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MDM2, an E3 ubiquitin ligase, is an important negative regulator of p53. In turn the MDM2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited by the binding of some ribosomal proteins, including RPL5 and RPL11, and that the resulting increase in p53 levels may be important in the red cell aplasia seen in DBA and in 5q- MDS. DBA and 5q- MDS are associated with inherited (DBA) and acquired (5q-MDS) haploinsufficiency of ribosomal proteins. A mutation in MDM2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation, retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we inadvertently noticed that Mdm2 C305F/ C305F mice had perturbed hematopoiesis. These mice had a mild macrocytic anemia with reticulocytosis and high erythropoietin levels. In the bone marrow, these mice showed a significant decrease compared to wildtype (WT) littermates in Ter119+ cells, while no decrease in the number of immature erythroid cells (Ter119+CD71+) was found in the spleen, which showed significantly increased hematopoiesis. In methyl cellulose cultures BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls(p < 0.01). Further investigation revealed that there was a decrease in Lin-Sca-1+c-Kit+(LSK)cells, accompanied by significant decreases in short-term hematopoietic stem cells(ST-HSC) (p < 0.05) and multipotent progenitor(MPP) cells(p < 0.05). These results suggest that control of the cell cycle through interactions between ribosomal proteins and MDM2 is important in the regulation of normal hematopoiesis as well as in the pathogenesis of 5q- MDS and DBA. Disclosures No relevant conflicts of interest to declare.

Telomerase is a ribonucleoprotein that adds telomeric repeats onto the chromosome ends, preventing the replication-dependent loss of telomere repeats and cellular senescence in highly proliferative germ-line cells and in stem cells and cancer cells. Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome, which affects tissues that need constant renewal by stem cell activity. So far 8 genes have been found whose mutation causes DC and they all encode products that play a role in telomere maintenance. About 35% of DC patients show X-linked-recessive inheritance due to mutations in the DKC1gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Mutant dyskerin can destabilize telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known and some results suggest dyskerin may play a more direct role in telomerase action. So far studies of the cell biology of DC stem cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in both telomere biology and hematopoiesis between mouse and human. In this study, to investigate disease pathogenesis of X-linked DC, we generated induced pluripotent stem cells (iPSC) from patients’ skin fibroblast cell carrying DKC1 mutations Q31E, Δ37 and A353V. The recurrent A353V mutation accounts for about 40% of DKC1 mutations and usually causes a severe clinical phenotype while patients carrying Q31E and Δ37 mutations show a milder phenotype. We found that dyskerin protein expression in all of these dyskerin mutant iPS cells is decreased in agreement with our mouse studies that show mutant proteins are relatively unstable. These iPS cells show dramatically decreased TERC RNA levels and telomerase activity. Telomere length measurement revealed that mutant iPS cells lose the ability to elongate telomeres during the reprogramming processing; telomere erosion was particularly rapid in A353V cells. To further investigate whether dyskerin protein could play direct role in regulating telomerase activity other than stabilization of TERC RNA during the processing of assembly, we tested if the defect in telomerase function in these iPS cells could be rescued by expressing wild type dyskerin or TERC RNA. We expressed the rescuing genes by using the zinc-finger nuclease-mediated method to insert them into the safe harbor AAVs1 site, initially of DKC1Δ37 cells. After testing the telomerase function, we found that expressing WT-dyskerin protein in Δ37 iPS cells fully restores the mature Terc RNA expression level and the telomerase activity to normal levels. However, although Δ37 iPS with over-expressed WT TERC RNA can accumulate normal level of TERC RNA, these cells fail to increase telomerase activity. These results suggest that defective telomerase activity cause by DKC1 mutations can only reversed by expressing WT dyskerin but not by TERC RNA. These data suggest that, as one of three core components of the telomerase complex, dyskerin may play a direct role in telomerase activity. Disclosures: No relevant conflicts of interest to declare.

Abstract 50 Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome showing considerable genetic and clinical heterogeneity. The most common form is the X-linked form due to mutations in the DKC1 gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Six other genes, all of whose products are involved in telomere maintenance, have been shown to be mutated in DC, together the seven genes accounting for about half of the known cases. The X-linked form can cause severe disease for which therapeutic options are limited. It is known that mutant dyskerin destabilizes telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known. So far studies of the cell biology of DC stem and progenitor cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in telomere biology between mouse and human. An alternative approach that has recently become feasible is the production of induced pluripotent stem cells (iPSC) from patient fibroblasts that can then be used to investigate disease pathogenesis. Accordingly we generated iPSC from skin fibroblast from X-linked DC patients carrying DKC1 mutations Q31E, δ37A and 353V, and by using the classical OCT4, KLF4, SOX2 and cMYC 4-transcription factor system. Of particular interest is the A353V mutation since this is a recurrent mutation and accounts for about 40% of DKC1 mutations. In total, we obtained two Q31E clones, three δ37 clones and eight A353V clones. We found that all these DKC1 mutant iPS cells express decreased levels of dyskerin, in agreement with our mouse studies that show mutant proteins are relatively unstable. Mutant iPSC have very low levels of TERC (only 20–30% of the levels in WT iPSC) while TERT expression is the same as in WT cells. By using the TRAP assay, we found that both A353V and δ37 iPSC showed dramatically decreased telomerase activity; only 10–20 % compared to WT iPSC. After measuring the telomere length of both patient skin fibroblast cells and DKC1 mutant iPSC, we found A353V and δ37 iPSC lost the ability to elongate the telomere end during iPSC reprogramming while WT iPSC showed significantly increased telomere length compared to WT skin fibroblast cells. These results indicated that DKC1 iPSC are defective in telomere maintenance. In terms of ribosome biogenesis, we found that some snoRNA expression was slightly decreased including H/ACA snoRNAs E2, E3, U69, ACA10 and scaRNAs U90 and U93 while all C/D snoRNA we investigated were unchanged compared with WT iPS cells. We also found that DKC1 mutant iPS cells did not show significantly changes in ribosomal profiles or in the kinetics of rRNA processing. Together these results suggest that the iPSC faithfully reproduce the molecular features of the human disease and will prove to be a useful tool in investigations of the pathogenesis and treatment of DC. Disclosures: Bessler: Alexion Phamaceutical: Membership on an entity's Board of Directors or advisory committees; National Organization for Rare Dieases: Speakers Bureau.

Abstract 1163 Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (BMF) associated with a predisposition to cancer. It is diagnosed by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. X-linked DC, accounting for around one third of DC patients, is due to the mutations in the DKC1 gene which encodes the protein dyskerin. Dyskerin is a component of small nucleolar RNA particles (H/ACA snoRNP) active in the pseudouridylation of specific residues in nascent ribosomal RNA (rRNA). It also plays an important role in maintaining the telomere repeats at the ends of chromosomes by forming and stabilizing the telomerase complex. Patients with Dkc1 mutations show a wide variation of severity emphasizing the variable expression of the disease and the influence of other genetic and environmental factors. Most mutations are clustered, in the 3D structure, in a region important for RNA binding and few mutations are present in the pseudouridine synthesis activity domain. The relationship between the severity of DC and the position of the mutation in dyskerin is still unclear. It will be interesting to compare the pathogenic effects of Dkc1 mutations in different positions of dyskerin protein. Since the production of mutant lines of mouse is time consuming, expensive, and may be difficult, here we established a suitable in vitro system to express mutant dyskerin in MEF cells. We began with MEF cells containing a Dkc1 gene that is deleted in the presence of Cre. Mutated dyskerin introduced by a retroviral vector is expressed and wild type (WT) dyskerin is deleted by retrovirally introduced Cre-ERT2 and subsequent tamoxifen treatment, which activates the Cre-ERT2 protein. MEF cells are then single cell cloned and selected cells that grew well and contained for the mutant dyskerin gene but not the WT gene. We obtained cell lines with several mutations including I38T, H68Q, S121G and A353V as well as WT control. All of these mutations are recurrent ones and cause severe bone marrow failure in patients. Interestingly, we also obtained a D125A mutant cell line. This mutation has not been found in human patients but is an alteration of a crucial catalytic aspartic acid residue that is essential for pseudouridylation activity. All of these Flag-tagged dyskerin proteins showed a normal nucleolar sub-cellular distribution pattern. We chose the cells expressing similar levels of flag-tagged dyskerin to WT dyskerin to do further experiments. The exception to this was the D125A mutation because all the clones express tiny amounts of flag-tagged dyskerin. First, we measured the Dkc1 mRNA level of these mutant cells and found the expression of Dkc1 mRNA in these mutant cells is 3–8 fold higher than in WT cells (25 fold higher for D125A). This result indicates that the mutant dyskerin proteins in these cells are very unstable, so more mRNA has to be produced to maintain growth. Then, we studied the growth curves of these cells and found that all mutant cells showed more or less slower growth rate than WT. Notably, the D125A mutant cells showed extremely slow growth. We find that the expression of Terc is lower than WT in all mutant cells (10∼50% compared to WT) while telomerase activity was not significantly decreased except for the D125A mutant. By Q-FISH, we showed the telomere length of all mutant cells is shorter than WT control cells and the number of telomere free ends is increased in these mutant cells. These findings indicate that all Dkc1 mutations affect the telomerase function and cause rapidly shortening telomeres. In ribosome biogenesis we found that, although I38T, H68Q, S121G and A353V mutant cells did not show significant changes in the ribosomal profile and ribosomal processing, D125A mutant cell showed significantly delayed maturation of rRNA as well as dramatically decreased amounts of mature 80S ribosomes. Further investigation revealed that the D125A mutation abolished in vivo pseudouridylation of rRNA. Together with our previous results this study further indicates that compromised telomerase function is the major effect of the pathogenic Dkc1 mutations. We also show, surprisingly, that mammalian cells can grow and survive with no pseudouridine in their rRNA. Disclosures: Bessler: Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

Abstract 2614 X-linked Dyskeratosis Congenita (DC) is due to mutations in the DKC1 gene, which encodes the protein dyskerin. Dyskerin is a highly conserved nucleolar protein that, as part of a specialized nucleolar RNP, catalyzes the pseudouridylation of specific residues in newly synthesized ribosomal RNAs and spliceosomal snRNAs. Dyskerin also associates with telomerase and is involved in telomere maintenance. In addition to the well known effect of telomere homeostasis on cancer, it is evident that telomere maintenance may also be important in replicative aging because of telomere shortening due to the limited expression of telomerase activity in dividing somatic cells. Accumulating evidence suggests that dysfunctional telomeres resulting in premature cellular senescence is the primary cause of bone marrow failure in dyskeratosis congenita. It is important to determine the mechanism whereby Dkc1 mutations lead to premature cellular senescence in bone marrow. We have produced a line of mice containing a mutation, Dkc1Δ15, which is a copy of a pathogenic human mutation. Male Dkc1Δ15 mice showed a decrease in the proportion of B and T lymphocytes in peripheral blood and reduced body weight with age but no overt bone marrow failure syndrome phenotypes. Our previous competitive bone marrow transplantation experiments showed that the Dkc1Δ15 mutation caused decay of stem cell function with age. Bone marrow from older Dkc1Δ15 mice was markedly inefficient in repopulation studies compared with bone marrow from age matched wild type mice. We also found that N-acetyl cysteine (NAC) could at least partially rescue the growth disadvantage of dyskerin mutant spleen cells or fibroblasts which was associated with accumulation of DNA damage and reactive oxygen species. To determine if NAC, or other antioxidants might be useful therapeutically it is important to determine their effects on stem cell function, which is defective in DC. To this end we established a cohort of mice that were given NAC in their drinking water (1mg/ml) from 3-weeks of age and maintained on NAC for 1 year. We found that long term NAC treatment did not show significant side effects on the mice. They had slightly increased neutrophils, but no difference in life span and body weight compared with the untreated group. Impressively, old male Dkc1Δ15 mice showed corrected B and T cell proportions in peripheral blood after treatment with NAC. Competitive bone marrow transplantation experiments were carried out in which a 1:1 mixture of BM cells from mutant and WT mice was used to repopulated lethally irradiated recipient mice. These experiments showed that, when taken from NAC treated animals, old Dkc1Δ15 BM cells could compete with age matched WT cells with 40–45% of Dkc1Δ15 cells in primary recipients compared with only 20% for the untreated group. Moreover, after secondary transplantation, cells from the NAC treated group still represent 15–20% of Dkc1Δ15 cells in recipients while those from the untreated group could not be detected. These results strongly suggest that NAC treatment can partially restore the bone marrow repopulating ability of Dkc1Δ15 stem cells. Together with our previous results these data suggest that a pathogenic Dkc1 mutation, through its effect on telomerase, initiates stem cell aging before telomeres are short and that increased oxidative stress might play a role in this process. Moreover the effects of the mutation may be prevented or delayed by antioxidant treatment, although the precise mechanism will be the subject of future investigation. Disclosures: Bessler: Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

Abstract 498 X-linked dyskeratosis congenita (DC) caused by mutations in DKC1, encoding the protein dyskerin, is the most common form of DC, a severe inherited bone marrow failure (BMF) syndrome associated with a predisposition to malignancy. Dyskerin is a component of small nucleolar ribonucleoprotein particles(snoRNPs) that modify specific residues in nascent ribosomal RNA(rRNA) molecules and also forms part of the telomerase complex responsible for synthesizing the telomere repeats at the ends of chromosomes. Strong evidence suggests that compromised telomerase function is the major cause of DC but defects in ribosome biogenesis may contribute to the disease. Excessive telomere shortening resulting in premature cellular senescence is thought to be the primary cause of bone marrow failure in dyskeratosis congenita. Our previous data showed that, in mice, cells expressing a Dkc1 mutation (Dkc1Δ15) had a telomerase dependent but telomere length independent growth defect. Here we show that the growth rate of Δ15 MEF cells was lower when cultured at both ambient oxygen (21%) and low (3%) oxygen. In 21% oxygen both Δ15 and WT cells stopped growing and entered senescence after 8-10 population doublings, with the Δ15 cells growing more slowly than the WT cells. In 3% oxygen Δ15 cells grew more slowly and entered senescence earlier than WT cells. Further investigations reveal that both γ-H2AX foci number and reactive oxygen species (ROS) levels in Δ15 cells were significantly higher than in WT cells with increased passage number even when cultured in low oxygen. Increased levels of γ-H2AX and p53 in Dkc1Δ15 mice, particularly in older mice, were also detected in liver, spleen and bone marrow. To study the effect of the mutation on stem cell function during aging, we carried out competitive repopulation experiments using the CD45.1/CD45.2 congenic system. Irradiated mice were injected with a 1:1 mixture of Dkc1Δ15 and Dkc1+ bone marrow from old (77-88w) or young (10w) animals. Old Dkc1Δ15cells are less able to compete with age matched WT cells in primary recipients, making up only 20% of cells after 12 weeks compared with 40% for the young cells. Moreover, serial transplantation results show that, in secondary recipients, BM cells from old Dkc1Δ15 mice were not detectable while Dkc1Δ15 cells from young mice still comprise 10-30% of the bone marrow after 12 weeks. These results strongly indicate the Dkc1Δ15 mutation causes decay of stem cell function with age. Because of the association with ROS we asked whether treatment with an antioxidant could rescue the growth disadvantage of Δ15 cells. We grew primary MEF cells from Dkc1Δ15/+ female mice in the presence or absence of 100 M N-acetyl cysteine (NAC), a clinically approved antioxidant. These cultures consist in early passages of 50% cells expressing WT and 50% expressing Δ15 dyskerin, reflecting random X-chromosome inactivation, Without NAC the WT cells almost completely outgrew the Δ15 cells after 11 population doublings but in the presence of NAC the Δ15 cells are still clearly present after 15 population doublings, suggesting that NAC at least partially rescues the growth disadvantage of dyskerin mutant cells. More impressively, the growth disadvantage of the Δ15 cells is also rescued in vivo in Dkc1Δ15/+ female mice given the NAC (1mg/ml) in their drinking water. Although the precise mechanism will be the subject of further investigation, these results point to a functional link between increased oxidative stress, defective telomere maintenance and stem cell aging in the pathogenesis of BMF in dyskeratosis congenita. Disclosures: Bessler: Alexion: Membership on an entity's Board of Directors or advisory committees.

NUP98 has been involved in multiple recurrent chromosome rearrangements in leukemia. We identified a novel fusion between NUP98 and IQ motif containing G (IQCG) from a de novo acute T/myeloid leukemia harboring t(3;11)(q29q13;p15). IQCG has 2 putative coiled-coil domains and an IQ domain. The FG repeat from NUP98 and the coiled-coil domain from IQCG were retained in the fusion protein. We demonstrated that NUP98-IQCG could form homodimer or heterodimerize with either NUP98 or IQCG, bind Co-activators and/or Co-repressors, and show transcriptional activity in vitro. Expression of NUP98-IQCG inhibited 32Dcl3 cell apoptosis induced by Ara-C, and partially blocked granulocyte differentiation induced by G-CSF. Colony forming assay also indicated that NUP98-IQCG was able to stimulate proliferation and partially block differentiation of hematopoietic stem/progenitor cells. We conclude that NUP98-IQCG may play an essential role in leukemogenesis by stimulating progenitor cell proliferation and inhibiting differentiation and apoptosis.

Acquisition of additional genetic and/or epigenetic abnormalities other than BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast phase. To gain insights into the underlying mechanisms, we screened DNA samples from CML patients during acute transformation for alterations in a number of transcription factor genes crucial to myeloid-lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. L359V within zinc finger domain (ZF) 2 of GATA-2 was found in 8 cases with myelo-monoblastic features, while an in-frame deletion of six amino acids (D341–346) across the border of ZF1 was detected in 1 patient at blast crisis with eosinophilia. Further studies showed that L359V not only increased transactivation activity, but also enhanced inhibitory effects on the major myelopoietic regulator PU.1. Consistent with the myelo-monoblastic features of CML patients with GATA-2 L359V mutant, transduction of GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring mouse model disturbed myelo-monocytic differentiation/proliferation in vitro and in vivo, respectively. These data suggest that GATA-2 mutations may be involved in acute myeloid transformation in some CML patients.

X-linked Dyskeratosis Congenita (DC) is a rare recessive disorder caused by mutations in the DKC1 gene that encodes dyskerin. Dyskerin is part of ribonucleoprotein complexes that participate in two different pathways: ribosome biogenesis and telomere maintenance. It is the subject of intense debate whether disease manifestations in DC are due to dysfunctional telomere maintenances or are caused by a defect in ribosome biogenesis. Pathogenic mutations in dyskerin cause telomere shortening and patients with X-linked DC have critically short telomeres, However, whether there is an additional defect in ribosome biogenesis is difficult to investigate. To dissect the impact of a pathogenic dyskerin mutation on telomeres from the possible additional impact on ribosome biogenesis in an in vivo model, we generated mice expressing a mutant dyskerin protein. Because laboratory mice have very long telomeres a short telomere phenotype requires several generations of inbreeding, whereas a phenotype seen in the first generation is likely to be caused by the defect in ribosome biogenesis. To delete the last 21 amino acids of dyskerin (Del15) we used homologous recombination followed by conditional gene deletion in murine embryonic stem (ES) cells and in mice. Six independent ES cell clones with the deleted Dkc1 gene were obtained. In vitro analysis of the ES cells showed that the Del15 mutation led to dramatically decreased expression of a truncated dyskerin protein with decreased accumulation of the telomerase RNA. In addition, both reduction in telomerase activity and significant telomere shortening after 65 passages were observed. These findings indicate that the Del15 mutation impairs the telomerase maintenance pathway. After testing the accumulation of a series of mouse H/ACA snoRNA in Del15 ES cells, we found a decrease of the mU68 and mE1 snoRNAs suggesting the mutation may also confer effects which are outside the telomerase pathway. We therefore went on to produce a line of mice expressing the truncated Dkc1 protein and were able to obtain male mice hemizygous for the mutant Dkc1 gene as well as female heterozgotes. The male mice express the truncated dyskerin protein and show no gross abnormality up to 6 months of age. Interestingly, heterozygous female mice were healthy as well but the truncated dyskerin protein was dramatically decreased in expression compared to the wild type dyskerin in spleen, thymus, and bone marrow, but not in liver and brain. This result must derive from preferential proliferation of cells expressing wild type dyskerin after random X-inactivation in early embryogenesis. Our analysis indicates that the mutant dyskerin impairs the proliferation in hematopoietic tissues while it does not affect cells which are not rapidly proliferating such as those in liver and brain. Because of the early appearance of the skewed X-inactivation phenotype we conclude that skewing in these mice is caused by a telomere independent mechanism. Interestingly, the lack of overt DC-like abnormalities in the male hemizygous mice indicates that this proliferative disadvantage is insufficient to cause bone marrow failure but in combination with impaired telomere maintenance may accelerate the onset and severity of disease and thus explain the earlier and more severe manifestation in X-linked DC compared to autosomal dominant DC which only affects the telomerase pathway.

The NUP98 gene has been reported to be fused with at least 17 partner genes in leukemia with 11p15 translocation. An adult patient with de novo acute myeloid/T lymphocytic leukemia harboring t(3;11)(q29q13;p15) has been investigated to characterize the genes involved in that translocation. Through molecular cytogenetic analysis, we identified a fusion transcript between NUP98 gene and a novel partner gene named as NUP98 related gene (NRG) at 3q29. Further molecular analysis showed that exon 13 of NUP98 was fused in-frame to exon 10 of NRG. Moreover, the segment from 3q13 to 3q29 translocated at 11p15 had been inverted and accompanied by the deletion of the distal portion of breakpoint at chromosome 3q29. Interestingly, the NUP98-NRG protein showed nuclear and cytoplasmic distribution, a pattern different from that of wild type NUP98 or NRG. When assayed in a GAL 4 reporter system, the fusion gene showed an aberrant trans-regulatory activity. Transfection in HL-60 cells demonstrated that NUP98-NRG could promote cell proliferation, survival and arrest differentiation. Therefore, NUP98-NRG may exert transforming effects by interfering with the cellular mechanism of transcriptional regulation. Our data provide thus new evidence that NUP98-related molecular abnormality is a recurrent genetic event in leukemogenesis.

PURPOSE: In this study, we systemically compare the molecular response by regular reverse transcript polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR in newly diagnosed patients with acute promyelocyte leukemia (APL). PATIENTS AND METHODS: A total of 31 newly diagnosed patients with APL who entered remission by induction therapy with ATRA plus As2O3 were included in this study with median follow-up of 38 months (27–46 months). Serial bone marrow samples were obtained before and after the induction, consolidation chemotherapy and every 3 to 6 months during maintenance therapy. Regular RT-PCR and real-time RT-PCR were carried out to evaluate the molecular response. The PML-RARa fusion transcript level was calculated in two ways: normalized PML-RARa level as DoseN and log-reduction compared to the pre-treatment level. RESULTS: A total of 29 patients remained disease free for a median of 27 to 46 months. Comparing two PCR assays, the sensitivity limit for real-time RT-PCR was 5.7 log DoseN reduction while the limit for regular RT-PCR was about 3 log. Furthermore, we established criteria for evaluation of molecular response by real-time RT-PCR: ≥3.0 log reduction of PML-RARa transcript level as minor molecular response; ≥5.0 log reduction as major molecular response and undetectable level of PML-RARa transcript as complete molecular responses. Minor molecular response by real-time RT-PCR was achieved in 22.6% and 96.8% of patients after induction and consolidation chemotherapy, which was equivalent to molecular response by regular RT-PCR (35.5% and 96.8% respectively). The major molecular response rate was 12.9% and 90.3% after induction and consolidation whereas the complete response was at 3.23% and 77.4% respectively. All patients achieved major and complete molecular remission during and after maintenance therapy. Loss of either major or complete molecular response occurred in two patients while both patients experienced subsequent loss of minor molecular response, which coincided with CNS leukemia and bone marrow relapse. CONCLUSION: Real-time RT-PCR was more sensitive than regular RT-PCR and the evaluation criteria for real-time RT-PCR was established and proved to be clinically relevant.

OBJECTIVE: To evaluate the safety and efficacy of Glivec in combination with As4S4 in the treatment of advanced CML patients. PATIENTS AND METHODS: 9 Ph+ CML patients in advanced phase were entered into the study. The first 3 patients received Glivec 400mg/d plus As4S4 150mg/kg/d (day 1–14, each month). If no patient were to experience grade 3/4 CTC toxicity after one month of treatment, the dose of Glivec would be increased to 600mg. All the patients were treated and followed up for 6 months. Adverse events, hematological, cytogenetic, molecular response and real-time RT-PCR were evaluated in each regular visit. RESULTS: From Dec.2003 to June.2004, 9 patients consisting of 6 males and 3 females were enrolled in this trial, 5 in accelerated phase and 4 in blast crisis, aged 22–53 years (median 33). The first 3 patients received Glivec 400mg/d plus As4S4, and other 6 patients received increased dosage of Glivec to 600mg/d. By the time of report, 6 patients had finished the study. 7/9 patients (77.8%) achieved complete hematological response (CHR). 2 patients relapsed after 4 and 5 months of treatment, respectively, and 2 patients withdrew from the study after 1 and 2 months respectively to receive chemotherapy due to progression of the disease. Notably, 3 of these 4 patients had complex cytogenetic abnormalities. Cytogenetic response was observed in 3 patients (33.3%), including 2 major cytogenetic response (1 complete response) and 1 minor response. No molecular response was obtained so far in any patients. Bcr-Abl fusion transcript copy number was measured by real-time RT-PCR in 3 patients, 1 patient with CCR had 3140 fold reduction of BCR-ABL DoseN. Non-hematologic toxicities such as gastrointerstinal discomfort, edema, liver dysfunction and myalgia/bone pain were common and mild (grade 1/2). Grade 3/4 hematological toxicities including neutropenia, thrombocytopenia and anemia occurred in 3 patients. CONCLUSIONS: Glivec in combination with As4S4 has potential effect in advanced CML with acceptable toxicities. Further investigation will be performed to compare the efficacy of Glivec/As4S4 combinative therapy with Glivec mono-therapy in advanced CML.