PML-RARA Fusion Gene Research Articles

BCOR as a Novel Fusion Partner of Retinoic Acid Receptor Alpha In a t(X;17)(p11;q12) Variant of Acute Promyelocytic Leukemia

AbstractAbstract 1703The majority of acute promyelocytic leukemia (APL) cases are characterized by the presence of a PML-RARA fusion gene. In a small subset, RARA is fused to a different partner including PLZF, NPM1, NuMA, STAT5b, PRKAR1A and FIP1L1. Here we identified a novel RARA fusion transcript, BCOR-RARA in a t(X;17)(p11;q12) variant of APL. The patient was a 45-year-old man. Although the patient was clinically responsive to ATRA, repeated standard chemotherapy with ATRA did not effect a cure. The bone marrow promyelocytes had unique morphologic features, including rectangular and round cytoplasmic inclusion bodies. They were more granular than those of AML M2 but less granular than the classical t(15;17) APL. Flow cytometric analysis revealed strong expression of CD13, CD33, CD56, weak expression of CD11c and lack of HLA-DR and CD7. The karyotype analysis detected a novel chromosomal translocation described as 45,-Y, t(X;17)(p11.2;q12)[19]/ 46,XY[1]. FISH analysis indicated one intact and two split signals of RARA and two intact signals of PML. To amplify unknown chimeric fusion transcripts, we performed 5'-RACE. The sequence revealed that BCL6 co-repressor, BCOR cDNA from exons 9 to 12 to be fused to RARA exon 3. By RT-PCR, we confirmed full length chimeric fusion transcripts spanning from the start codon to 4,948 nt of BCOR cDNA (NM_001123384) fused to RARA cDNA from exon 3 to the stop codon. The chimeric cDNA had an in-frame codon from BCOR through RARA, creating a 1,931 amino acid fusion protein. One of the consistent features in all known RARA fusion partners is self-association. To determine whether BCOR-RARA self-associate, we performed co-immunoprecipitation assays. These results showed that BCOR-RARA is able to self-associate both through the region of BCOR-S and the ankyrin repeat domain of BCOR. In addition, BCOR-RARA associated with BCL6. RXR recruitment is a critical determinant of transforming potential of oligomeric RARA fusion proteins. To investigate how BCOR-RARA associates with RARE in vitro, we performed EMSA. These results showed that BCOR-RARA/RXRA complex associates with RARE in an alternative manner compared to RARA and PML-RARA. Deregulation of RARA transcriptional activations has a central role in pathogenesis of APL. Therefore, we evaluated ATRA-induced transcriptional activation of 4× RAREs with a reporter assay in HepG2 cells. Without ATRA, BCOR-RARA repressed the reporter activity. With addition of ATRA, BCOR-RARA induced transcriptional activation very weakly. Subsequently, we evaluated dominant-negative effects of the samples in the RARA/RXRA pathway. In contrast to BCOR, BCOR-RARA clearly inhibited ATRA-induced RARA transcriptional activation as well as PML-RARA. Furthermore, we asked which domains are sufficient for the dominant-negative effects with the deletion mutants. The results indicated that the region spanning from 999 to 1,409 aa of BCOR-RARA has pivotal roles in the dominant-negative effects. Correct protein function is highly dependent on intracellular localization. To investigate subcellular localization of BCOR-RARA, we performed immunofluorescence analysis in 293T cells. In BCOR-RARA-expressing cells, BCOR-RARA localized as two patterns; (I) diffusely in the nucleus as well as PML-RARA in 82% of the cells, (II) diffusely in the nucleus and aggregately in the cytoplasm in 18% of the cells. The subcellular localization of BCOR-RARA was clearly distinguishable from the punctuate pattern as shown in the nucleus of BCOR-expressing cells. Moreover, co-immunofluorescence analysis between BCOR-RARA and BCL6 indicated that the subcellular localization of BCOR-RARA/BCL6 is distinct from BCOR/BCL6. BCOR-RARA was found to possess common features with other RARA fusion proteins. These included: (I) the same break point in RARA cDNA; (II) self-association; (III) RXRA is necessary for BCOR-RARA to associate with the RARA responsive element; (IV) action in a dominant-negative manner on RARA transcriptional activation; (V) aberrant subcellular relocalization. It should be noted that there was no intact BCOR found in the 45,-Y,t(X;17)(p11;q12) APL cells because they featured only a rearranged × chromosome. These results highlight essential features of pathogenesis in APL in more detail. BCOR appears to be involved not only in human congenital diseases but also in a human cancer. Disclosures:No relevant conflicts of interest to declare.

Gain of MYC underlies recurrent trisomy of the MYC chromosome in acute promyelocytic leukemia

Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia (AML). It has been hypothesized that gain of the MYC protooncogene is of central importance in trisomy 8, but the experimental data to support this are limited and controversial. In a mouse model of promyelocytic leukemia in which the MRP8 promoter drives expression of the PML-RARA fusion gene in myeloid cells, a Myc allele is gained in approximately two-thirds of cases as a result of trisomy for mouse chromosome 15. We used this model to test the idea that MYC underlies acquisition of trisomy in AML. We used a retroviral vector to drive expression of wild-type, hypermorphic, or hypomorphic MYC in bone marrow that expressed the PML-RARA transgene. MYC retroviruses cooperated in myeloid leukemogenesis and suppressed gain of chromosome 15. When the PML-RARA transgene was expressed in a Myc haploinsufficient background, we observed selection for increased copies of the wild-type Myc allele concomitant with leukemic transformation. In addition, we found that human myeloid leukemias with trisomy 8 have increased MYC. These data show that gain of MYC can contribute to the pathogenic effect of the most common trisomy of human AML.

Utilization of Molecular Phenotypes to Detect Relapse and Optimize the Management of Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is characterized by a unique genetic aberration, the t(15;17) chromosome translocation. Translocation breakpoints are located within the promyelocytic leukemia (PML) locus on chromosome 15 and the retinoic acid receptor alpha (RARA) locus on chromosome 17. In the past 2 decades, critical advances have been made in understanding the molecular pathogenesis of APL. APL represents a paradigm for molecularly targeted therapy in cancer and an extraordinary model for translational research in medicine. In fact, the release of differentiation block upon treatment of APL with all-trans-retinoic acid (ATRA) has represented the first example of targeted therapy in human cancer. More recently, the advent of arsenic trioxide (ATO) has allowed further progress in the management of this disease through improved outcomes in patients receiving this agent in combination with ATRA. Finally, optimization of therapy and minimization of toxicity is feasible in this disease through careful monitoring of residual disease using polymerase chain reaction-based approaches targeting the PML-RARA fusion gene.

Tratamiento con trióxido de arsénico en paciente con recaída de leucemia promielocítica aguda

Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia (AML) with M3 morphology and specific chromosomal translocation t(15;17) with resultant leukemogenic PML-RARα fusion gene. Its main characteristics are therapy response using all-trans-retinoic acid (ATRA) and its high rate of recovery; higher than other subtypes of acute myeloid leukemia. Arsenic trioxide induces a high complete remission rate in patients having an APL relapse. Experience in the use of this drug is still limited in the field of Paediatrics.

Dynamic of morphologic and molecular remission in patients (pts) with APL treated with frontline ATRA and arsenic trioxide (ATO).

6548 Background: APL is associated almost in all pts with t (15; 17) (q21; q22) leading to the fusion gene PML-RARα which can be identified by reverse transcriptase polymerase chain reaction (RT-PCR). Minimal residual disease (MRD) monitoring based on the detection of PML-RARα transcripts by PCR technology has a well-demonstrated role in APL. Purpose: To describe the dynamics of response in pts with APL on our frontline study of ATRA and ATO. Methods: From February 2002 to January 2010, 111 pts with newly diagnosed APL have been treated with ATRA plus ATO. The first cohort of 65 pts received ATRA and ATO (beginning on day 10 of ATRA). High risk pts (WBC > 10 x 109/L) received GO (gemtuzumab) on the first day. The second cohort of 46 pts (38 evaluable pts) received ATRA and ATO concomitantly on day one. They also received GO on day one, if high risk, and if their WBC increased to more than 30 x 109/L during induction. Quantitative PCR (Q-PCR) for PML-RARα fusion gene was conducted after induction and throughout consolidation and follow up for the latter cohort. Results: The median time (range) to CR was 29 days (19-70) in the first cohort, 29 days (25-73) in the second cohort; the median time to complete molecular response (CMR) was 117.5 days (26-323) in the first cohort, was 101.5 days (25-240) in the second cohort. There was no difference of time to CMR between two cohorts (p = 0.15). Three pts in the first and 2 pts in the second cohort have relapsed, all having previously achieved CMR. Two pts with CNS relapse remained PCR-negative at the time of relapse. All other relapsed pts had positive peripheral blood (PB) or bone marrow (BM) PCR tests prior to relapse. There was strong correlation (Spearman correlation: 0. 86, p < 0.0001) between PB and BM Q-PCR on 75 concomitant samples (within 1 day of each other) from 25 pts; no significant difference between BM and PB (p = 0.76, paired Wilcoxon test). Conclusions: There was no significant difference between ATO at day 1 of ATRA or day 10 regarding time to CR and time to CMR. Relapse occurred in 5% and was preceded by a positive PCR except for CNS relapse. Among the available paired samples there is a strong correlation between BM and PB PCR suggesting that PB may be used to monitor pts for impending relapse. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cepahlon Cepahlon

Double Genetic Mutations in PML-Rara Fusion Gene Confirmed in a Patient Showing Resistance to All-Trans Retinoic Acid and Arsenic-Trioxide Therapy.

Abstract 1743Poster Board I-769 BackgroundMolecular targeting drugs, all-trans retinoic acid (ATRA)and arsenic trioxide (ATO), have major advances in the treatment of acute promyelocytic leukemia (APL). However, resistance to these drugs has been also observed in clinical practice. ATRA acts as a ligand for retinoic acid receptor alpha (RAR) and restores the aberrant transcription repression by PML-RARA fusion protein in APL cells. Previous reports demonstrate that amino-acids substitution, resulting from genetic mutations, in ligand binding domain (LBD) of RARA region of PML-RARA were closely related to drug resistance to ATRA therapy. In contrast, for ATO therapy, the molecular mechanisms of the effectiveness and also the resistance are still unclear. Here we identified a PML-RARA that holds double genetic missense mutations in RARA and PML regions, respectively, from an APL patient, who showed clinically resistance to ATRA and ATO therapy. These mutations were observed as his disease progression, and we are interested in the relationship between these mutations with drug resistance to ATRA and/or ATO. AimsAnalyses of the molecular and clinical significance of the double missense mutations of PML-RARA for disease progression and resistance to ATRA and ATO therapy. ResultsEight APL patients were treated with ATO in Nagoya University Hospital, Japan, during ∼5 years from Apr. 1, 2000 to Dec. 31, 2004. One out of 8 patients showed clinically ATO resistance. The patient showing ATO resistance firstly diagnosed as APL (M3 variant) from cytogenetic and chromosomal analyses, and complete remission was obtained after combination chemotherapy with ATRA. Molecular CR was confirmed by RT-PCR analysis, but after 3 month from the induction therapy, ATRA-resistant relapse was observed. After treatment with ATO therapy, response was observed, but the effectiveness was gradually decreased, resulting finally into the resistance. The patient died of disease progression. During his 7 years clinical course, leukemia cells were harvested repeatedly from his bone marrow and peripheral blood. RT-PCR using the total RNA from his tumor cells followed by DNA sequencing was performed, with the result of PML-RARA fusion gene with the bcr3 breakpoint in the intron 3 of PML. When using the tumor cells that were harvested at his terminal stage, a missense point mutation in the LBD of the RARA region of PML-RARA was confirmed. Furthermore, missense point mutation in the PML-B2 domain was also confirmed in the same cDNA clones. Interestingly, these mutations were not observed in the leukemia cells obtained at the onset. These mutations were analyzed in each sample that was obtained as his disease progressed, and some correlation between disease progression and/or the drug resistance and the timing of appearance of these two mutations were suggested. These mutated fusion transcripts were cloned into expression vectors, and we are now analyzing the function relating to the drug resistance and disease progression. ConclusionsDouble genetic missense mutations in the RARA-LBD and PML-B2 of PML-RARA were confirmed in ATRA and ATO resistant patient. These genetic mutations were confirmed in the leukemia cells during his disease progression, and the relationship between those mutations and drug resistances were suggested from the clinical features. Mutations in the PML-B2 domain has not been reported previously, thus, it may be important to show whether this type of mutations are related to the drug resistance, especially to ATO therapy. DisclosuresKiyoi:Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

Masked t(15;17) APL with the insertion of PML–RARα fusion gene in 4q21

Most cases of acute promyelocytic leukemia (APL) are characterized by the reciprocal translocation t(15;17); however, several complex variant translocations have also been reported. Here we report complex cytogenetic abnormalities without t(15;17) assayed by the G-banding method in a 62-year-old woman with the typical morphology and clinical features of APL. Based on spectral karyotyping and FISH analyses, we confirm the insertion of a cryptic chromosomal segment containing the PML/RARα fusion gene. The patient achieved complete remission after treatment with all-trans retinoic acid (ATRA) alone. Although the mechanism of this cryptic variant insertion is not known, we conclude that the insertion of PML–RARα fusion into 4q21 seems not to alter the effectiveness of treatment with ATRA.

Effective treatment of acute promyelocytic leukemia with all-trans-retinoic acid, arsenic trioxide, and gemtuzumab ozogamicin.

We examined the outcome of patients with newly diagnosed acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) with or without gemtuzumab ozogamicin (GO) but without traditional cytotoxic chemotherapy. From February 2002 to March 2008, 82 patients with APL were treated with a combination of ATRA plus ATO. The first cohort of 65 patients received ATRA and ATO (beginning on day 10 of ATRA). High-risk patients (WBCs >or= 10 x 10(9)/L) received GO on the first day. From July 2007, the second cohort of 17 patients received ATRA and ATO concomitantly on day 1. They also received GO on day 1, if high risk, and if their WBC increased to more than 30 x 10(9)/L during induction. Monitoring for PML-RARA fusion gene was conducted after induction and throughout consolidation and follow-up. Overall, 74 patients achieved complete remission (CR) and one achieved CR without full platelet recovery after the induction, for a response rate of 92%. Seven patients died at a median of 4 days (range, 1 to 24 days) after inclusion in the study from disease-related complications. The median follow-up is 99 weeks (range, 2 to 282 weeks). Among the responding patients, three experienced relapse at 39, 52, and 53 weeks. Three patients died after being in CR for 14, 21, and 71 weeks, all from a second malignancy. The estimated 3-year survival rate is 85%. The combination of ATRA and ATO (with or without GO) as initial therapy for APL was effective and safe and can substitute chemotherapy-containing regimens.

The seventh pathogenic fusion gene FIP1L1-RARA was isolated from a t(4;17)-positive acute promyelocytic leukemia

The majority of acute promyelocytic leukemia (APL) cases are characterized by the expression of the chimeric fusion gene PML-RARA . Although the PML-RARA fusion gene is detected in more than 95% of APL cases, RARA has also been found to fuse with other partner genes in some APL variants. To date,

5′ RARA submicroscopic deletion from new variant translocation involving chromosomes 15, 17, and 18, in a case of acute promyelocytic leukemia

Submicroscopic deletions of the PML-RARA fusion genes constitute rare rearrangements in acute promyelocytic leukemia (APL). We describe a rare case of APL carrying a novel complex translocation involving chromosomes 15, 17, and 18 associated with a submicroscopic deletion of the 5′ part of the RARA gene, as evidenced by fluorescence in situ hybridization (FISH). A PML/RARA dual-fusion probe did not reveal the RARA-PML fusion signal on the der(17q), usually detected in the typical t(15;17). The RARA break-apart probe showed a deletion hybridization pattern with loss of the signal corresponding to the 5′ portion of the RARA gene. Reverse transcriptase–polymerase chain reaction confirmed the absence of the fusion RARA-PML transcript. The patient achieved complete remission, but died during consolidation therapy, 2 months after diagnosis. To our knowledge, this is the first reported case of APL with a complex variant t(15;17) involving chromosome 18 at band q12 and one of the very rare described cases displaying a submicroscopic deletion of the RARA 5′ region. Further cases are needed to delineate the incidence of submicroscopic deletions in APL and elucidate their prognostic impact.

Ectopic Expression of CDX2 Perturbs HOX Gene Expression in Murine Progenitors Depending on Its N-Terminal Transactivation Domain and Is Closely Correlated with Deregulated HOX Gene Expression in Human Acute Myeloid Leukemia.

Deregulated homeobox gene expression characterizes more than 60% of all acute myeloid leukemia (AML) patients, particular in patients with normal karyotype (NK). So far it is largely unknown how the aberrant expression of homeobox genes is initiated in the malignant clone. The ParaHox gene Cdx2 was shown to act as positive upstream regulator of Hox genes in embryogenesis. We have previously shown that ectopic Cdx2, which normally is not expressed in hematopoietic cells, can be the key event in the development of AML in mice (Rawat et al., PNAS 2004). In our present study we now demonstrate that ectopic expression of Cdx2 in murine hematopoietic progenitors induced significant up-regulation of Hox genes with leukemogenic potential such as HoxA9, Hoxa10, HoxA5, Hoxa7, Hoxb6, Hoxb8. Deletion of the N-terminal transactivation domain of Cdx2 abrogated its ability to perturb Hox gene expression and eliminated its leukemogenic potential in vivo (n=13), whereas inactivation of the putative Pbx binding site of the protein did not prevent Cdx2 induced leukemogenesis. As we showed that Cdx2 upregulated leukemogenic Hox genes and caused AML in the murine model we analyzed 115 AML patients for a correlation between the expression levels of CDX2 and deregulated HOX gene expression. A total of 71 patients with normal karyotype (AML NPMc+ = 45 cases; NPMc– = 26 cases) was analyzed for CDX2 expression. 89% of the AML NPMc+ cases showed ectopic expression of CDX2 as well as 88% of the cases without the NPM1 mutation. We extended this analysis to 44 patients with abnormal karyotype and detected ectopic CDX2 expression in 64% (28 out of 44) of the cases: 12 of 24 patients with t(8;21), 10 of 10 patients with t(15;17). Importantly, when the expression level of CDX2 was compared between AML cases with normal and abnormal karyotype, there was 14fold higher expression level in the patient group with NK (n=63) compared to the group with aberrant karyotype (n=28) (ØΔCT 7.72 vs. ØΔCT 11.62, respectively; p>0.001). By using oligonucleotide microarray analysis, we confirmed that high Cdx2 expressing AML-NK patients with (n=12) or without NPM1 mutation (n=12) were characterized by aberrant expression of multiple HOXA and B cluster genes such as HOXA10, HOXA9 and HOXB3, HOXB6 in contrast to AML cases expressing the PML-RARA or AML1-ETO fusion gene or normal healthy donors. Three NPMc- cases with normal karyotype showed the same low level of expression of CDX2 (ΔCT 10.55–11.55) as AML cases with aberrant karyotype. Of note, these three cases did not show any perturbation of HOX gene expression and thereby fell into the same cluster as AML cases with t(8;21) or t(15;17) in the microarray data set evaluating HOX gene expression in the different AML subtypes. All AML-NK patients tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression.

Development of a PML-RARα-IL-2 Recombinant Plasmid DNA for APL.

Specific active immunotherapy with DNA vaccines is one of the best approaches to eradicate minimal residual disease in cancer patients, when appropriate tumor-specific antigens are identified for this disease. However, little data are regarding the DNA vaccines in acute leukemia, recent reports has shown that a human PML-RARα breakpoint-drived DNA vaccine can prevent APL in mice model, but the host immune response were weakly, due to the weak immunogenicity. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we develop a vector coexpressing PML-RARα gene fused to human Interleukin- 2 (hIL-2) gene. PML-RARα fusion gene segment and the full-lenght hIL-2 gene were amplified from NB4 cells or Jurkat cells. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hIL-2. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/hIL-2 mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same sequence of PML-RARα gene and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/hIL-2 mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Sal I/Not I) and sequence analysis, the PML-RARα/hIL-2 mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. Our results indicated that the recombinant plasmid expressing PML-RARα and hIL-2 was successfully constructed, which can induce more effective immune response and anti-APL cells effect in animal models. The data suggest that effective vaccination approaches should be possible against APL. It could be farther used in the research as PML-RARα DNA vaccine for APL.

A Vector Expressing PML-RARα Fused to GM-CSF Is an Effective DNA Vaccine for Inducing Specific Immune Response to APL Cells.

Despite combinations of different therapeutic strategies have significantly increased survival, acute leukemia is still not curable. To further improve outcome, specific immunotherapy might be one of the best choice. DNA vaccines have been showed leading to strong and persistent cell-mediated and humoral immune response to the antigen encoded by the plasmid. However, little data exist regarding the DNA vaccines in acute leukemia, there are few studies reported that PML-RARα DNA vaccines were developed, but the host immune response were weakly, due to the weak immunogenicity of tumor antigens. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we have used a full-length human GM-CSF (hGM-CSF) sequence fused to PML-RARα breakpoint-drived sequence and develop a vector coexpressing PML-RARα gene and hGM-CSF gene, which was expected to to promote T cells response in host. PML-RARα fusion gene segment and the hGM-CSF gene were amplified from NB4 cells or pORF-hGM-CSF plasmid. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hGM-CSF. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/GM-CSF mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same PML-RARα segment and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/GM-CSF mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Xba I/Sal I) and sequence analysis, the PML-RARα/GM-CSF mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. In conclusions, the vector expressing PML-RARα and hGM-CSF was successfully constructed, which can more effective immune response and anti-APL cells effect in animal models than that from plasmid containing single PML-RARα segment. It could be farther used in the research as PML-RARα DNA vaccine for APL.

Increased Global Gene Promoter Methylation after Relapse (Rel) of Acute Promyelocytic Leukemia (APL) from All-trans Retinoic Acid (ATRA)-Containing Treatment Is Dissociated from Concurrent Gene Expression Changes.

Increased methylation of gene-regulatory DNA sequences has been associated with the progression of malignancies, and, in APL, the pathogenetic PML-RARα fusion gene has been associated with DNA methyltransferase activity, which might contribute to this process. We, thus, hypothesized that promoter methylation levels would be increased in APL samples at disease Rel and that this might be contributory to gene expression changes related to Rel. We investigated this hypothesis using matched pretreatment (PTx) and Rel DNA and RNA samples from 5 patients of whom 3 had received combined chemotherapy(CT)-ATRA (A-set) and 2 had received only CT (C-set); one additional RNA matched pair was available from another CT-only patient. The specimens used for DNA/RNA extraction had ≥ 85% blasts. The HELP assay (Khulan, et al, Genome Res. 16, 1046, 2006), which is based on comparative hybridization of HpaII (methylation-sensitive) versus MspI (methylation-insensitive) digested genomic DNA to a microarray representing the promoters of ∼24,000 human genes, was used to distinguish hypermethylated loci (HR) from hypomethylated loci (HO). Gene expression analysis utilized the Affymetrix Human Genome U-133 Plus 2.0 chip. Unsupervised cluster analysis, performed on subsets of HELP gene probes (hgp), selected for variation of ≥1 sample by ≥2-fold from the mean methylation value of all samples, demonstrated segregation by patient in 4/5 of the paired DNA sets but by treatment type (A-set vs C-set) at Rel. A subset of 6,220 Affymetrix gene probe sets (gps), selected for a mean expression level of ≥100 and a mean absolute change from PTx to Rel of ≥100 among the 3 pairs of A-set and C-set RNA samples, also segregated by treatment type at Rel. In an overall analysis of methylation based on >23,000 hgp, the average HR increased by 5% in A-set and decreased by 6% in C-set patients. Conversely, the average HO decreased by 4.3% in the A-set and increased by 7% in the C-set patients. In order to more critically evaluate the methylation changes and their possible relationship to gene expression changes, we selected a subset of hgp within −500 to +200nt of the transcription start site (TSS) in each gene promoter. Further selecting these hgp for an ≥1.5-fold change from PTx to Rel for each matched sample pair from the A- or C-sets and performing a scatter plot analysis of this selected hgp subset versus the change in expression of all 6,220 Affymetrix gps, there was a predominant increase in the HR hgp in the A-set and of HO hgp in the C-set, paralleling the overall hgp results. However, the distribution of methylation changes was random with respect to changes in gene expression. Similar results were observed by a reverse selection analysis starting from Affymetrix gps that were changed by ≥1.5-fold in all samples from each set (559, A-set; 361, C-set) and plotting versus all near-TSS hgp (15,782 hgp). Within the limits of the small sample size analyzed, we conclude that treatment type affects both gene promoter methylation and gene expression at Rel, and, most confidently, that Rel from ATRA-containing treatment was associated with increased gene promoter methylation that was not broadly correlated with gene expression changes. Further studies are required to assess whether coordinated methylation/ expression changes occur in key genes with pathogenetic potential.

Kinetics of Human and Murine Mobilization of Acute Myeloid Leukemia in Response to AMD3100.

The CXCR4-SDF-1 axis possesses a central role in the trafficking and retention of both normal and malignant stem cells in the bone marrow. Previous work from our laboratory established that in a murine model, a single dose of the CXCR4 antagonist, AMD3100, sensitizes AML blasts to chemotherapy supporting the premise that the interaction between AML blasts and the marrow microenvironment confers resistance to genotoxic stress (Nervi et al., ASH 2006). Here we examine the effects of repetitive dosing of AMD3100 on the kinetics of normal and leukemic mobilization. Following SQ injection of AMD3100 5mg/kg into B6/129 F1 mice daily for 5 days (n=8), we observed a 2.4 fold increase in total leukocyte counts with a 12.4 increase in CFU-GM when compared to 3 hours post injection (Fig 1A). No differences were seen in the degree of mobilization between d1 and d5 with WBC and CFU-GM counts returning to baseline after 24 hours. We next tested repetitive doses of AMD3100 in our mouse model of AML in which 106 blasts derived from leukemic mice carrying the PML-RARα fusion gene in the murine cathepsin G locus are adoptively transferred into genetically compatible secondary recipients. AMD3100 at 5mg/kg was then administered to these AML mice for 4 consecutive days. At 3 hrs post AMD3100 injection, we observed a 1.8 fold increase in peripheral leukocyte counts with a 4.5 fold increase in circulating blasts compared to baseline (n=3). Again, no significant differences are seen in the degree of mobilization from d1 to d4 (Fig 1B). Based on these preclinical data, we have initiated a phase I/II trial of AMD3100 plus mitoxantrone, etoposide and cytarabine (MEC) in relapsed or refractory AML in which AMD3100 is administered 4 hours prior to MEC daily for 5 consecutive days. To study the kinetics of human AML mobilization, we administered AMD3100 by SQ injection followed by 24hr observation period prior to chemotherapy. Two patients have been treated at the first dose level of AMD3100, 80 μg/kg. In pt #1 following AMD3100 mobilization, total WBC increased from 3 × 103/mm3 to a peak of 17 × 103/mm3 at 6 hours post-AMD3100 representing a 5.7 fold increase in total white count (Fig 2). In addition, the blasts (CD45dim, SSlow) increased by 7.3 fold. Similarly in pt #2, we observed a 2 fold increase in the total WBC from 2.5 to 5.1 × 103/mm3 with a 2.3 fold increase in blasts (CD45dim, SSlow). Mobilization of AML was confirmed in both patients through informative FISH for 11q23 (MLL). No adverse events have been observed during mobilization. These data provide the preclinical rationale for repetitive dosing of AMD3100 and direct clinical evidence that AMD3100 mobilizes human AML blasts into the peripheral circulation. Our trial of AMD3100 plus MEC in relapsed or refractory AML is ongoing. Figure 1. AMD3100 induced mobilization of (A) normal progenitors and (B) AML blasts Figure 1. AMD3100 induced mobilization of (A) normal progenitors and (B) AML blasts Figure 2. AMD3100 mobilization of human AML Figure 2. AMD3100 mobilization of human AML

Studies of the interaction between Aloe-emodin and DNA and preparation of DNA biosensor for detection of PML-RARα fusion gene in acute promyelocytic leukemia

The interaction of Aloe-emodin (AE) with salmon sperm DNA in 0.1 M Tris–HCl buffer (pH 4.4) and at the DNA-modified glassy carbon electrode (GCE) was systemically studied with voltammetry and ultraviolet–visible (UV–vis) spectroscopy. AE had excellent electrochemical activity on the GCE with a couple of redox peaks. We propose that AE can intercalate into DNA strands forming a nonelectroactive complex, which results in the decrease of the reduction peak current of AE. The Langmuir adsorption constants of AE at ss- and dsDNA/GCE were (2.1 ± 0.4) × 10 5 and (2.7 ± 0.2) × 10 5 M −1, respectively. The difference between AE at ss- and dsDNA has been used for the preparation of a sequence-specific DNA electrochemical biosensor for detection of PML-RARα fusion gene in acute promyelocytic leukemia (APL) with a detection limit of 6.7 × 10 −8 M and a linear range from 1.5 × 10 −8 to 1.5 × 10 −7 M. The selectivity of ssDNA-modified electrode was also described.

Morphological characteristics of leukemia cells in acute myeloblastic leukemia with t(8;21)(q22;q22): possible predictability of t(8;21)

The laboratory systems for chromosomal analysis or the detection of fusion genes are generally not available in Indonesia. Therefore, bone marrow (BM) morphological analysis should be developed and applied to get an accurate diagnosis. In this study the BM smears of eight (8) cases of acute myeloblastic leukemia (AML) which had already been known to have t(8;21)(q22;q22), were morphologically evaluated in order to find out the characteristics, which might be used to predict t(8;21)(q22;q22) or the presence of AML1-ETO(MTG8) fusion gene. All of the cases belonged to AML-M2. The morphological characteristics, indicative of t(8;21) were pink colored cytoplasm in mature neutrophil (75%), neutrophilic myelocytes or metamyelocytes without granules or with scarce granules (2.3%), eosinophilia (eosinophilic myelocytes and metamyelocytes) (above 5%), myelocytes with abundant granules 8.5%, and low percentage of type I blasts (below 10%). These characteristics were not observed in AML-M2 cases without t(8;21) or AML1-ETO(MTG8). The myelocytes with abundant granules have not been described so far, while other characteristics were in line with the findings of Nakamura et al (Leukemia 1997;11:651-55). (Med J Indones 2007; 16:84-8) Keywords: PML-RARA fusion gene, AML1-ETO (MTG8), myelocytes, abundant granul

Investigation of PML-RARα fusion gene in acute promyelocytic leukemia by real-time quantitative reverse transcription PCR

目的 利用PML-RARα融合基因作为标志物,应用实时荧光定量PCR技术,监测急性早幼粒细胞白血病(APL)微小残留病(minimal residual disease,MRD)患者体内病毒的状态,探讨预测APL复发风险的方法.方法 利用本实验室建立的定量检测APL患者PML-RARα融合基因的方法,对25例处于初诊、完全缓解(complete remission,CR)、复发等不同阶段的APL患者进行了PML-RARα融合基因的定量检测,并对其中6例患者的MRD状态进行了动态监测.结果 应用荧光定量PCR技术分析患者初诊、CR和复发状态的PML-RARα基因对数值(lg)水平,结果 显示有统计学意义(x2=6.847,P＜0.05);随访期患者的PML-RARα对数值(lg)水平变化较大:初诊时较高(-0.61±0.79),CR时下降(-1.31±0.62),复发时又出现上升(-0.57±0.44),提示若该患者在CR时的对数值呈现上升趋势,患者复发的机率大.结论 应用实时荧光定量(RQ)-PCR技术定量检测PML-RARα融合基因,对监测APL患者MRD状态具有临床适用性,随访期的MRD动态追踪监测具有预后价值。

Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations

We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFβ-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 106 normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

Novel PML-RARA Fusion Gene on Chromosome 17 in Acute Promyelocytic Leukemia with Normal Chromosome 15 and 17

We describe a patient with acute promyelocytic leukemia (APL) with no detectable cytogenetic abnor- mality of either chromosomes 15 or 17 who nevertheless had juxtaposition of promyelocytic leukemia (PML) and retinoic acid receptor-α (RARA) and expressed a chimeric transcript. Conventional cyto- genetics showed the 46, XX. The metaphase fluorescence in situ hybridization (FISH) with a 5' PML and 3' RARA probe showed a juxtaposed PML-RARA fusion signal on one chromosome 17 homologue, an RARA signals on the other chromosome 17 homologue, and one PML signal on each chromosome 15 homologue. Our patient is presently in remission and doing well after chemotherapy with idarubicin and all trans retinoic acid (ATRA) treatment. Our results show that APL patients with cytogenetically normal chromosome 15 and 17 may, nevertheless, have involvement of both PML and RARA genes and as the prognostic outcome in APL is associated with the presenc e of a PML-RARA fusion, it is necessary to perform RT-PCR or FISH to aid diagnosis. (Korean J Hematol 2007;42:296-300.)