bluuebunny/crossref_metadata_2025 huggingface dataset:
Release of Polymorphonuclear Leukocytes From the Bone Marrow by Interleukin-8
Authors: Terashima Takeshi , English Dean , Hogg James C. , vanEeden Stephan F.
Published: October 2019

Several studies have shown that interleukin-8 (IL-8) causes a rapid granulocytosis with the release of polymorphonuclear leukocytes (PMN) from the bone marrow (BM) partially responsible for the granulocytosis. This study was designed to quantitate the release of PMN from the BM by IL-8 and measure the transit time of PMN through the marrow after IL-8 administration. The thymidine analogue, 5'-bromo-2'-deoxyuridine (BrdU), was used to label dividing PMN in the marrow and follow their release into the circulation after intravenous IL-8. This allowed us to calculate the transit time of PMN through the mitotic and postmitotic pools of BM. BrdU was infused intravenously into rabbits 24 hours before IL-8 (2.5 μg/kg). IL-8 caused a rapid, transient granulocytopenia (5.9 ± 0.4 at baseline v 0.2 ± 0.06 × 10/9L at 5 minutes, P < .05) followed by granulocytosis (8.4 ± 0.1 at 30 minutes, P < .05) associated with an increased number (0.3 ± 0.1 at baseline v1.2 ± 0.6 × 109/L at 30 minutes, P < .05) and percentage of band cells (P < .05), as well as a rapid increase in the number of BrdU-labeled PMN (PMNBrdU) in the circulation (0.09 ± 0.05 at baseline to 1.5 ± 0.6 × 109/L at 60 minutes, P < .05). The transit time of PMN through both the mitotic and postmitotic pools of BM was not affected by IL-8. To determine the marrow compartment from which the PMN were mobilized by IL-8, we quantitated PMN movement from the hematopoietic and sinusoidal compartments into the circulation. The fraction of PMNBrdU in both compartments was higher than in the circulating blood (P < .05) and the fraction and number of PMNBrdU in the sinusoids decreased with IL-8 treatment (P < .05). We conclude that the pool of PMN residing in the BM venous sinusoids are rapidly released into the circulation after administration of IL-8.

© 1998 by The American Society of Hematology.
Expression of Cyclin E and the Cyclin-Dependent Kinase Inhibitor p27 in Malignant Lymphomas—Prognostic Implications
Authors: Erlanson Martin , Portin Cajsa , Linderholm Barbro , Lindh Jack , Roos Göran , Landberg Göran
Published: October 2019

Cyclin E and the cyclin-dependent kinase inhibitor p27 are two important regulators of the G1-S transition modulating the activity of cyclin-dependent kinases. Aberrations in the cell cycle control are often observed in tumors and might even be mandatory in tumor development. To investigate the importance of cell-cycle defects in malignant lymphomas we have characterized the expression of cyclin E and p27 in 105 newly diagnosed lymphomas using immunohistochemistry. A significant, inverse correlation between p27 and cyclin E expression was observed (rs = −.24, P = .02) and both proteins correlated with the S-phase fraction (rs = −.35, P < .001 andrs = .45, P < .001, respectively). The inverse relationship between p27 expression and proliferation was abrogated in some lymphomas, suggesting that p27 downregulation can represent a genuine aberration. Survival analysis was performed in 105 patients with a median observation time of 86 months. Low p27 and high cyclin E expression were significantly associated with a poor prognosis (P = .0001 and .03, respectively). In a multivariate Cox analysis, p27 expression, stage, serum lactate dehydrogenase level, grade, and age were independent prognostic factors, in contrast to S-phase fraction and cyclin E expression. This is the first report showing that p27 expression in malignant lymphomas has independent prognostic significance, which necessitates future studies regarding its more precise biological role in lymphoid tumorogenesis.

© 1998 by The American Society of Hematology.
Allelotype Analysis of Adult T-Cell Leukemia
Authors: Hatta Yoshihiro , Yamada Yasuaki , Tomonaga Masao , Said Jonathan W. , Miyosi Isao , Koeffler H. Phillip
Published: October 2019

Allelotype analysis of adult T-cell leukemia (ATL) was undertaken for the first time to identify chromosomal loci relevant to the development of acute/lymphomatous ATL. Loss of heterozygosity (LOH) was screened using 94 highly polymorphic microsatellite markers, distributed among all nonacrocentric, autosomal chromosomes. In each of the 22 cases, DNA obtained from their leukemic cells in acute/lymphomatous phase was compared with their constitutional DNA from mononuclear cells in chronic or remission phase. Allelic losses of at least on one chromosome arm occurred in 91% of the cases (20 individuals). Among 39 chromosome arms, allelic losses were observed on 31 arms at least for one sample. A high frequency of allelic loss (>30%) was seen on chromosome arms 6q (41%) and 17p (48%). The mean fractional allelic loss (FAL) was 0.109. These findings suggest that a novel tumor suppressor gene on chromosome arm 6q, as well as the p53 gene on chromosome arm 17p, probably have an important role in the development of acute/lymphomatous ATL.

© 1998 by The American Society of Hematology.
Decreased Rejection and Improved Survival of First and Second Marrow Transplants for Severe Aplastic Anemia (A 26-Year Retrospective Analysis)
Authors: Stucki Anne , Leisenring Wendy , Sandmaier Brenda M. , Sanders Jean , Anasetti Claudio , Storb Rainer
Published: October 2019

Between 1970 and 1996, 333 patients with severe aplastic anemia underwent HLA-matched related marrow transplant after conditioning with cyclophosphamide (CY). Thirty-five percent of patients transplanted between 1970 and 1976 (group 1), 12% of those transplanted between 1977 and 1981 (group 2), and 9% of patients transplanted between 1982 and 1997 (group 3) had graft rejection. Graft rejection occurred later among group 3 patients (median, 180 days) than among those in groups 1 and 2 (medians, 28 and 47 days, respectively; P < .001 group 3 v 2). In group 3, 92% of rejecting patients underwent a second transplant, compared with 78% and 77% in groups 1 and 2, respectively. Group 1 patients received various conditioning regimens before second transplant, whereas most patients of groups 2 and 3 received CY combined with antithymocyte globulin (ATG). Graft-versus-host disease (GVHD) prophylaxis after second transplant consisted of methotrexate (MTX) for all group 1 and 2 patients, whereas group 3 patients received MTX combined with cyclosporine (CSP). Over the three time periods studied, first graft rejection decreased from 35% to 9%, and the proportion of rejecting patients undergoing second transplants increased from 77% to 92%. The 10-year probability of survival after second transplants increased from 5% to 83%. Multivariate analysis showed MTX/CSP GVHD prophylaxis to be a significant factor accounting for the increase in patient survival after second transplant.

© 1998 by The American Society of Hematology.
The Relationship Between Thiopurine Methyltransferase Activity and Genotype in Blasts From Patients With Acute Leukemia
Authors: Coulthard Sally A. , Howell Christopher , Robson Jill , Hall Andrew G.
Published: October 2019

The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of thiopurines used in the treatment of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Studies in red blood cells (RBC) have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. The genetic basis for this polymorphism has now been elucidated and polymerase chain reaction (PCR)-based assays described for the most common mutations accounting for reduced activity. In previous studies, genotype has been correlated with red blood cell activity. In this report, we describe the relationship between genotype and TPMT activity measured directly in the target of drug action, the leukemic cell. We have demonstrated that the TPMT activity in lymphoblasts from 38 children and adults found by PCR to be homozygotes (*1/*1) was significantly higher than that in the five heterozygotes (*1/*3) detected (median, 0.25 v 0.08, P < .002, Mann-Whitney U). Similar results were obtained when results from children were analyzed separately. However, comparison of activity in blasts from AML and ALL showed a higher level in the former (0.35 v 0.22 nU/mg,P < .002, n = 17, 35), suggesting that factors other than genotype may also influence expression.

© 1998 by The American Society of Hematology.
Abnormal Microsomal Detoxification Implicated in Fanconi Anemia Group C by Interaction of the FAC Protein With NADPH Cytochrome P450 Reductase
Authors: Kruyt Frank A.E. , Hoshino Taizo , Liu Johnson M. , Joseph Pius , Jaiswal Anil K. , Youssoufian Hagop
Published: October 2019

The FAC protein encoded by the Fanconi anemia (FA) complementation group C gene is thought to function in the cytoplasm at a step before DNA repair. Because FA cells are susceptible to mitomycin C, we considered the possibility that FAC might interact with enzymes involved in the bioreductive activation of this drug. Here we report that FAC binds to NADPH cytochrome-P450 reductase (RED), a microsomal membrane protein involved in electron transfer, in both transfected COS-1 and normal murine liver cells. FAC-RED interaction requires the amino-terminal region of FAC and the cytosolic, membrane-proximal domain of the reductase. The latter contains a known binding site for flavin mononucleotide (FMN). Addition of FMN to cytosolic lysates disrupts FAC-reductase complexes, while flavin dinucleotide, which binds to a distinct carboxy-terminal domain, fails to alter FAC-RED complexes at concentrations similar to FMN. FAC is also functionally coupled to this enzyme as its expression in COS-1 cells suppresses the ability of RED to reduce cytochrome c in the presence of NADPH. We propose that FAC plays a fundamental role in vivo by attenuating the activity of RED, thereby regulating a major detoxification pathway in mammalian cells.

© 1998 by The American Society of Hematology.
Reversal of Lethal - and β-Thalassemias in Mice by Expression of Human Embryonic Globins
Authors: Russell J. Eric , Liebhaber Stephen A.
Published: October 2019

Genetic mutations that block - or β-globin gene expression in humans can result in severe and frequently lethal thalassemic phenotypes. Homozygous inactivation of the endogenous - or β-globin genes in mice results in corresponding thalassemic syndromes that are uniformly fatal in utero. In the current study, we show that the viability of these mice can be rescued by expression of human embryonic ζ- and -globins, respectively. The capacity of embryonic globins to fully substitute for their adult globin homologues is further demonstrated by showing that ζ- and -globins reverse the hemolytic anemia and abnormal erythrocyte morphology of mice with nonlethal forms of - and β-thalassemia. These results illustrate the potential therapeutic utility of embryonic globins as substitutes for deficient adult globins in thalassemic individuals. Moreover, the capacity of embryonic globins to functionally replace their adult homologues brings into question the physiologic basis for globin gene switching.

© 1998 by The American Society of Hematology.
Beneficial Effect of Intravenous Dexamethasone in Children With Mild to Moderately Severe Acute Chest Syndrome Complicating Sickle Cell Disease
Authors: Bernini Juan Carlos , Rogers Zora R. , Sandler Eric S. , Reisch Joan S. , Quinn Charles T. , Buchanan George R.
Published: October 2019

Acute chest syndrome (ACS) in patients with sickle cell disease (SCD) has historically been managed with oxygen, antibiotics, and blood transfusions. Recently high-dose corticosteroid therapy was shown to reduce the duration of hospitalization in children with SCD and vaso-occlusive crisis. Therefore, we chose to assess the use of glucocorticoids in ACS. We conducted a randomized, double-blind placebo-controlled trial to evaluate the efficacy and toxicity of intravenous dexamethasone (0.3 mg/kg every 12 hours × 4 doses) in children with SCD hospitalized with mild to moderately severe ACS. Forty-three evaluable episodes of ACS occurred in 38 children (median age, 6.7 years). Twenty-two patients received dexamethasone and 21 patients received placebo. There were no statistically significant differences in demographic, clinical, or laboratory characteristics between the two groups. Mean hospital stay was shorter in the dexamethasone-treated group (47 hours v 80 hours; P = .005). Dexamethasone therapy prevented clinical deterioration and reduced the need for blood transfusions (P < .001 and = .013, respectively). Mean duration of oxygen and analgesic therapy, number of opioid doses, and the duration of fever was also significantly reduced in the dexamethasone-treated patients. Of seven patients readmitted within 72 hours after discharge (six after dexamethasone; P = .095), only one had respiratory complications (P = 1.00). No side effects clearly related to dexamethasone were observed. In a stepwise multiple linear regression analysis, gender and previous episodes of ACS were the only variables that appeared to predict response to dexamethasone, as measured by lengh of hospital stay. Intravenous dexamethasone has a beneficial effect in children with SCD hospitalized with mild to moderately severe acute chest syndrome. Further study of this therapeutic modality is indicated.

© 1998 by The American Society of Hematology.
Caspase 2 and Caspase 3 Protein Levels as Predictors of Survival in Acute Myelogenous Leukemia
Authors: Estrov Zeev , Thall Peter F. , Talpaz Moshe , Estey Elihu H. , Kantarjian Hagop M. , Andreeff Michael , Harris David , Van Quin , Walterscheid Monika , Kornblau Steven M.
Published: October 2019

Because caspase activation is an essential step in programmed cell death (apoptosis) and cytotoxic drug-induced apoptosis is mediated by caspase 2 and caspase 3, we hypothesized that caspase 2 and 3 levels predict clinical outcome in acute myelogenous leukemia (AML). Using quantitative Western blot analysis, we studied the levels of nonactivated (uncleaved) caspase 2 and 3 in peripheral blood low-density cells from 185 patients with newly diagnosed AML. We also measured the level of activated (cleaved) caspase 3 in 41 randomly selected samples from the 185 patients. Finally, we analyzed the effect of caspase 2 and 3 levels and other prognostic variables on patient survival using a multivariate Cox model. We found that median levels of nonactivated caspase 2 and 3 were higher in AML than in normal peripheral blood cells (P < .001 and P <.02, respectively). There was no association between caspase level and either the percentage of peripheral blasts or any specific type of leukemia cell cytogenetic abnormalities. When the effect of each uncleaved caspase was considered individually, a high level of uncleaved caspase 3 (P = .04), but not of caspase 2 (P = .16), was associated with decreased survival. Conversely, a high level of cleaved caspase 3 denoted improved survival and correlated with the inactivation of the DNA-repair enzyme poly(ADP-ribose) polymerase. Thus, cleaved caspase 3 could stimulate the apoptotic cascade further, and lack of its activation likely caused an accumulation of the uncleaved caspase. Although uncleaved caspase 2 level per se had no prognostic significance, the interactive effect of high levels of both uncleaved caspase 2 and 3 denoted very poor survival (P < .001) and had the largest effect of all prognostic variables (P < .001; estimated relative risk, 2.49; 95% confidence interval, 1.59 to 3.90). Taken together, caspase 2 and caspase 3 protein levels obtained at diagnosis may constitute a reliable prognostic factor in AML.

© 1998 by The American Society of Hematology.
Successful Use of Extracorporeal Photochemotherapy in the Treatment of Severe Acute and Chronic Graft-Versus-Host Disease
Authors: Greinix Hildegard T. , Volc-Platzer Beatrix , Rabitsch Werner , Gmeinhart Bernd , Guevara-Pineda Carlos , Kalhs Peter , Krutmann Jean , Hönigsmann Herbert , Ciovica Marina , Knobler Robert M.
Published: October 2019

Extracorporeal exposure of peripheral blood mononuclear cells to the photosensitizing compound 8-methoxypsoralen and ultraviolet A radiation has been shown to be effective in the treatment of several T-cell–mediated diseases, including cutaneous T-cell lymphoma and rejection after organ transplantation. We present 21 patients (10 men and 11 women) with hematological malignancies with a median age of 36 years (range, 25 to 55 years) who had received marrow grafts from sibling (n = 12) or unrelated (n = 9) donors. Six patients had acute graft-versus-host disease (GVHD) grade II to III not responding to cyclosporine A (CSA) and prednisolone when referred to extracorporeal photochemotherapy (ECP). In 15 patients, 2 to 24 months after bone marrow transplantation (BMT), extensive chronic GVHD with involvement of skin (n = 15), liver (n = 10), oral mucosa (n = 11), ocular glands (n = 6), and thrombocytopenia (n = 3) developed and was unresponsive to conventional therapy, including steroids. All patients were treated with ECP on 2 consecutive days every 2 weeks for the first 3 months and thereafter every 4 weeks until resolution of GVHD. ECP was tolerated excellently without any significant side effects. After a median of 14 cycles of ECP, acute GVHD resolved completely in 4 of 6 patients (67%) and partially in another 2 patients. Cutaneous chronic GVHD completely resolved in 12 of 15 (80%) patients. Contractures of knees and elbows due to scleroderma resolved partially. Oral mucosal ulcerations resolved in all patients. Seven of 10 patients (70%) with liver involvement had complete responses after ECP. After discontinuation of ECP, no severe infections were observed. Our findings suggest that ECP is a safe and effective adjunct therapy for both acute and extensive chronic GVHD with skin and visceral involvement and resistance to conventional therapy.

© 1998 by The American Society of Hematology.
Relationship Between Cleaved L-Selectin Levels and the Outcome of Acute Myeloid Leukemia
Authors: Extermann M. , Bacchi M. , Monai N. , Fopp M. , Fey M. , Tichelli A. , Schapira M. , Spertini O.
Published: October 2019

High plasma levels of the shed form of L-selectin (sL-selectin) are frequently detectable in acute myeloid leukemia (AML). sL-selectin can inhibit blast cell adhesion to vascular endothelium and may thereby influence the phenotype of AML. In this study, we have investigated the relationship between sL-selectin levels and clinical presentation or disease outcome in 100 patients with AML. Fifty-eight patients were found to have sL-selectin levels ≥3.12 μg/mL (≥3 SD above the mean of healthy controls: “increased”). Patients with extramedullary disease such as lymphadenopathies, splenomegaly, hepatomegaly, and/or muco-cutaneous infiltration had significantly increased sL-selectin levels (P < .001). sL-selectin levels were significantly heterogeneous in the French-American-British subtypes (P = .0003). Patients with “normal” sL-selectin levels had higher probability of achieving complete remission (CR) than with “increased” levels: 81% versus 64%, respectively (P = .06). When adjusting for clinically relevant covariates predictive for CR (sex, age, Auer rods), “normal” sL-selectin levels were significantly associated with CR (odds ratio, 3.08; 95% confidence interval [CI], 1.10 to 8.58;P = .03). Moreover, patients with “increased” sL-selectin levels (≥3.12 μg/mL) had shorter event-free survival (EFS) (median 7.3 v 12 months, P = .008) and overall survival (median 1 v 2.05 years, P = .03) than patients with sL-selectin <3.12 μg/mL. Multivariate statistical analysis (adjusted for age and presence of Auer rods) indicated that sL-selectin was an independent prognostic factor for EFS (hazard ratio [HR], 1.96; 95% CI, 1.21 to 3.17, P = .006) and overall survival (HR, 1.80; 95% CI, 1.09 to 2.98; P = .02). Thus, plasma sL-selectin may be a useful prognostic marker in the evaluation of AML at diagnosis.

© 1998 by The American Society of Hematology.
Clinical Relevance of BCL2, BCL6, and MYC Rearrangements in Diffuse Large B-Cell Lymphoma
Authors: Kramer M.H.H. , Hermans J. , Wijburg E. , Philippo K. , Geelen E. , van Krieken J.H.J.M. , de Jong D. , Maartense E. , Schuuring E. , Kluin P.M.
Published: October 2019

Diffuse large B-cell lymphoma (DLCL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 156 patients with de novo DLCL for rearrangements of the BCL2, BCL6, and MYC oncogenes by Southern blot analysis and BCL2 protein expression. We related these data to the primary site of presentation, disease stage, and other clinical risk factors. Structural alterations of BCL2, BCL6, and MYC were detected in 25 of 156, 36 of 116, and 10 of 151 patients, respectively. Three cases showed a combination of BCL2 and BCL6 rearrangements, and two cases had a combination of BCL6 and MYC rearrangements. BCL2 rearrangement was found more often in extensive (39%) and primary nodal (17%) lymphomas than in extranodal cases (4%) (P = .003). BCL2 rearrangement was present in none of 40 patients with stage I disease, but in 22% of patients with stage II to IV (P = .006). The presence of BCL2 rearrangements did not significantly affect overall survival (OS) or disease-free survival (DFS). In contrast, high BCL2 protein expression adversely affected both OS (P = .008) and DFS (P = .01). BCL2 protein expression was poorly correlated with BCL2 rearrangement: only 52% of BCL2-rearranged lymphomas and 37% of BCL2-unrearranged cases had high BCL2 protein expression. Rearrangement of BCL6 was found more often in patients with extranodal (36%) and extensive (39%) presentation versus primary nodal disease (28%). No significant correlation was found with disease stage, lymphadenopathy, or bone marrow involvement. DFS and OS were not influenced by BCL6 rearrangements. MYC rearrangements were found in 16% of primary extranodal lymphomas, versus 2% of primary nodal cases (P = .02). In particular, gastrointestinal (GI) lymphomas (5 of 18 cases, 28%) were affected by MYC rearrangements. The distinct biologic behavior of these extranodal lymphomas was reflected by a high complete remission (CR) rate: 7 of 10 patients with MYC rearrangement attained complete remission and 6 responders remained alive for more than 4 years, resulting in a trend for better DFS (P = .07). These data show the complex nature of molecular events in DLCL, which is a reflection of the morphologic and clinical heterogeneity of these lymphomas. However, thus far, these genetic rearrangements fail as prognostic markers.

© 1998 by The American Society of Hematology.
Role of Members of the Wnt Gene Family in Human Hematopoiesis
Authors: Van Den Berg David J. , Sharma Arun K. , Bruno Edward , Hoffman Ron
Published: October 2019

The hematopoietic system is derived from ventral mesoderm. A number of genes that are important in mesoderm development have been identified including members of the transforming growth factor-β (TGF-β) superfamily, the fibroblast growth factor (FGF) family, and the Wnt gene family. Because TGF-β plays a pleiotropic role in hematopoiesis, we wished to determine if other genes that are important in mesoderm development, specifically members of theWnt gene family, may play a role in hematopoiesis. Three members of the Wnt gene family (Wnt-5A, Wnt-2B, and Wnt-10B) were identified and cloned from human fetal bone stromal cells. These genes are expressed to varying levels in hematopoietic cell lines derived from T cells, B cells, myeloid cells, and erythroid cells; however, only Wnt-5A was expressed in CD34+Lin− primitive progenitor cells. The in vitro biological activity of these Wnt genes on CD34+Lin− hematopoietic progenitors was determined in a feeder cell coculture system and assayed by quantitating progenitor cell numbers, CD34+ cell numbers, and numbers of differentiated cell types. The number of hematopoietic progenitor cells was markedly affected by exposure to stromal cell layers expressing Wnt genes with 10- to 20-fold higher numbers of mixed colony-forming units (CFU-MIX), 1.5- to 2.6-fold higher numbers of CFU-granulocyte macrophage (CFU-GM), and greater than 10-fold higher numbers of burst-forming units-erythroid (BFU-E) in the Wnt-expressing cocultures compared with the controls. Colony formation by cells expanded on theWnt-expressing cocultures was similar for each of the three genes, indicating similar action on primitive progenitor cells; however, Wnt-10B showed differential activity on erythroid progenitors (BFU-E) compared with Wnt-5A and Wnt-2B. Cocultures containing Wnt-10B alone or in combination with all three Wnt genes had threefold to fourfold lower BFU-E colony numbers than the Wnt-5A– or Wnt-2B–expressing cocultures. The frequency of CD34+ cells was higher inWnt-expressing cocultures and cellular morphology indicated that coculture in the presence of Wnt genes resulted in higher numbers of less differentiated hematopoietic cells and fewer mature cells than controls. These data indicate that the gene products of theWnt family function as hematopoietic growth factors, and that they may exhibit higher specificity for earlier progenitor cells.

© 1998 by The American Society of Hematology.
Erythropoietin mRNA Expression in Human Fetal and Neonatal Tissue
Authors: Dame Christof , Fahnenstich Hubert , Freitag Patricia , Hofmann Dietmar , Abdul-Nour Thair , Bartmann Peter , Fandrey Joachim
Published: October 2019

Based on animal experiments, a switch of the erythropoietin (EPO) production site from the liver in the fetus to the kidneys in the adult has been postulated. To study the switch in humans, we have quantitated EPO mRNA expression in liver, kidney, spleen, and bone marrow of human fetuses and neonates by means of a competitive polymerase chain reaction (PCR). Tissue samples from 66 routine postmortem examinations were obtained. EPO mRNA was expressed in 97% of the tissue specimen derived from the liver (n = 66) and in 93% of those from the kidneys (17 weeks of gestation until 18 months after birth; n = 59). For the first time the EPO gene was found expressed in vivo in human spleen (96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly beyond 30 weeks of gestation (P < .05). Although there was a slight decrease in EPO mRNA content per g liver tissue towards birth, the liver accounted for about 80% of the total body EPO mRNA. The contribution of the spleen and bone marrow were minor compared with liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch.

© 1998 by The American Society of Hematology.
Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers
Authors: Sansonno Domenico , Lotesoriere Claudio , Cornacchiulo Vito , Fanelli Massimo , Gatti Pietro , Iodice Giuseppe , Racanelli Vito , Dammacco Franco
Published: October 2019

Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production.

© 1998 by The American Society of Hematology.
The Presence of Typical and Atypical BCR-ABL Fusion Genes in Leukocytes of Normal Individuals: Biologic Significance and Implications for the Assessment of Minimal Residual Disease
Authors: Bose Shikha , Deininger Michael , Gora-Tybor Joanna , Goldman John M. , Melo Junia V.
Published: October 2019

The number of genetic lesions necessary to generate leukemia in humans is unknown, but it is possible that certain specific abnormalities, eg, fusion genes, known to be associated with acute and chronic leukemia are produced relatively frequently in human cells but require other events to occur before the leukemia becomes manifest. We investigated this possibility by studying peripheral blood leukocytes from normal individuals and various hematopoietic cell lines for the presence and expression of the p210 and the p190 types of the BCR-ABL gene associated with chronic myeloid leukemia (CML) and acute lymphoblastic leukemia. We used two-step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in which batches of 108 cells per sample were tested in 40 replicate reactions. We estimate that this assay is 1.5 logs more sensitive than the two-step RT-PCR assays that we use routinely to assess minimal residual disease. BCR-ABL fusion gene transcripts of various configurations were found in circulating leukocytes from 12 of the 16 healthy adults analyzed. Transcripts with an e1a2 junction (p190 BCR-ABL) were present in 11 and p210-type transcripts with b2a2 and/or b3a2 junctions were detected in 4 individuals. The same RT-PCR assays in non-CML cell lines showed the presence of classical or aberrant p210-type mRNA in 3 of 7 lines and of p190-type transcripts in all 7 lines of hematopoietic origin (HL60, KG1, U937, Kasumi, Jurkat, JVM13, and JVM25), whereas the NIH3T3 murine fibroblast line was reproducibly negative for these fusion genes. These findings confirm and extend previous reports on the detection of leukemia-associated genes in normal leukocytes and suggest that certain fusion genes are generated relatively frequently in hematopoietic cells, but only infrequently do the cells acquire the additional changes necessary to produce leukemia in humans. Although there is only a small probability that such innocent BCR-ABL–carrying leukocytes are detected by conventional RT-PCR assays, they may be the source of some sporadically positive tests in leukemia patients in long-term remission.

© 1998 by The American Society of Hematology.
Prognostic Significance of a Polymerase Chain Reaction–Detectable Dominant T-Lymphocyte Clone in Cutaneous Lesions of Patients With Mycosis Fungoides
Authors: Delfau-Larue Marie-Hélène , Dalac Sophie , Lepage Eric , Petrella Tony , Wechsler Janine , Farcet Jean-Pierre , Bagot Martine
Published: October 2019

Although mycosis fungoides (MF) is considered to be an indolent lymphoma, survival is highly influenced by TNM stage. At diagnosis, most MF patients present with early stage disease and a high probability of long-term survival. Treatment is generally directed towards skin lesions, and achievement and duration of complete responses are variable. A dominant T-cell clone is detectable in the cutaneous lesions of 60% of patients. The aim of this study was to determine whether the presence of a T-cell clonal population influences the clinical course of the disease after topical therapy. Cutaneous biopsies from 68 patients were histologically diagnosed as MF and T-cell clonality was analyzed by in vitro amplification of TCR-γ chain gene rearrangements (polymerase chain reaction γ [PCRγ]). After a median follow-up of 48 months, response to treatment was clinically assessed. Age, sex, duration of symptoms before diagnosis, type of cutaneous lesions (T stage), TNM stage, and PCRγ were evaluated as predictive factors of response to treatment in univariate and multivariate analyses. Univariate analysis demonstrated that T1 cutaneous lesions (P = .05) and PCRγ negativity (P = .007) were associated with a higher complete remission rate. Using multivariate analysis, T stage (relative risk, 3.13; P = .06) and PCRγ (relative risk, 4.4; P = .01) remained independent significant predictive parameters of response. In conclusion, T stage and cutaneous PCRγ at diagnosis are the two predictive parameters of treatment response for MF. Therefore, the cutaneous PCRγ findings should be considered in the analysis of future therapeutic trials.

© 1998 by The American Society of Hematology.
A Multicenter Prospective Study on the Risk of Acquiring Liver Disease in Anti–Hepatitis C Virus Negative Patients Affected From Homozygous β-Thalassemia
Authors: Prati Daniele , Zanella Alberto , Farma Elena , De Mattei Claudia , Bosoni Patrizia , Zappa Manuela , Picone Alessandra , Mozzi Fulvio , Rebulla Paolo , Cappellini Maria Domenica , Allain Jean-Pierre , Sirchia Girolamo
Published: October 2019

Although the risk of transfusion-transmitted hepatitis has been recently reduced, transfusion-dependent β-thalassemia patients may still develop liver disease due to viral infection or iron overload. We assessed the frequency and causes of liver dysfunction in a cohort of anti–hepatitis C virus (HCV) negative thalassemics. Of 1,481 thalassemics enrolled in 31 centers, 219 (14.8%) tested anti-HCV− by second-generation assays; 181 completed a 3-year follow-up program consisting of alanine-aminotransferase (ALT) measurement at each transfusion and anti-HCV determination by third-generation enzyme-immunoassay (EIA-3) at the end of study. Serum ferritin levels were determined at baseline and at the end of follow-up. Ten patients were anti-HCV+ by EIA-3 at the end of follow-up. Of them, seven were already positive in 1992 to 1993 when the initial sera were retested by EIA-3, one tested indeterminate by confirmatory assay, and two had true seroconversion (incidence, 4.27/1,000 person years; risk of infection, 1/7,100 blood units, 95% confidence interval [CI], 1 in 2,000-1 in 71,000 units). At baseline, 67 of 174 thalassemics had abnormal ALT. Of those with normal ALT, seven subsequently developed at least one episode of moderate ALT increase (incidence, 24.6/1,000 person-years). All of the 20 patients with ferritin values ≥3,000 ng/mL had clinically relevant ALT abnormalities, as compared with 53 of 151 with <3,000 ng/mL (P< .005). Hepatic dysfunction is still frequent in thalassemics. Although it is mainly attributable to siderosis and primary HCV infection, the role of undiscovered transmissible agents cannot be excluded.

© 1998 by The American Society of Hematology.
Osteoclast-Mediated Bone Resorption Is Stimulated During Short-Term Administration of Granulocyte Colony-Stimulating Factor But Is Not Responsible for Hematopoietic Progenitor Cell Mobilization
Authors: Takamatsu Yasushi , Simmons Paul J. , Moore Robert J. , Morris Howard A. , To Luen B. , Lévesque Jean-Pierre
Published: October 2019

The cellular and molecular mechanisms responsible for hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) into peripheral blood after administration of cytokines such as granulocyte colony-stimulating factor (G-CSF) are still unknown. In this study we show that high concentrations of soluble calcium induce the detachment of BM CD34+ HPC adherent on fibronectin, a major component of BM extracellular matrix. Because G-CSF has been shown to induce osteoporosis in patients with congenital neutropenia and in G-CSF–overexpressing transgenic mice, we hypothesized that short-term G-CSF administration may be sufficient to induce bone resorption, resulting in the release of soluble calcium in the endosteum leading in turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced after 3 days of G-CSF administration. Furthermore, in patients mobilized with G-CSF either alone or in association with stem cell factor or interleukin-3, the reduction of serum osteocalcin is significantly correlated with the number of HPC mobilized in peripheral blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of bone resorption, gradually elevated during the time course of G-CSF administration until day 7 after cessation of G-CSF, showing a simultaneous stimulation of bone degradation during G-CSF–induced HPC mobilization. In an in vivo murine model, we found that the number of osteoclasts was dramatically increased paralleling the elevation of DPyr after G-CSF administration. When pamidronate, an inhibitor of osteoclast-mediated bone resorption, was administered together with G-CSF in mice, the G-CSF–induced increase of DPyr levels was completely abolished whereas the numbers of colony-forming cells mobilized in peripheral blood were not decreased, but unexpectedly increased relative to the numbers elicited by G-CSF alone. Collectively, our data therefore show that short-term administration of G-CSF induces bone degradation by a simultaneous inhibition of bone formation and an enhanced osteoclast-mediated bone resorption. This increased bone resorption is inhibited by pamidronate without reducing G-CSF–induced HPC mobilization, suggesting that the activation of bone resorption after G-CSF administration is not the direct cause of HPC mobilization as initially hypothesized, but a parallel event.

© 1998 by The American Society of Hematology.
Age Is Not a Prognostic Variable With Autotransplants for Multiple Myeloma
Authors: Siegel D.S. , Desikan K.R. , Mehta J. , Singhal S. , Fassas A. , Munshi N. , Anaissie E. , Naucke S. , Ayers D. , Spoon D. , Vesole D. , Tricot G. , Barlogie B.
Published: October 2019

Multiple myeloma (MM) typically afflicts elderly patients with a median age of 65 years. However, while recently shown to provide superior outcome to standard treatment, high-dose therapy (HDT) has usually been limited to patients up to 65 years. Among 550 patients with MM and a minimum follow-up of 18 months, 49 aged ≥65 years were identified (median age, 67; range, 65 to 76 years). Their outcome was compared with 49 younger pair mates (median, 52; range, 37 to 64 years) selected among the remaining 501 younger patients (<65 years) matched for five previously recognized critical prognostic factors (cytogenetics, β2-microglobulin, C-reactive protein, albumin, creatinine). Nearly one half had been treated for more than 1 year with standard therapy and about one third had refractory MM. All patients received high-dose melphalan-based therapy; 76% of the younger and 65% of the older group completed a second transplant (P = .3). Sufficient peripheral blood stem cells to support two HDT cycles (CD34 > 5 × 106/kg) were available in 83% of younger and 73% of older patients (P = .2). After HDT, hematopoietic recovery to critical levels of granulocytes (>500/μL) and of platelets (>50,000/μL) proceeded at comparable rates among younger and older subjects with both first and second HDT. The frequency of extramedullary toxicities was comparable. Treatment-related mortality with the first HDT cycle was 2% in younger and 8% among older subjects, whereas no mortality was encountered with the second transplant procedure. Comparing younger/older subjects, median durations of event-free and overall survival were 2.8/1.5 years (P = .2) and 4.8/3.3 years (P = .4). Multivariate analysis showed pretransplant cytogenetics and β2-microglobulin levels as critical prognostic features for both event-free and overall survival, whereas age was insignificant for both endpoints (P = .2/.8). Thus, age is not a biologically adverse parameter for patients with MM receiving high-dose melphalan-based therapy with peripheral blood stem cell support and, hence, should not constitute an exclusion criterion for participation in what appears to be superior therapy for symptomatic MM.
Granulocyte Colony-Stimulating Factor–Mobilized Allogeneic Stem Cell Transplantation Maintains Graft-Versus-Leukemia Effects Through a Perforin-Dependent Pathway While Preventing Graft-Versus-Host Disease
Authors: Pan Luying , Teshima Takanori , Hill Geoffrey R. , Bungard David , Brinson Yani S. , Reddy Vijay S. , Cooke Kenneth R. , Ferrara James L.M.
Published: October 2019

Minimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)–mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF–mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 → B6D2F1). B6 mice (H-2b) were injected subcutaneously with human G-CSF (100 μg/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v0%, P < .001). Systemic levels of lipopolysaccharide and tumor necrosis factor- were markedly reduced in recipients of allogeneic G-CSF–mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2d) at the time of transplantation, all surviving recipients of G-CSF–mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell–depleted (TCD) splenocytes from G-CSF–mobilized B6 donors died of leukemia. When splenocytes from G-CSF–mobilized perforin-deficient (pfp−/−) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF–mobilized PBSCT. These data illustrate that G-CSF–mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.
Presenting White Blood Cell Count and Kinetics of Molecular Remission Predict Prognosis in Acute Promyelocytic Leukemia Treated With All-Trans Retinoic Acid: Result of the Randomized MRC Trial
Authors: Burnett Alan K. , Grimwade David , Solomon Ellen , Wheatley Keith , Goldstone Anthony H.
Published: October 2019

All-trans retinoic acid (ATRA) is an essential component of the treatment of acute promyelocytic leukemia (APL), but the optimal timing and duration remain to be determined. Molecular characterization of this disease can refine the diagnosis and could be potentially useful in monitoring response to treatment. Patients defined morphologically to have APL were randomized to receive a 5-day course of ATRA before commencing chemotherapy or to receive daily ATRA commencing with chemotherapy and continuing until complete remission (CR). The chemotherapy was that used in current MRC Leukaemia Trials. Outcome comparisons were by intention to treat with additional analysis for relevant risk factors. Patients were characterized by molecular techniques for the fusion products of the t(15;17) and monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) during and after treatment. Two hundred thirty-nine patients were randomized. Treatment with extended ATRA resulted in a superior remission rate (87% v 70%, P < .001), due to fewer early and induction deaths (12% v 23%, P = .02), and less resistant disease (2% v 7%, P = .03), which was associated with a significantly more rapid recovery of neutrophils and platelets. Extended ATRA reduced relapse risk (20%v 36% at 4 years, P = .04) and resulted in superior survival (71% v 52% at 4 years, P = .005). Presenting white blood cell count (WBC) was a key determinant of outcome. The 70% of patients who presented with a WBC less than 10 × 109/L had a better CR (85% v62%, P = .0001) and reduced relapse risk (22% v42%, P = .002) and superior survival (69%v 43%, P < .0001). Within the low count group, extended ATRA resulted in a better CR (94% v 76%, P= .001), reduced relapse risk (13% v 35%, P = .04), and improved survival (80% v 57%, P = .0009). There was no evidence of benefit in patients presenting with a higher WBC (>10 × 109/L). Molecular monitoring after the third chemotherapy course had a correlation with risk of relapse. The relapse risk was 57% if the RT-PCR was positive versus 27% if the RT-PCR was negative (P = .006). APL patients who present with a low WBC derive substantial benefit from combining ATRA with induction chemotherapy until remission is achieved, whereas patients with a higher WBC did not benefit. Molecular characterization of disease can improve diagnostic precision and a positive RT-PCR after consolidation identifies patients at a higher risk of relapse.
Defective Internalization and Sustained Activation of Truncated Granulocyte Colony-Stimulating Factor Receptor Found in Severe Congenital Neutropenia/Acute Myeloid Leukemia
Authors: Ward Alister C. , van Aesch Yvette M. , Schelen Anita M. , Touw Ivo P.
Published: October 2019

Acquired mutations truncating the C-terminal domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) are found in about 20% of severe congenital neutropenia (SCN) patients, with this cohort of patients predisposed to acute myeloid leukemia (AML). In myeloid cells, such mutations act in a dominant-negative manner leading to hyperproliferation and lack of differentiation in response to G-CSF. However, why these truncated receptors are dominant in function over wild-type receptors has remained unclear. We report that ligand-induced internalization of truncated G-CSF-R is severely impaired compared with the wild-type receptor, which results in sustained activation of STAT proteins. Strikingly, in cells coexpressing both truncated and wild-type forms, the truncated receptors acted dominantly with regard to both internalization and sustained activation. Site-directed mutagenesis of the C-terminus showed that receptor tyrosines in this region were dispensable for internalization, whereas a di-leucine–containing motif in Box B3 played some role. However, loss of the di-leucine motif was not the critical determinant of the sustained activation status of truncated receptors. These data suggest that defective internalization, leading to extended receptor activation, is a major cause of the dominant hyperproliferative effect of truncated G-CSF receptors, which is only partially due to the loss of a di-leucine motif present in the Box B3 region of the full-length receptor.