Spontaneous Erythroid Colony Formation Research Articles

Metformin Reduces Tumor Burden and Splenomegaly in JAK2V617F Murine Models

Introduction : The recurrent gain-of-function JAK2V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). Metformin (1,1-dimethylbiguanide) is a biguanide widely prescribed for the treatment of type II diabetes and metabolic syndromes. In recent years, studies using cancer cell lines and murine models have provided evidence for potential anticancer activity of metformin. Previous results from our research group indicate that metformin reduces cell viability, proliferation, clonogenicity and JAK2/STAT activation in human JAK2V617F cell lines and spontaneous erythroid colony formation in primary cells from patients with polycythemia vera (Machado-Neto et al. ASH Annual Meeting Abstracts #1960, 2016). Objectives : To investigate the effects of metformin treatment in tumor formation and MPN phenotype using Ba/F3 JAK2V617F cells and Jak2V617F knockin-induced MPN mice. Methods : For tumor formation, experimental groups consisted of 8-10 week-old NSG female mice that received a 100 µL (50 µL PBS plus 50 µL matrigel) dorsal subcutaneous injection of 2×106 Ba/F3 JAK2V617F cells and daily treatment by intraperitoneal injection of vehicle (PBS, n=9) or metformin (125 mg/kg, n=9) for 13 days after initial tumor growth (7 days). Tumor volume (V) was obtained using the formula (V = W2 × L × 0.52), where W and L are the smaller and larger diameters, respectively. For MPN phenotype induction, 5×106 bone marrow cells from Jak2V617F knockin (Jak2WT/V617F) mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry in peripheral blood. Mice with >70% CD45.2 cells were randomized and treated daily by intraperitoneal injection of vehicle (PBS, n=4) or metformin (125 mg/kg, n=5) for 6 weeks. At the conclusion of the experiment, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen were evaluated by CD71 and Ter119 markers (Becton-Dickinson) by flow cytometry. All experiments were approved by the Animal Ethics Committee. For comparisons, Mann-Whitney test or ANOVA test and Bonferroni post-test was used. A p value <0.05 was considered as statistically significant. Results : In NSG mice inoculated with Ba/F3 JAK2V617F, metformin treatment (125 mg/kg/day) was able to significantly reduce the tumor burden after 13 days of treatment. At the endpoint, the mean±SD of tumor volume for vehicle (n=4) and metformin (n=4) treated mice was 1120.7±289.1 mm3 and 510.3±244.4 mm3, respectively (p <0.001). A second independent experiment including five mice in each group presented similar results (vehicle versus metformin: 1307.7±511.9 mm3versus 725.1±380.6 mm3; p <0.01). In Jak2V617F knockin-induced MPN animals, metformin treatment (125 mg/kg/day) was well-tolerated, reduced splenomegaly (p=0.02) and improved splenic architecture. Erythoid progenitors in the spleen and bone marrow did not differ between control and metformin-treated groups (p >0.05), but the smaller spleens presented with a very low percentage of erythroid progenitors. Hemoglobin and hematocrit were not modulated by metformin treatment. Conclusions : Metformin treatment at a dose of 125 mg/kg/day effectively reduced tumor burden in Ba/F3 JAK2V617F allograft and splenomegaly in Jak2V617F knockin-induced MPN murine models. In humans, this dose represents a pharmacological dose similar to that which is used for type II diabetes treatment. Our results provide additional insights for metformin in preclinical models of MPN. DisclosuresDruker:Millipore: Patents & Royalties: Royalties from Dana-Farber Cancer Institute, which has an exclusive commercial license with Millipore for monoclonal antiphosphotyrosine antibody 4G10, which I developed while employed at DFCI.; Henry Stewart Talks: Patents & Royalties; Novartis: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties: #843 Mutated ABL Kinase Domains (licensed to various companies); #0996 Detection of Gleevec Resistant Mutations (licensed to various companies, including MolecularMD); #0606 Treatment of Gastrointestinal Stromal Tumors (exclusively licensed to Novartis); Beta Cat: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Baxalta US Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MED-C: Membership on an entity's Board of Directors or advisory committees; GRAIL: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; The Leukemia & Lymphoma Society: Other: Joint Steering Committee of AML Master Protocol, Research Funding; Third Coast Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche TCRC: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Bristol-Myers Squibb: Research Funding.

Metformin exerts multitarget antileukemia activity in JAK2V617F-positive myeloproliferative neoplasms

The recurrent gain-of-function JAK2V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor ruxolitinib indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that exerts selective antineoplastic activity in hematological malignancies. In the present study, we investigate and compare effects of metformin and ruxolitinib alone and in combination on cell signaling and cellular functions in JAK2V617F-positive cells. In JAK2V617F-expressing cell lines, metformin treatment significantly reduced cell viability, cell proliferation, clonogenicity, and cellular oxygen consumption and delayed cell cycle progression. Metformin reduced cyclin D1 expression and RB, STAT3, STAT5, ERK1/2 and p70S6K phosphorylation. Metformin plus ruxolitinib demonstrated more intense reduction of cell viability and induction of apoptosis compared to monotherapy. Notably, metformin reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice and spontaneous erythroid colony formation in primary cells from polycythemia vera patients. In conclusion, metformin exerts multitarget antileukemia activity in MPN: downregulation of JAK2/STAT signaling and mitochondrial activity. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action and provides insights for development of alternative/complementary therapeutic strategies for MPN.

Multitarget Antileukemic Effects of Metformin in Myeloproliferative Neoplasm Cells: Inhibition of JAK2/STAT Signaling and Mitochondrial Activity

Background: The recurrent JAK2V617F gain-of-function mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that inhibits the mitochondrial complex I and metformin antineoplastic activity has been described in leukemia, lymphoma and multiple myeloma. More importantly, metformin antineoplastic effects appear to act selectively in cancer cells, since cytotoxicity was not observed for normal hematopoeitic cells. Considering that metformin has been widely used in clinical practice, preclinical studies in an attempt to determine its effects, alone or in combination with ruxolitinib, in MPN, may be of interest, since these findings have a translational potential. Objectives: To investigate the effects of metformin and/or ruxolitinib on cell signaling and cellular functions of JAK2V617F cells. Metformin and/or ruxolitinib effects were also evaluated on spontaneous erythroid colony formation from polycythemia vera (PV) patients. Materials and methods: HEL, SET2 (JAK2V617F) and Ba/F3 JAK2V617F cell lines treated or not with metformin (5 and 10 mM) and/or ruxolitinib (300 nM) were submitted for cell viability (MTT), clonogenicity (colony formation), cell cycle (flow cytometry), cell proliferation (Ki-67 staining), apoptosis (annexin-V/PI and caspase 3/PARP1 cleavage), PCR array for PI3K/AKT-related genes and oxygen consumption (high-resolution respirometry). For spontaneous erythroid colony formation assays, mononuclear cells from peripheral blood or bone marrow of PV patients (n=3) were cultured on methylcellulose supplied with hematopoietic growth factors except EPO, in the presence or not of metfomin (2.5 mM) and/or ruxolitnib (50 nM). Gene and protein expressions were evaluated by qPCR and Western blot. Statistical analyses were performed by ANOVA test or Student’s t-test. Results: In HEL, SET2 and Ba/F3 JAK2V617F cells, metformin or ruxoltinib treatment significantly reduced cell viability and clonogenicity (p<0.05). Ruxolitinib plus metformin significantly decreased cell viability and induced apoptosis when compared with monotherapy (p<0.05). Metformin and/or ruxolitinib delay cell cycle progression (p<0.05). In HEL cells, PCR-array identified 11, 14 and 17 genes modulated by metformin, ruxolitinib, and combined treatment, respectively. Reduced cyclin D1 (CCND1) and upregulated p27 (CDKN1B) were validated in HEL and SET2 cells (p<0.05). We also observed downregulation of retinoblastoma (RB) phosphorylation, a cyclin D1 target and a key cell cycle progression-related protein, in both JAK2V617F cell lines upon all treatment conditions. Ruxolitinib treatment reduced STAT3, STAT5, ERK1/2, 4EBP1 and P70S6K phosphorylation. Metformin also reduced activation of same proteins, but combined treatment presented more intense inhibition of ERK1/2 and P70S6K activation compared to metformin or ruxolitinib monotherapy. A strong reduction of cellular oxygen consumption supported by exogenous substrates in culture media (ROUTINE state), at the non-phosphorylating state (LEAK) and at the maximum respiratory capacity (ETS state) was observed in metformin- and metformin plus ruxolitinib-treated HEL, SET2 and Ba/F3 JAK2V617F cells (p<0.05). Ruxolitinib significantly reduced oxygen consumption at routine, leak and ETS states only in SET2 and Ba/F3 JAK2V617F cells and at LEAK state in HEL cells. In primary cells from PV patients, a reduction of 71±21%, 94±4%, 98±1% was observed on spontaneous erythroid colony formation upon metformin, ruxolitinib and metformin plus ruxolitinib treatment, respectively. Conclusion: Metformin exerts an anticancer activity and downregulates JAK2/STAT and MAPK signaling in JAK2V617F cells. PCR-array identified cyclin D1 and p27 as target of metformin and ruxolitinib, corroborating cell cycle and proliferation findings. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action on JAK2V617F aberrant signaling and provides insights for development of alternative/complementary therapeutic strategies for MPN. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia inhibits JAK2V617F activation via suppression of SHP-2 function in myeloproliferative neoplasm cells

The hypoxic microenvironment of the bone marrow, known as the hypoxic niche, supports hematopoietic stem cell quiescence and maintains long-term repopulation activity. Hypoxia also affects the expansion of progenitor cells and enhances erythropoiesis and megakaryopoiesis. In contrast to the known effects of hypoxia on normal hematopoiesis, the effects of the hypoxic environment of the bone marrow on the pathogenesis of myeloproliferative neoplasms (MPNs) have not been well studied. In the present study, we investigated the role of the hypoxic environment in the pathophysiology of MPNs, focusing on JAK2V617F, a major driver of mutation in Philadelphia-negative MPNs. We found that the activity of JAK2V617F was suppressed in hypoxic conditions not only in JAK2V617F-positive leukemia cells, but also in primary peripheral blood mononuclear cells from patients with polycythemia vera. Concomitant with the inhibition of JAK2V617F activity, hypoxia increased the expression of p27/KIP1, the primary negative regulator of the cell cycle, and inhibited cell cycle progression in JAK2V617F-positive leukemia cell lines. The spontaneous erythroid colony formation of primary cells from polycythemia vera patients was also suppressed under hypoxic conditions. We also revealed that the hypoxia-induced overproduction of reactive oxygen species played a crucial role in the inhibition of JAK2V617F through the oxidation and inhibition of SHP-2, a protein tyrosine phosphatase that contains SH-2, which is required for JAK2 activation. In conclusion, a hypoxic environment may modulate JAK2-positive MPN cell fate and disease progression through the suppressionofSHP-2 function and the subsequent suppression of JAK2V617F activity.

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

AbstractPolycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.

Prognostic utility of spontaneous erythroid colony formation and JAK2 mutational analysis for thrombotic events in essential thrombocythaemia

Thrombotic events in essential thrombocythaemia (ET) are difficult to predict with current risk stratification based on age and prior history of thrombosis. We aimed to assess the predictive value of the JAK2 V617F mutation (JAK2) and spontaneous erythroid colony (SEC) growth for the development of thrombotic events post diagnosis in patients with ET. Consecutive patients with ET were retrospectively identified, and clinical and laboratory correlates were evaluated. Thrombotic events were categorized according to their occurrence at or prior to diagnosis (prior thrombosis), and any time post diagnosis of ET (subsequent thrombosis). JAK2 analysis was performed by allele-specific PCR on whole blood or bone marrow. A total of 62 patients was identified, median age 63 years; 67% (41/61) JAK2-positive and 47% (25/53) SEC-positive. Median follow-up was 33 months (range, 1 to 137). JAK2-positive patients showed a trend to increased prior thrombosis (27% vs 5%, P= 0.08), and a significant increase in the development of subsequent thrombosis (5-year event rate 31% vs 6%, P= 0.04), which persisted when stratified for a history of prior thrombosis (P= 0.04). Survival was not affected by JAK2 status. The SEC assay predicted an increased rate of baseline thrombosis (16% vs 0%, P= 0.04), but was not found to be predictive of any subsequent thrombotic events. Patients with ET who are JAK2-positive by whole blood allele-specific PCR appear to be at increased risk of thrombotic complications, which is independent of a prior history of thrombosis.

JAK2 Gene Is Mutated in Patients with Myeloproliferative Disorders and Spontaneous Erythroid Colony Formation but Not in Patients with Spontaneous Megakaryocyte Growth Only.

In the past two decades spontaneous erythroid (BFU-E) and megakaryocytic (CFU-Meg) colony formation have proved to be useful diagnostic tools in diagnosing myeloproliferative disorders (MPD). Recently a point mutation in the JAK2-gene was discovered to be a pathogenetic event in polycythemia vera (PV) and essential thrombocythemia (ET). JAK2 mutation analysis has been recommended to be used as a primary diagnostic method for these disorders. However, so far only a few studies comparing the in vitro growth pattern of hematopoietic progenitors and JAK2 mutation status have been published and, to the best of our knowledge, no studies describing the association between spontaneous CFU-Meg growth and the JAK2 status have been conducted. Therefore, the aim of this study was to compare the results obtained with these methods in PV and ET. 52 ET and 33 PV patients were studied. Allele-specific PCR based JAK2 mutation analysis and hematopoietic colony forming assays were done from bone marrow (BM) aspirate samples. Morphology of BM aspirates was analyzed in our routine diagnostic laboratory.30/33 (91%) PV patients and 35/52 (67%) ET patients showed spontaneous BFU-E growth. Spontaneous CFU-Meg growth was found in 23/33 (70%) of PV patients and in 29/52 (56%) of ET patients. JAK2 mutation was seen in 26 (79%) PV patients and in 31 (60 %) ET patients. All JAK2 mutated PV and ET patients were found to have spontaneous BFU-E growth. In addition, 4 of 7 (57%) JAK2 mutation negative PV patients and 4 of 21 (19%) JAK2 mutation negative ET patients had spontaneous BFU-E colony formation. JAK2 mutated ET patients had spontaneous CFU-Meg growth more often than JAK2 mutation negative patients (71% vs. 33%), while in PV patients there was no clear difference between the two groups (JAK2 mutation positive 69% vs. negative 71%). Interestingly, 9 patients (6 with ET, 3 with PV) had only spontaneous CFU-Meg growth but no spontaneous BFU-E growth. They were all JAK2 mutation negative. In addition, 11/52 ET patients (21%) were JAK2 mutation negative and had neither spontaneous BFU-E nor spontaneous CFU-Meg growth. BM morphology was considered suggestive of ET or PV in 83% and 70% of the cases respectively. No significant differences in BM morphology were found between JAK2 mutation negative and positive patients.In conclusion, in this group of 88 MPD patients spontaneous BFU-E colony growth was the most sensitive diagnostic assay. All patients with JAK2 mutation also had spontaneous BFU-E growth and an additional 8 patients without the mutation also had spontaneous BFU-E growth. Although recently the JAK2 mutation has been described in megakaryocytes as well, none of the present patients with spontaneous CFU-Meg growth but without spontaneous BFU-E growth were JAK2 mutation positive. This suggests that JAK2 mutation may not be the only pathogenetic event causing spontaneous CFU-Meg growth and further studies are needed to define genetic alterations behind this phenomenon.

Spontaneous erythroid colony formation in Brazilian patients with sickle cell disease

The ability of circulating progenitor cells to develop erythroid colonies was studied in vitro in the presence or absence of growth factors (5637-CM and erythropoietin) in 63 patients with sickle cell disease (SCD) (36 homozygotes for hemoglobin [Hb] S, 13 double heterozygotes for Hb S and β thalassemia, and 14 SC patients) in Southeast Brazil. In the presence of growth factors, SCD patients (all genotypes) presented significantly higher numbers of circulating burst-forming unit-erythroid (BFU-E/5 × 105 MNC), when compared with control subjects. However, when the progenitor cells were cultured in the absence of added stimulus, high numbers of BFU-E were observed only in the genotypes SS and S/β thalassemia. SC patients presented a similar response to the control subjects. Moreover, there was an inverse correlation between spontaneous (without stimulus) BFU-E and Hb levels in SCD patients. These results suggest that the formation of spontaneous BFU-E observed in SCD may be due to an expanded erythropoiesis secondary to hemolysis. Am. J. Hematol. 61:40–45, 1999. © 1999 Wiley-Liss, Inc.

Spontaneous erythroid colony formation in Brazilian patients with sickle cell disease.

The ability of circulating progenitor cells to develop erythroid colonies was studied in vitro in the presence or absence of growth factors (5637-CM and erythropoietin) in 63 patients with sickle cell disease (SCD) (36 homozygotes for hemoglobin [Hb] S, 13 double heterozygotes for Hb S and beta thalassemia, and 14 SC patients) in Southeast Brazil. In the presence of growth factors, SCD patients (all genotypes) presented significantly higher numbers of circulating burst-forming unit-erythroid (BFU-E/5 x 10(5) MNC), when compared with control subjects. However, when the progenitor cells were cultured in the absence of added stimulus, high numbers of BFU-E were observed only in the genotypes SS and S/beta thalassemia. SC patients presented a similar response to the control subjects. Moreover, there was an inverse correlation between spontaneous (without stimulus) BFU-E and Hb levels in SCD patients. These results suggest that the formation of spontaneous BFU-E observed in SCD may be due to an expanded erythropoiesis secondary to hemolysis.

Spontaneous erythroid colony formation as the clue to an underlying myeloproliferative disorder in patients with Budd-Chiari syndrome or portal vein thrombosis.

Budd-Chiari syndrome is a severe disease characterized by occlusion of large hepatic veins leading to death if untreated. Using the classical criteria for the diagnosis of polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF), overt PV was the underlying cause in about 10% of the cases and ET or IMF in only a very few. Using spontaneous endogenous erythroid colony (EEC) formation in vitro and/or bone marrow biopsies, a primary myeloproliferative disorder (PMD) was present in 78% of the patients with apparently idiopathic Budd-Chiari syndrome and in about half of the patients with portal, splenic, and/or mesenteric vein thrombosis. The diagnoses in 40 reported cases with hepatic vein thrombosis and spontaneous EEC were overt PV in 25 and latent unclassified PMD in 15 patients. The diagnoses of 40 reported cases with splanchnic vein thrombosis and spontaneous EEC were overt PV in 12, ET in 2, IMF in 2, and latent unclassified PMD with the presence of EEC in 24 patients. Thrombocytosis as a manifestation of myeloproliferative disease was recorded in 34 of 80 (42.5%) patients with spontaneous EEC and Budd-Chiari syndrome or portal vein thrombosis. Thrombocythemia was present in 15 of 41 patients with a proven and in 19 of 39 patients with a latent myeloproliferative disorder. Patients with hepatic vein or splanchnic vein thrombosis associated with a PMD are predominantly females younger than 45. It is concluded that both spontaneous EEC and histopathology from bone marrow biopsy provide specific information as sensitive clues to the diagnosis of all variants of overt and latent myeloproliferative disorders. The association of hepatic and splanchnic vein thrombosis and PMD is not fully understood. Therapeutic options of Budd-Chiari syndrome include anticoagulation with heparin, fibrinolysis followed by oral anticoagulation, and appropriate treatment of the underlying PMD. In case of failure, invasive options include local procedures such as angioplasty or stenting, venous decompression by portal-systemic shunts, or liver transplantation.

The effects of IL-1 and IL-4 on the Epo-independent erythroid progenitor in polycythaemia vera.

Human recombinant interleukin-1 (IL-1) was studied for its effects on the erythroid progenitors from normal subjects and from patients with polycythaemia vera (PV). No supportive effect of IL-1 was noticed on the normal, erythropoietin (Epo) dependent, erythroid burst-forming unit (BFU-E) using peripheral blood or bone marrow. In contrast, the Epo-independent BFU-E from peripheral blood of PV patients could be stimulated significantly. This enhancing effect of IL-1 was not only observed with unsorted but also with sorted CD34+ cells. In addition, it was shown that IL-1 indirectly stimulated the Epo-independent BFU-E because anti-GM-CSF could abrogate the supportive effects of IL-1. In contrast to the Epo-independent BFU-E, the Epo-dependent erythroid colony formation from PV patients could not be augmented by IL-1. Finally, we studied the effects of IL-4 on the Epo-independent BFU-E, because IL-4 can affect the erythroid colony formation and modulate the effects of IL-1. IL-4 suppressed the Epo-independent BFU-E. This effect could be counteracted by the addition of IL-1 to the culture medium. However, the suppressive effect of IL-4 was not related to a decline in spontaneous release of IL-1, because an anti-IL-1 antibody did not modify the spontaneous erythroid colony formation. These data indicate that IL-1 and IL-4 exert separate influences on the Epo-independent erythroid colony formation in PV.

Spontaneous erythroid colony formation in the differential diagnosis of erythrocytosis.

Erythroid colony formation in vitro was studied in 80 patients with erythrocytosis. 43 of the patients had polycythaemia vera (PV), 18 patients had secondary erythrocytosis, 6 had normal red cell mass, and 13 patients were regarded as unclassified. Spontaneous erythroid colony formation, in the absence of exogenous erythropoietin in the cultures, was discovered in all patients with PV, whereas no patient with secondary erythrocytosis or with normal red cell mass showed this phenomenon. 8 of the 13 patients with unclassified erythrocytosis spontaneously formed erythroid colonies. 7 patient with unclassified erythrocytosis have been followed for 5 yr. 3 of the 4 patients with spontaneous colony growth but none of the 3 without it can now be classified as PV. Thus, spontaneous erythroid colony formation indicates PV even in early and atypical cases. Therefore, the culture of erythroid progenitors is very useful in the differential diagnosis of problematic cases with erythrocytosis.

Erythrocytosis associated with spontaneous erythroid colony formation and idiopathic hypererythropoietinemia

Erythrocytosis associated with spontaneous erythroid colony formation and idiopathic hypererythropoietinemia

Colony formation by megakaryocytic progenitors in essential thrombocythaemia.

Colony formation by megakaryocytic progenitors (CFU-M) from the blood or bone marrow of 12 patients with essential thrombocythaemia was studied in vitro with the methyl cellulose assay. When the cultures were stimulated with plasma from a patient with aplastic anaemia and with phytohaemagglutinin stimulated conditioned medium (PHA-LCM), the patients showed a significant trend towards higher circulating CFU-M numbers when compared with the controls; three of the patients exceeded our normal range. In the bone marrow cultures there were no differences in the number or morphology of megakaryocytic colonies between the patients and the controls. When normal human plasma was the only source of colony stimulating activity, 11 out of 12 patients, but none of the controls, showed megakaryocytic colony formation. The same patients also had 'spontaneous' erythroid colony growth without the addition of exogenous erythropoietin into the cultures. Only one of the patients with essential thrombocythaemia (ET) had normal megakaryocytic and erythroid colony formation. The present study shows that, in most patients with ET 'spontaneous' CFU-M colony formation occurs in suboptimal culture conditions, a phenomenon obviously analogous to the spontaneous erythroid colony formation seen in the myeloproliferative disorders.

Spontaneous erythroid colony formation in erythrocytosis.

Erythroid colony formation from the bone marrow and blood of 30 patients with erythrocytosis has been studied. Seventeen patients formed spontaneous erythroid colonies. They included patients with polycythaemia vera (PV) and 4 patients who did not quite fulfil the Polycythemia Vera Study Group criteria for PV, but had suggestive evidence of a myeloproliferative disorder. Thirteen patients did not form spontaneous colonies. They included patients with secondary polycythaemia, patients with normal total red cell volume and patients with absolute pure erythrocytosis of unknown origin. All patients with PV, and none with secondary polycythaemias, had spontaneous erythroid colony formation. Three of 4 untreated and 2 of 9 treated PV patients had increased number of CFU-E colonies in bone marrow. From these results it can be concluded that erythroid progenitor cell culture is a useful differential diagnostic method in erythrocytosis patients who cannot be classified clinically under PV or secondary polycythaemias.

Circulating haematopoietic progenitors in myelofibrosis.

We studied circulating erythroid and granulocyte-monocyte progenitors in 18 patients with idiopathic myelofibrosis and in healthy controls, using the methyl cellulose assay. 9 of the patients had been splenectomized prior to the study. The median number of circulating erythroid burst-forming units (BFU-E) was 8 times higher than that of the controls. 12 patients also had CFU-E (colony-forming unit, erythroid) in the blood. 10 patients had spontaneous BFU-E colony formation, and 8 patients had spontaneous CFU-E colony growth. Granulocyte-monocyte progenitors (CFU-GM) were increased 47 times compared to the controls. There were no differences in colony numbers between splenectomized and non-splenectomized patients. We conclude that in myelofibrosis, circulating erythroid and granulocyte-monocyte progenitors are usually markedly increased in number, but erythroid precursors to a lesser extent than granulocyte-monocyte precursors. Many patients, but not all, show spontaneous erythroid colony formation.