Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Molecular biology of polycythemias.

Polycythemia is literally translated as "many cells in the blood". Only erythrocytosis (an alternative term for these disorders) produces polycythemia since leukocytes and platelets are present in blood in far smaller proportions. Polycythemia may be due to increased proliferation or decreased apoptosis of erythroid progenitors, or to delayed erythroid differentiation with an increased number of progenitor cell divisions. Prolonged red cell survival, another theoretical cause of polycythemia, has not yet been described and with intact regulatory mechanisms is unlikely to occur. Primary polycythemias result from abnormalities expressed in hematopoietic progenitors. In contrast, circulating factors cause secondary polycythemia (1). There are acquired and congenital causes of both primary and secondary polycythemia (1).

Pathogenetic mechanisms of polycythemia vera and congenital polycythemic disorders.

The absolute polycythemias--those with increased red blood cell mass--can be divided into two groups: primary, caused by acquired or inherited mutations leading to a "gain-of-function" abnormalities expressed within the erythroid progenitors; and secondary, due to circulating serum factors, typically erythropoietin, stimulating erythropoiesis. This overview concentrates on the molecular biology of polycythemia vera (PV) discussed in the context of other polycythemic disorders. Recent advances in the regulation of erythropoiesis, as they may relate to polycythemic states, are discussed as a background for those well-defined polycythemic states wherein the molecular defect has not yet been elucidated. A number of cellular abnormalities associated with PV, including the hyperresponsiveness of PV progenitors to many cytokines as well as decreased expression of the thrombopoietin receptor on platelets and increased expression of Bcl-xL, suggest that the PV defect alters a number of cellular functions and is not restricted to cytokine receptor signal transduction. The increasing number of recognized instances of familial incidence of PV suggests that in these families the predisposition for PV is inherited as a dominant trait, and that PV is acquired as a new mutation that leads to a clonal hematopoiesis and may be due to loss of heterozygosity. The existence of these families provides a unique opportunity for isolation of the mutations in the gene leading to PV. Semin Hemaol 38(suppl 2):10-20.

Development of a novel trans-lentiviral vector that affords predictable safety.

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) hold great promise for gene therapy. However, the possibility of generating replication-competent retrovirus (RCR) through genetic recombination raises concerns for safety. Here we describe a novel HIV-based packaging system (trans-lentiviral) that splits gag/gag-pol into two parts: One that expresses gag/gag-pro and another that expresses reverse transcriptase and integrase as fusion partners of viral protein R (Vpr). Using a sensitive assay developed to specifically detect recombinant lentiviral DNA mobilization, we demonstrated that the trans-lentiviral vector prevents the generation of recombinants that contain a functional gag-pol structure, while the lentiviral vector generates env-minus recombinant lentivirus that mobilizes recombinant genomes to other cells when pseudotyped with an exogenous envelope. Since an intact gag-pol structure is absolutely required for retroviral DNA mobilization and RCR, the trans-lentiviral vector design significantly reduces this risk. Moreover, it makes it possible to assess the risk of RCR and DNA mobilization using an in vitro assay that monitors trans-lentiviral vector stocks for the regeneration of the gag-pol structure. Therefore, the trans-lentiviral vector design will ensure the greatest predictable level of safety for the clinical application of retroviral vectors, including HIV-based vectors.

A polymorphism of the X-linked gene IDS increases the number of females informative for transcriptional clonality assays.

Studies of clonality have been essential for understanding the hierarchy of hematopoiesis and the biology of malignancies. Most clonality assays are based on the X chromosome inactivation phenomenon in females; these assays detect protein polymorphisms, differences in DNA methylation, or transcripts of the active X chromosome. Assays based on protein polymorphisms or DNA methylation have significant shortcomings. The major disadvantage of transcriptional assays is their limited applicability since only approximately half of females are informative for these studies. We have developed a new transcriptional assay based on an exonic polymorphism of the X-chromosome gene IDS. This gene is located in the same X-chromosome region (Xq28) as G6PD and p55, two genes with exonic polymorphisms for which we previously developed transcriptional assays. We developed non-radioactive PCR-based assays for rapid screening of genotype and determination of clonality. We also report reaction conditions for a quantitative ligase detection assay of IDS allelic transcripts. The frequency of the IDS polymorphism is 46% in Caucasian females and 39% in African-American females; in combination with G6PD and p55, 76% of Caucasian females and 62% of African-American females are informative for these assays. While this gene is highly polymorphic in Caucasian and African-American females, it is not informative in Oriental females. We established that the IDS gene is in linkage equilibrium with G6PD and p55. Unlike methylation-based assays, this assay is suitable for studying clonality in non-nucleated cells such as platelets and reticulocytes. With the discovery of exonic polymorphisms of other X-chromosome genes, all females should eventually be suitable for X-chromosome transcriptional clonality analysis.

Congenital and inherited polycythemia.

Absolute polycythemia is a condition with increased red blood cell mass. There are a number of primary and secondary polycythemic disorders leading to absolute polycythemia. Primary polycythemias are caused by a defect intrinsic to the erythroid progenitor cells. The best characterized primary polycythemia is the autosomal dominant primary familial and congenital polycythemia (PFCP). Familial or childhood occurrence of the myeloproliferative disorder polycythemia vera are also discussed, emphasizing the importance of distinction between polycythemia vera and PFCP. Congenital or familial secondary polycythemic conditions are characterized by increased red cell mass, which is caused by circulating serum factors, typically erythropoietin.

Effect of recombinant human erythropoietin combined with granulocyte/ macrophage colony-stimulating factor in the treatment of patients with myelodysplastic syndrome. GM/EPO MDS Study Group.

This randomized, placebo-controlled trial was designed to assess the efficacy and safety of therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (epoetin alfa) in anemic, neutropenic patients with myelodysplastic syndrome. Sixty-six patients were enrolled according to the following French-American-British classification: refractory anemia (20), refractory anemia with excess blasts (35), refractory anemia with ringed sideroblasts (9), and refractory anemia with excess blasts in transformation (2). Patients were stratified by their serum erythropoietin levels (less than or equal to 500 mU/mL, n = 37; greater than 500 mU/mL, n = 29) and randomized, in a 2:1 ratio, to either GM-CSF (0.3-5.0 microg/kg.d) + epoetin alfa (150 IU/kg 3 times/wk) or GM-CSF (0.3-5.0 microg/kg.d) + placebo (3 times/wk). The mean neutrophil count rose from 948 to 3831 during treatment with GM-CSF +/- epoetin alfa. Hemoglobin response (increase greater than or equal to 2 g/dL, unrelated to transfusion) occurred in 4 of 45 (9%) patients in the GM-CSF + epoetin alfa group compared with 1 of 21 (5%) patients with GM-CSF + placebo group (P = NS). Percentages of patients in the epoetin alfa and the placebo groups requiring transfusions of red blood cells were 60% and 92%, respectively, for the low-endogenous erythropoietin patients and 95% and 89% for the high-endogenous erythropoietin patients (P = NS). Similarly, the average numbers of units of red blood cells transfused during the 12-week study in the epoetin alfa and the placebo groups were 5.9 and 9.5, respectively, in the low-endogenous erythropoietin patients and 9.7 and 8.6 in the high-endogenous erythropoietin patients (P = NS). GM-CSF +/- epoetin alfa had no effect on mean platelet count. Treatment was well tolerated in most patients, though 10 withdrew from the study for reasons related predominantly to GM-CSF toxicity. (Blood. 2000;95:1175-1179)

Molecular basis for polycythemia.

This overview concentrates on familial and congenital polycythemias in the context of other polycythemic disorders, with emphasis on those with established molecular lesions. Recent advances in the regulation of erythropoiesis, as they may relate to polycythemic states, are discussed as a background for those well-defined polycythemic states wherein the molecular defect has not yet been elucidated. Primary familial congenital polycythemias and congenital and familial secondary polycythemias, including hemoglobin mutants, methemoglobinemias and congenital 2,3-bisphosphoglycerate deficiency, are discussed. The most common primary polycythemia, polycythemia vera, as well as the only likely endemic congenital secondary polycythemia, known as Chuvash polycythemia, are discussed.

Human hematopoietic progenitors express erythropoietin.

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Haematopoietic progenitors and signal transduction in polycythaemia vera and primary thrombocythaemia.

While significant progress has been made in understanding the cellular defect and molecular basis of polycythaemia vera (PV), elucidation of the primary mutation leading to PV remains elusive. While clinically useful, the PV diagnostic criteria put forward by the Polycythemia Vera Study Group are not based on the pathophysiology of this disorder and in some instances may lead to false diagnosis or may not be sufficient to diagnose an early PV. In diagnostically unclear situations, clinical and laboratory findings must take into account the acquired nature of PV, its clonality, and the presence of endogenous erythroid colony formation in serum-containing media. It is likely that other simpler assays may be developed based on the rapidly emerging knowledge of the cellular pathology of PV. Several intriguing observations of abnormalities pertaining to the erythroid signal transduction have been recently reported; these remain to be validated in other laboratories and to be proven specific for PV. The clinical concept of primary thrombocythaemia (PT) lags behind what we know about PV. While the diagnosis of PT is still based on the exclusion of other known causes of thrombocytosis, new knowledge is emerging. Recent clonality studies of a large number of PT females show that the majority are clonal. It is our belief that thrombocythaemic subjects who are not found to be clonal are those with secondary thrombocytosis. Multiple in vitro-based assays of megakaryocytic and erythroid progenitors have been developed and conflicting data published. It is likely that standardized assays of megakaryocytic progenitors will soon become available and a reproducible PT specific defect will be found. Such a specific test would be of immense diagnostic value in this most elusive of all myeloproliferative disorders.

Angiotensin II stimulates proliferation of normal early erythroid progenitors.

Angiotensin II exerts a mitogenic effect in several in vitro models, but a direct effect on erythroid progenitors has not been documented. Angiotensin-converting enzyme inhibitors and losartan, an angiotensin II type 1 receptor (AT1) antagonist, ameliorate posttransplant erythrocytosis, without altering serum erythropoietin levels. We studied erythroid differentiation and the effect of angiotensin II on proliferation of erythroid progenitors by culturing CD34+ hematopoietic progenitor cells in liquid serum-free medium favoring growth of erythroid precursors. Aliquots of cells were collected every third day, and were used for RNA preparation. AT1 mRNA was detected after 6 d. In these same samples, erythroid-specific mRNA (erythropoietin receptor) was also detected. AT1 protein was detected in 7-d-old burst-forming units-erythroid colonies by Western blotting. The CD34+ cell liquid cultures were used to incubate erythroid precursors with angiotensin II from days 6-9. After incubation, cells were transferred to semisolid medium and cultured with erythropoietin. Angiotensin II increased proliferation of early erythroid progenitors, defined as increased numbers of burst-forming units-erythroid colonies. Losartan completely abolished this stimulatory effect of angiotensin II. Moreover, we observed increased numbers of erythroid progenitors in the peripheral blood of posttransplant erythrocytosis patients. Thus, activation of AT1 with angiotensin II enhances erythropoietin-stimulated erythroid proliferation in vitro. A putative defect in the angiotensin II/AT1 pathway may contribute to the pathogenesis of posttransplant erythrocytosis.

Two new EPO receptor mutations: truncated EPO receptors are most frequently associated with primary familial and congenital polycythemias.

Primary polycythemias are caused by an acquired or inborn mutation affecting hematopoietic/erythroid progenitors that results in an abnormal response to hematopoietic cytokines. Primary familial and congenital polycythemia (PFCP; also known as familial erythrocytosis) is characterized by elevated red blood cell mass, low serum erythropoietin (EPO) level, normal oxygen affinity of hemoglobin, and typically autosomal dominant inheritance. In this study we screened for mutations in the cytoplasmic domain of the EPO receptor (EPOR; exons 7 and 8 of the EPOR gene) in 27 unrelated subjects with primary or unidentified polycythemia. Two new EPOR mutations were found, which lead to truncation of the EPOR similarly to previously described mutations in PFCP subjects. The first is a 7-bp deletion (del5985-5991) found in a Caucasian family from Ohio. The second mutation (5967insT) was found in a Caucasian family from the Czech Republic. In both cases the EPO dose responses of the erythroid progenitors of the affected subjects were examined to confirm the diagnosis of PFCP. In one of these families, the in vitro behavior of erythroid progenitors in serum-containing cultures without the addition of EPO mimicked the behavior of polycythemia vera progenitors; however, we show that antibodies against either EPO or the EPOR distinguish the in vitro growth abnormality of polycythemia vera erythroid progenitors from that seen in this particular PFCP family. We conclude that PFCP is a disorder that appears to be associated in some families with EPOR mutations. So far, most of the described EPOR mutations (6 out of 8) associated with PFCP result in an absence of the C-terminal negative regulatory domain of the receptor.

Trilineage extramedullary myeloid cell tumor in myelodysplastic syndrome.

A patient with a 17-month history of myelodysplastic syndrome (refractory anemia with excess blasts that evolved into chronic myelomonocytic leukemia), which was treated with transfusions and erythropoietin, developed abdominal and inguinal lymphadenopathy. Biopsies of the abdominal nodes revealed virtual obliteration of the architecture by myeloid blasts admixed with maturing granulocytic, erythroid, and megakaryocytic precursors. The lymph node findings appeared to represent extramedullary dyshematopoiesis undergoing a tissue phase blast transformation. Four months later, the patient developed rising peripheral blast counts consistent with acute leukemia. Although the development of granulocytic sarcoma (also called extramedullary myeloid cell tumor) is well known to occur in patients with myelodysplastic syndromes, to our knowledge this is the first description of an extramedullary myeloid cell tumor associated with trilineage differentiation.

Rapid determination of clonality by detection of two closely-linked X chromosome exonic polymorphisms using allele-specific PCR.

We reported two specific, reproducible, and quantitative clonality assays based on detection of exonic polymorphisms of the X chromosome genes p55 and G6PD using rtPCR-LDR. These assays are inconvenient for screening purposes. This study sought to develop a simple, reproducible assay, practical for screening genomic DNA samples for p55/G6PD genotypes, rapid clonality determination, and to determine the linkage relationship between these closely related loci. The salient feature of ASPCR is the performance of two PCR rounds. The first generates template; the second, using one aliquot of first-round products in two reaction tubes, each containing one allele-specific primer, detects each allele. ASPCR and rtPCR-LDR produced identical p55/G6PD results in 91 normal female genomic DNAs, and in 12 clonal hematopoietic disorder cDNAs, confirming assay validity. 209 female and 207 male genomic DNA samples were analyzed for p55/G6PD genotype by ASPCR; 60% of females were heterozygous at one or both loci. G6PD and p55 allelic frequencies were significantly different among African-American men and women, but were not significantly different among Caucasian men and women. These loci were in linkage equilibrium among African Americans, but not among Caucasians. ASPCR is a rapid, sensitive, and specific method for screening large numbers of genomic DNAs, and for rapid clonality determination.

"Benign erythrocytosis" and other familial and congenital polycythemias.

The term familial and congenital polycythemia encompasses a heterogeneous group of disorders with the common characteristic of an absolute increased red cell mass since birth and/or similar phenotype also present in relatives. In the last 2 decades the differential diagnosis between primary and secondary familial polycythemias became more physiologically relevant as new sensitive techniques, such as accurate measurements of serum erythropoietin (S-EPO) concentration by radioimmunoassay (RIA) or ELISA, and assessment of growth of erythroid progenitor cells in vitro became available. Consequently, correct classification of many older previous reports of familial polycythemias is difficult. While familial secondary polycythemias due to high oxygen affinity hemoglobin mutants are not infrequent and have been well delineated in terms of molecular pathophysiology and phenotype during the last 3 decades, those secondary familial polycythemias due to 2,3 DPG deficiency are very rare. Familial and congenital polycythemias with increased EPO concentration and normal arterial oxygen saturation and oxygen dissociation kinetics represent an intriguing group of disorders wherein the molecular lesions remain obscure; however, in some instances a possibility of abnormal oxygen sensing pathway involving hypoxia inducible factor-1 (HIF-1) open an intriguing yet unexplored area of hematology and biology. In contrast the primary familial and congenital polycythemia (PFCP) has been only recently recognized (the first report published in 1977). Various designations have been used in the past to describe PFCP, a rare clinical syndrome, including: benign familial erythrocytosis, polycythemia vera of childhood, primary polycythemia, pure erythrocytosis, etc. Some of these terms stressed the relatively benign, non-progressive course of the disease with a normal lifespan of affected subjects; however, the apparent benignity of some of these disorders has been questioned. These disorders are familial and/or congenital, and the clinical and laboratory evidence of secondary polycythemias must be excluded. Only about 2 dozen familial and sporadic cases with PFCP have been reported. However, the mutations of erythropoietin receptor (EPOR) found in some of families with PFCP represent the only defined molecular defect of primary polycythemic phenotypes. All reported PFCP associated EPOR mutations result in truncation of its intracytoplasmic C-terminal domain which negatively regulates the EPO/EPOR signal transduction pathway. Subjects with these mutations have decreased or normal S-EPO and increased sensitivity of erythroid progenitor cells to low EPO concentrations in in vitro assays. Mutations of other genes involved in post EPOR signaling pathway such as JAK-2, HCP and STAT 5 may also play a causative role in pathogenesis of some of PFCP families where mutation of EPOR was not found.

Stable transduction of recombinant adeno-associated virus into hematopoietic stem cells from normal and sickle cell patients.

Stable introduction of genes into human hematopoietic stem cells with self-renewing potential is a necessary requirement for gene therapy strategies. We have developed an adeno-associated virus (AAV) vector and a partial packaging cell line that produces recombinant AAV at a titer of 10(8) transducing particles per milliliter. A high-titer viral stock containing the CMV/lacZ gene was used to transfer lacZ sequences into CD34+ Lin-Thy+ hematopoietic stem cells purified from normal and homozygous sickle cell patients. After infection, the cells were cultured in two ways. In the first set of experiments, the cell were expanded 300-fold in liquid culture for 21 days and plated in methylcellulose. Burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte/macrophage (CFU-GM) were then analyzed for lacZ sequences. In the second set of experiments, infected cells were cultured for 6 weeks under conditions that maintain long-term culture-initiating cells (LTC-IC). Progenitors were plated in methylcellulose, and BFU-E were analyzed for lacZ DNA. Stable transduction of lacZ sequences was observed in 25% of the colonies in both sets of experiments. These results demonstrate for the first time that LTC-IC can be transduced stably with a recombinant AAV vector. The results suggest that AAV may be a useful vector for genetic therapy of sickle cell disease and other hematopoietic disorders.