Rona S. Weinberg

Hematotoxicity in Workers Exposed to Low Levels of Benzene

Benzene is known to have toxic effects on the blood and bone marrow, but its impact at levels below the U.S. occupational standard of 1 part per million (ppm) remains uncertain. In a study of 250 workers exposed to benzene, white blood cell and platelet counts were significantly lower than in 140 controls, even for exposure below 1 ppm in air. Progenitor cell colony formation significantly declined with increasing benzene exposure and was more sensitive to the effects of benzene than was the number of mature blood cells. Two genetic variants in key metabolizing enzymes, myeloperoxidase and NAD(P)H:quinone oxidoreductase, influenced susceptibility to benzene hematotoxicity. Thus, hematotoxicity from exposure to benzene occurred at air levels of 1 ppm or less and may be particularly evident among genetically susceptible subpopulations.

MPD-RC 101 prospective study of reduced-intensity allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis

From 2007 to 2011, 66 patients with primary myelofibrosis or myelofibrosis (MF) preceded by essential thrombocythemia or polycythemia vera were enrolled into a prospective phase 2 clinical trial of reduced-intensity allogeneic hematopoietic stem cell transplantation (AHSCT), Myeloproliferative Disorder Research Consortium 101 trial. The study included patients with sibling donors (n = 32) receiving fludarabine/melphalan (FluMel) as a preparative regimen and patients with unrelated donors (n = 34) receiving conditioning with FluMel plus anti-thymocyte globulin (ATG). Patient characteristics in the 2 cohorts were similar. Engraftment occurred in 97% of siblings and 76% of unrelated transplants, whereas secondary graft failure occurred in 3% and 12%, respectively. With a median follow-up of 25 months for patients alive, the overall survival (OS) was 75% in the sibling group (median not reached) and 32% in the unrelated group (median OS: 6 months, 95% confidence interval [CI]: 3, 25) (hazard ratio 3.9, 95% CI: 1.8,8.9) (P < .001). Nonrelapse mortality was 22% in sibling and 59% in unrelated AHSCT. Survival correlated with type of donor, but not with the degree of histocompatibility match, age, or JAK2(V617F) status. In patients with MF with sibling donors, AHSCT is an effective therapy, whereas AHSCT from unrelated donors with FluMel/ATG conditioning led to a high rate of graft failure and limited survival. This trial was registered at www.clinicaltrials.gov as #NCT00572897.

Polymorphisms in Cytokine and Cellular Adhesion Molecule Genes and Susceptibility to Hematotoxicity among Workers Exposed to Benzene

Benzene is a recognized hematotoxin and leukemogen but its mechanism of action and the role of genetic susceptibility are still unclear. Cytokines, chemokines, and cellular adhesion molecules are soluble proteins that play an important regulatory role in hematopoiesis. We therefore hypothesized that variation in these genes could influence benzene-induced hematotoxicity. We analyzed common, well-studied single-nucleotide polymorphisms (SNPs) in 20 candidate genes drawn from these pathways in a study of 250 workers exposed to benzene and 140 unexposed controls in China. After accounting for multiple comparisons, SNPs in five genes were associated with a statistically significant decrease in total WBC counts among exposed workers [IL-1A (-889C>T), IL-4 (-1098T>G), IL-10 (-819T>C), IL-12A (8685G>A), and VCAM1 (-1591T>C)], and one SNP [CSF3 (Ex4-165C>T)] was associated with an increase in WBC counts. The adhesion molecule VCAM1 variant was particularly noteworthy as it was associated with a decrease in B cells, natural killer cells, CD4+ T cells, and monocytes. Further, VCAM1 (-1591T>C) and CSF3 (Ex4-165C>T) were associated, respectively, with decreased (P = 0.041) and increased (P = 0.076) CFU-GEMM progenitor cell colony formation in 29 benzene-exposed workers. This is the first report to provide evidence that SNPs in genes that regulate hematopoiesis influence benzene-induced hematotoxicity.

Spleens of myelofibrosis patients contain malignant hematopoietic stem cells

Cancer stem cell behavior is thought to be largely determined by intrinsic properties and by regulatory signals provided by the microenvironment. Myelofibrosis (MF) is characterized by hematopoiesis occurring not only in the marrow but also in extramedullary sites such as the spleen. In order to study the effects of these different microenvironments on primitive malignant hematopoietic cells, we phenotypically and functionally characterized splenic and peripheral blood (PB) MF CD34+ cells from patients with MF. MF spleens contained greater numbers of malignant primitive HPCs than PB. Transplantation of PB MF CD34+ cells into immunodeficient (NOD/SCID/IL2Rγ(null)) mice resulted in a limited degree of donor cell chimerism and a differentiation program skewed toward myeloid lineages. By contrast, transplanted splenic MF CD34+ cells achieved a higher level of chimerism and generated both myeloid and lymphoid cells that contained molecular or cytogenetic abnormalities indicating their malignant nature. Only splenic MF CD34+ cells were able to sustain hematopoiesis for prolonged periods (9 months) and were able to engraft secondary recipients. These data document the existence of MF stem cells (MF-SCs) that reside in the spleens of MF patients and demonstrate that these MF-SCs retain a differentiation program identical to that of normal hematopoietic stem cells.

Switch from fetal to adult hemoglobin is associated with a change in progenitor cell population.

To examine the switch from fetal to adult hemoglobin at the cellular level, erythroid progenitor cells from newborn infants and adults were cultured in methyl cellulose with erythropoietin. Individual erythroid colonies were labeled with [3H]leucine at various times, and globin synthesis patterns examined by gel electrophoresis and fluorography. The percent gamma- or beta-globin synthesis was determined from the total of gamma + beta, and the percent G gamma from the total of G gamma + A gamma. The nonparametric correlation coefficients of percent G gamma with percent gamma or beta were obtained. Each group of colonies at each time point was examined separately. In colonies from adult blood, the proportion of G gamma-synthesis did not correlate with the proportion of gamma-synthesis. Colonies from newborn blood fell into two groups. Those that developed from relatively mature progenitor cells, and were seen on day 14, showed a strong negative correlation of G gamma with beta-globin synthesis. However, those newborn colonies that developed from immature progenitors, and were seen later in culture (days 17 and 21), showed no correlation of G gamma with beta-synthesis. These findings are compatible with a clonal model for hemoglobin switching. Fetal progenitors, in which G gamma- and beta-syntheses are negatively correlated, are gradually replaced during ontogeny by adult progenitors. The adult progenitors produce more beta (less gamma), and the proportions of G gamma- and gamma- or beta-synthesis are not correlated.

Comparison of erythroid progenitor cell growth in vitro in polycythemia vera and chronic myelogenous leukemia: Only polycythemia vera has endogenous colonies☆

The ability of erythroid cultures to distinguish among myeloproliferative disorders was examined. We studied 14 patients with polycythemia vera (PV), 11 with chronic myelogenous leukemia (CML), four with non-PV erythrocytosis, two with agnogenic myeloid metaplasia, as well as three normal fetuses and greater than 25 normal adults. Endogenous, i.e. grew without added erythropoietin, bone marrow CFU-E-derived colonies were observed in all but one PV patient. However, endogenous blood BFU-E-derived bursts were observed in only eight of 14 PV patients. Endogenous erythroid colonies were not seen in cultures from any normal adults or fetuses, or patients with CML, erythrocytosis, or myeloid metaplasia. In PV, relative HbF synthesis was always greater in cultures without erythropoietin, while in cultures from all other patients relative HbF synthesis was similar to that observed in cultures from normal individuals. We conclude that PV and CML are distinguishable in culture since CML patients do not have endogenous growth. Most important, endogenous bone marrow CFU-E-derived colonies are the only consistently unique observation in patients with PV, and endogenous CFU-E- and BFU-E-derived colonies and bursts are not uniformly observed in PV blood cultures. In-vitro studies of erythropoiesis to confirm the diagnosis of PV, therefore, require marrow when endogenous colonies and bursts are absent from blood cultures.

Erythropoiesis is Distinct at Each Stage of Ontogeny

In vitro erythropoiesis from fetuses, newborn infants, and adults was compared in methyl cellulose cultures. Fetal and newborn blood erythroid colony formation tended to be more sensitive to erythropoietin than adult. The day of maximal colony formation was earlier in fetal than in newborn or adult cultures. The number of colonies/100 000 mononuclear cells on d 13 of culture and on the day of peak growth was highest in fetal, intermediate in newborn, and lowest in adult cultures. Burst forming units-erythroid/mL of blood on culture d 13 and the day of peak growth were similar in fetuses and newborns, and both were significantly greater than in adults. The proportional synthesis of 7-globin in fetal colonies was 2-fold greater than in newborn colonies, and 6-fold greater than in adult colonies. Thus, fetal, newborn, and adult erythroid progenitor cultures are each unique with regard to erythropoietin sensitivity, growth time course, number of erythroid colonies, and the proportion of 7-globin synthesis.

Lipocalin produced by myelofibrosis cells affects the fate of both hematopoietic and marrow microenvironmental cells

Myelofibrosis (MF) is characterized by cytopenias, constitutional symptoms, splenomegaly, and marrow histopathological abnormalities (fibrosis, increased microvessel density, and osteosclerosis). The microenvironmental abnormalities are likely a consequence of the elaboration of a variety of inflammatory cytokines generated by malignant megakaryocytes and monocytes. We observed that levels of a specific inflammatory cytokine, lipocalin-2 (LCN2), were elevated in the plasmas of patients with myeloproliferative neoplasms (MF > polycythemia vera or essential thrombocythemia) and that LCN2 was elaborated by MF myeloid cells. LCN2 generates increased reactive oxygen species, leading to increased DNA strand breaks and apoptosis of normal, but not MF, CD34(+) cells. Furthermore, incubation of marrow adherent cells or mesenchymal stem cells with LCN2 increased the generation of osteoblasts and fibroblasts, but not adipocytes. LCN2 priming of mesenchymal stem cells resulted in the upregulation of RUNX2 gene as well as other genes that are capable of further affecting osteoblastogenesis, angiogenesis, and the deposition of matrix proteins. These data indicate that LCN2 is an additional MF inflammatory cytokine that likely contributes to the creation of a cascade of events that results in not only a predominance of the MF clone but also a dysfunctional microenvironment.

Adult‘fetal-like’erythropoiesis characterizes recovery from bone marrow transplantation

The transient fetal‐like erythropoiesis which appears during recovery from bone marrow transplantation has now been examined at the level of erythroid progenitor cells. A 7‐year‐old boy with β+‐thalassaemia major was studied during engraftment from his β‐thalassaemia trait sister. Hb F and i antigen rose as expected. Macrocytosis never developed, but red cell size distribution became very heterogeneous. Bone marrow CFU‐E and BFU‐E were detected by 30 d, prior to the appearance of reticulocytes. Marrow erythroid progenitor cell numbers were normal by 146 d, while those in the blood became normal by 360 d. After transplantation globin synthesis ratios in erythroid colonies were diagnostic of thalassaemia trait, indicating engraftment. Individual erythroid colonies derived from both blood and marrow at all times during reconstitution showed no correlation of Gγ and γ. Thus the fetal‐like stress erythropoiesis of marrow expansion following transplantation was derived from adult and not fetal progenitor cells.

Sickle and Thalassemic Erythroid Progenitor Cells are Different from Normal

Blood erythroid progenitors (BFU-E) from patients with sickle and thalassemic syndromes were compared with those from normal individuals. The day of maximal colony formation in methyl cellulose was slightly later in the cultures from the patients with hemoglobinopathies than in the normal cultures. The number of colonies/100,000 mononuclear cells was similar in all cultures on day 13, but was higher in the hemoglobinopathy cultures on the day of maximal growth. The number of BFU-E/mL of blood was significantly higher than normal at all times in both sickle cell anemia and thalassemia. The proportional synthesis of gamma globin was twice normal in all sickle cultures, and 4 times normal in those from beta+-thalassemia. Hemin and interleukin-3 increased the numbers of erythroid colonies in all cultures, but did not consistently alter the globin synthesis patterns. Each progenitor population has a unique pattern in terms of time course, number of BFU-E, and level of gamma globin synthesis. These features indicate distinct types of BFU-E, or differences in accessory cells, or both, which distinguish blood-borne erythropoiesis in normals and those with hemoglobinopathies.