative marrow transplant patients were randomized in a prospective controlled trial comparing the use of leukocyte-depleted platelets plus CMV-seronegative red blood cells with standard unscreened blood products for the prevention of primary CMV infection during the first 100 days after transplant. Eligible patients included CMV-seronegative patients undergoing autologous transplant or seronegative patients undergoing allogeneic transplant for aplastic anemia or non-hematologic malignancy who had seronegative marrow donors. Patients and marrow donors were serologically screened for CMV and randomized before conditioning for transplant and followed for CMV infection with weekly cultures of throat, urine, and blood and with weekly CMV serologies until day 100 after transplant. Leukocyte-depleted platelets were prepared by centrifugation, a procedure that removed greater than 99% of leukocytes. There were no CMV infections observed in 35 evaluable treatment patients compared with seven infections in 30 evaluable control patients (P = .0013). There was no statistically significant difference in the mean number of platelet concentrates in the treatment patients (164 concentrates) compared with the control patients (126 concentrates). Leukocyte-depleted platelets plus CMV-seronegative red blood cells are highly effective in preventing primary CMV infection after marrow transplant.

Description: Background: Angiogenesis has previously been shown to contribute to acute graft-versus-host disease (aGVHD) post-allogeneic hematopoietic cell transplantation (HCT), but its association with response to therapy is not known. We hypothesized that patients with abundant circulating angiogenic factors (AF) involved in repair/regeneration would have improved outcomes relative those with higher levels of AF involved in damage/inflammation. Patients and Methods: We measured by MILLIPLEX magnetic bead array circulating levels of AF known predominantly for repair/regeneration (epidermal growth factor [EGF], fibroblast growth factor (FGF)-1, FGF-2, heparin binding-EGF-like growth factor, follistatin [FS], vascular endothelial growth factor [VEGF]-A, VEGF-C, and VEGF-D) and those known predominantly for endothelial dysfunction/inflammation (angiopoietin-2 [Ang2], endothelin-1, endoglin [sEng], leptin, placental growth factor [PlGF]) in HCT recipients with grade III-IV acute GVHD (N=17) compared to recipients who did not experience aGVHD post-HCT (N=17) and healthy stem cell donors (HD, N=16). AF demonstrating 1.5-fold difference in median with p‰¤0.1 in the pilot study were validated in a cohort with aGVHD (N=158) enrolled in Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0802, with samples analyzed at aGVHD onset and day 28 post-GVHD treatment, compared to allogeneic HCT recipients from the University of Minnesota who did not experience aGVHD (N=53). Results: In the pilot study, AF associated with repair/regeneration were altered at aGVHD onset. Specifically, FS was 1.9-fold elevated in patients with aGVHD, while EGF and VEGF-A levels were lower compared to HCT recipients without aGVHD. Three AF that are indicative of damage/inflammation (PlGF, Ang2, and sEng) were elevated in aGVHD patients compared to HD. In the validation cohort (Table), levels of FS were 2-fold higher, and levels of sEng and PlGF were 1.7-fold higher in aGVHD compared to HCT controls. As with the pilot study, levels of EGF and VEGF-A were lower (6-fold and 2.6-fold, respectively) in aGVHD. None of the factors differed by grade or organ stage. Patients who received grafts from matched unrelated donors (URD) had a 50% increase in PlGF levels at aGVHD onset compared to sibling donors HCT with aGVHD (31.3 versus 21.8 pg/mL, p=0.03). Patients with aGVHD who had a complete response (CR) to therapy at day 28 had higher levels of EGF and VEGF-A compared to those who had a partial response or no response (Figure 1). Patients with FS levels 〉85th percentile (〉1,355 pg/mL, Figure 2) had an increased risk of death (RR 2.1, 95% CI 1.1 €“ 3.8, p=0.03). Conclusions: Three AFs were elevated at aGVHD onset: FS, sEng and PlGF. Elevated FS is associated with shortened survival. Although FS is critical for tissue regeneration after injury, excess FS may reflect more extensive tissue damage or loss and has recently been linked to delayed healing in a rodent model. EGF and VEGF-A levels are low in aGVHD and are higher with CR at day 28, which may reflect improved tissue production or reduced losses. PlGF levels are disproportionately elevated in aGVHD after URD HCT. Further studies are evaluating the impact of these 5 angiogenic factors on healing and the regulation of inflammation post-HCT, with a goal to identify novel, non-immunosuppressive treatments to improve outcomes of aGVHD. Table Six angiogenic factors in patients at aGVHD onset compared to those without aGVHD and day 28 post-treatment. Factor (median, pg/mL) Onset (N=158) No aGVHD (N=53) Day 28 (N=158) Follistatin 855 445* 1,022 EGF 18 112* 15 VEGF-A 117 309* 137 Endoglin 1,691 1,013* 1,722** Ang2 3,264 3,590 2,734** PlGF 26 14* 32 Compared to onset, *p

Description: In paroxysmal nocturnal hemoglobinuria (PNH), lack of the GPI-anchored terminal complement inhibitor CD59 from erythrocytes renders them susceptible to chronic hemolysis, which is central to the signs and symptoms of PNH. Patients are at elevated risk for thrombosis, experience anemia that may require transfusion support, and suffer from fatigue that can be severe. Patients often have a poor quality of life resulting from PNH related symptoms including pain, dyspnea, dysphagia and erectile dysfunction, which negatively impact quality of life. The prevalence and severity of symptoms were explored in the context of a multi-national content validation study, of patients not receiving eculizumab therapy, employing the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue and the European Organization for Research and Treatment of Cancer Quality of Life Core 30 (EORTC QLQ-C30) instruments. Symptom questions were asked of 29 PNH patients (19 men, 10 women, mean age 41.2±13.2 years) from the United Kingdom, United States, France and Spain. More than half (52%) had PNH for over 5 years. Most (76%) reported never having had a blood clot, 31% reported not receiving any medication for their PNH, and 59% reported either that they had never been transfused or had not received transfusion within the last year for PNH. Patients viewed overall quality of life, global health, functioning, fatigue, pain, and shortness of breath as important PNH-related signs/symptoms. Both the FACIT-Fatigue and EORTC instruments were relevant and adequate in assessing the level of fatigue and other quality of life measures in PNH. The burden of disease in this multicultural and diverse cohort of patients was significant: 76% were forced to modify their daily activities to manage their PNH and 17% were unemployed due to PNH. Nearly all (96%) complained of fatigue and more than half reported abdominal pain, headache and shortness of breath (Table). Patients also commonly reported dysphagia (41%) and erectile dysfunction (47% in males). Most patients reported these PNH-related symptoms as moderate to very severe, and a substantial majority reported distress associated with the symptoms. Significant disease burden was identified in a diverse population of PNH patients, most of which had minimal or no transfusion requirements and a low incidence of thrombosis. Therapy that controls hemolysis and thereby improves fatigue, pain, shortness of breath, dysphagia and erectile dysfunction may prove beneficial for PNH patients with these disease characteristics.

Description: This study describes the magnitude of risk of therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) in 578 individuals diagnosed with Ewing sarcoma and enrolled on Children's Oncology Group therapeutic protocol, INT-0091. Between 1988 and 1992, patients with or without metastatic disease were randomized to receive doxorubicin, vincristine, cyclophosphamide, and dactinomycin (regimen A) or these 4 drugs alternating with etoposide and ifosfamide (regimen B). Between 1992 and 1994, patients with metastatic disease were nonrandomly assigned to receive high-intensity therapy (regimen C: regimen B therapy with higher doses of doxorubicin, cyclophosphamide, and ifosfamide). Median age at diagnosis of Ewing sarcoma was 12 years, and median length of follow-up, 8 years. Eleven patients developed t-MDS/AML, resulting in a cumulative incidence of 2% at 5 years. While patients treated on regimens A and B were at a low risk for development of t-MDS/AML (cumulative incidence: 0.4% and 0.9% at 5 years, respectively), patients treated on regimen C were at a 16-fold increased risk of developing t-MDS/AML (cumulative incidence: 11% at 5 years), when compared with those treated on regimen A. Increasing exposure to ifosfamide from 90 to 140 g/m2, cyclophosphamide from 9.6 to 17.6 g/m2, and doxorubicin from 375 to 450 mg/m2 increased the risk of t-MDS/AML significantly.

Description: An association has been established between human Ia-like antigenic determinants, expression during DNA synthesis on multipotential (CFU- GEMM) and erythroid (BFU-E) progenitor cells, and the regulatory action of acidic isoferritins in vitro. Treatment of human bone marrow cells with monoclonal anti-Ia (NE1–011) plus complement inhibited colony formation of CFU-GEMM) and BFU-E by 50%-70%. Reduction of colonies was similar whether bone marrow cells were exposed to anti-Ia plus complement, high specific tritiated thymidine (3HTdr), or acidic isoferritins. No further decrease was apparent with 3HTdr or acidic isoferritins after Ia-antigen+ CFU-GEMM or BFU-E were removed, or with anti-Ia plus complement or acidic isoferritins after S-phase CFU-GEMM or BFU-E were removed. Anti-Ia, in the absence of complement, had no effect on colony formation but blocked the inhibition of CFU-GEMM and BFU-E by acidic isoferritins. Demonstration of Ia-antigens on BFU-E and inhibition of BFU-E by acidic isoferritins appeared to require the presence of phytohemmagglutinin leukocyte conditioned medium (PHA-LCM) in the culture medium during the 14-day incubation period. these results implicate Ia-antigen+ cells, acidic isoferritins, and PHA-LCM in the regulation of multipotential and erythroid progenitor cells in vitro.

Description: Fetal hemoglobin (Hb F) production in sickle cell (SS) disease and in normal individuals varies over a 20-fold range and is under genetic control. Previous studies suggested that variant Hb F levels might be controlled by genetic loci separate from the beta-globin complex on chromosome 11. Using microscopic radial immunodiffusion and flow cytometric immunofluorescent assays to determine the percentage of F reticulocytes and F cells in SS and nonanemic individuals, we observed that F-cell levels were significantly higher in nonanemic females than males (mean +/- SD, 3.8% +/- 3.2% v 2.7% +/- 2.3%). F-cell production as determined by F reticulocyte levels in SS females was also higher than in SS males (17% +/- 10% v 13% +/- 8%). We tested the hypothesis that F-cell production in both normal and anemic SS individuals was controlled by an X-linked locus with two alleles, high (H) and low (L). Using an algorithm to determine the 99.8% confidence interval of a normal distribution in nonanemic individuals, we estimated that males and females with at least one H allele had greater than 3.3% F cells. Comparisons of male-male or female-female SS sib pairs with discordant F reticulocyte levels distinguished two phenotypes in SS males (L, less than 12%; H, greater than 12%) and three phenotypes in SS females (LL, less than 12%; HL, 12% to 24%, HH greater than 24%). Linkage analysis using polymorphic restriction sites along the X chromosome in eight SS and one AA family localized the F-cell production (FCP) locus to Xp22.2, with a maximum lod score (logarithm of odds of linkage v independent assortment) of 4.6 at a recombination fraction of 0.04.

Description: Background: The HCT-CI is a recently developed comorbidity score which has been adapted to hematopoietic stem cell transplantation, and identified 3 risk groups with increased non-relapse mortality (NRM) and lower overall survival (OS) (Blood2005;106:2912). We determined the HCT-CI score in a cohort of patients who underwent myeloablative MUD transplantation in a single arm, institutional trial assessing the efficacy of a combination of cyclosporine, methotrexate and prednisone for GVHD prophylaxis. Methods: The analysis included all patients undergoing MUD transplant from 1996–2006 who received GVHD prophylaxis with cyclosporine 2 mg/kg iv BID from day −2, methotrexate 15 mg/m2 iv on day +1 and 10 mg/m2 iv on days +3 and +6, and methylprednisolone 0.25 mg/kg iv BID beginning on day +7 and tapering from day +28. Patients were stratified by disease risk per CIBMTR classification. The comorbidities were obtained by retrospective chart review and scored according to the HCT-CI score. Results: 150 patients (median age 40) received the 3 drug-regimen, including 38% with low-, 34% with intermediate- and 28% with high-risk disease. Diagnoses included acute leukemia in 50%, MDS in 12%, CML in 15%, lymphoma in 18%, and multiple myeloma in 3.0%. Conditioning regimens included Cy-TBI in 64% and Bu-Cy in 21%. Source of stem cells was PBSC in 47.3% and marrow in 50.7%. HCT-CI scores of 0, 1–2 or ≥3 were found in 17%, 30% and 53% of patients evaluated. The majority of comorbidities were pulmonary (72%). With a median follow-up of 46 weeks, day 100 and 5-year OS were 82.7 and 33%, with a 23% and 50.4% cumulative incidence of NRM. Five year relapse-related mortality was 15.8%. Although higher HCT-CI scores were associated with increased NRM and decreased OS, no statistically significant differences were detected when using the published HCT-CI grouping of 0, 1–2 and ≥3. Unadjusted hazard ratio (HR) for inferior survival were 0.9 (CI 0.47–1.85, P=.79) and 1.65 (CI 0.885–3.090, P=.11) for scores 1–2 and ≥3, respectively. We then determined an alternate prognostic model based on 2 groups. Statistical modeling separated patients with a score of 0–3 (n=97, 64%) and ≥4 (n=53, 35.6%), with a 3 month and 5 year OS of 84% and 45% versus 52% and 10%, respectively (P