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Tandutinib (MLN518/CT53518) is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases: FMS-like tyrosine kinase 3 (FLT3), platelet-derived growth factor receptor (PDGFR), and KIT. Because of the correlation between FLT3 internal tandem duplication (ITD) mutations and poor prognosis in acute myelogenous leukemia (AML), we conducted a phase 1 trial of tandutinib in 40 patients with either AML or high-risk myelodysplastic syndrome (MDS). Tandutinib was given orally in doses ranging from 50 mg to 700 mg twice daily The principal dose-limiting toxicity (DLT) of tandutinib was reversible generalized muscular weakness, fatigue, or both, occurring at doses of 525 mg and 700 mg twice daily. Tandutinib's pharmacokinetics were characterized by slow elimination, with achievement of steady-state plasma concentrations requiring greater than 1 week of dosing. Western blotting showed that tandutinib inhibited phosphorylation of FLT3 in circulating leukemic blasts. Eight patients had FLT3-ITD mutations; 5 of these were evaluable for assessment of tandutinib's antileukemic effect. Two of the 5 patients, treated at 525 mg and 700 mg twice daily, showed evidence of antileukemic activity, with decreases in both peripheral and bone marrow blasts. Tandutinib at the MTD (525 mg twice daily) should be evaluated more extensively in patients with AML with FLT3-ITD mutations to better define its antileukemic activity.

The transcription factor CEBPA is crucial for normal myeloid differentiation. CEBPA gene mutations have been reported in patients with acute myeloid leukaemia. The inevitable evolution of chronic myeloid leukaemia (CML) in chronic phase (CP) to a fatal blast crisis (BC) is assumed to result from the acquisition of additional genetic changes in the leukaemic clone. Gain of CEBPA mutations might represent a key event causing the differentiation block observed in myeloid CML-BC, but not in CML-CP. Here, no CEBPA mutation in 95 CML-BC patients was found, suggesting a limited role, if any, of CEBPA mutations in this disorder.

BACKGROUND Aberrant DNA methylation, which results in leukemogenesis, is frequent in patients with myelodysplastic syndromes (MDS) and is a potential target for pharmacologic therapy. Decitabine indirectly depletes methylcytosine and causes hypomethylation of target gene promoters. METHODS A total of 170 patients with MDS were randomized to receive either decitabine at a dose of 15 mg/m2 given intravenously over 3 hours every 8 hours for 3 days (at a dose of 135 mg/m2 per course) and repeated every 6 weeks, or best supportive care. Response was assessed using the International Working Group criteria and required that response criteria be met for at least 8 weeks. RESULTS Patients who were treated with decitabine achieved a significantly higher overall response rate (17%), including 9% complete responses, compared with supportive care (0%) (P < .001). An additional 12 patients who were treated with decitabine (13%) achieved hematologic improvement. Responses were durable (median, 10.3 mos) and were associated with transfusion independence. Patients treated with decitabine had a trend toward a longer median time to acute myelogenous leukemia (AML) progression or death compared with patients who received supportive care alone (all patients, 12.1 mos vs. 7.8 mos [P = 0.16]; those with International Prognostic Scoring System intermediate-2/high-risk disease, 12.0 mos vs. 6.8 mos [P = 0.03]; those with de novo disease, 12.6 mos vs. 9.4 mos [P = 0.04]; and treatment-naive patients, 12.3 mos vs. 7.3 mos [P = 0.08]). CONCLUSIONS Decitabine was found to be clinically effective in the treatment of patients with MDS, provided durable responses, and improved time to AML transformation or death. The duration of decitabine therapy may improve these results further. Cancer 2006. © 2006 American Cancer Society.

Among the drug combinations designed for the initial treatment of multiple myeloma, none has been unequivocally shown to be superior. However, a regimen leading to a high response rate and a low incidence of adverse events is highly desirable. We report the results of a phase II clinical trial involving 45 patients with Durie-Salmon stage II and III multiple myeloma. Doxorubicin and dexamethasone were given for 2 or 3 months followed by thalidomide and dexamethasone for 2 months (AD-TD regimen) with prophylactic antibiotics and daily aspirin (81 mg/d). Among the 42 patients whose response could be assessed, 38 responded to therapy (90.5%). The intent-to-treat response rate was 84.4% with seven complete responses (CR 15.5%), nine near complete responses (nCR 20.0%), and 22 partial responses (PR 48.9%). Two patients had stable disease (4.4%), and two progression of disease (4.4%). Normalization of the free light chain ratio after one or two cycles of treatment was highly predictive of achievement of CR or nCR. Patients tolerated the treatment well although five patients developed thromboembolic complications (11%). AD-TD administered with low dose aspirin for deep vein thrombosis prophylaxis was well tolerated and yielded a high response rate with minimal treatment-related morbidity.

Contains fulltext : 50832.pdf (Publisher’s version ) (Closed access) The myelodysplastic syndromes (MDSs) are heterogeneous with respect to clinical characteristics, pathologic features, and cytogenetic abnormalities. This heterogeneity is a challenge for evaluating response to treatment. Therapeutic trials in MDS have used various criteria to assess results, making cross-study comparisons problematic. In 2000, an International Working Group (IWG) proposed standardized response criteria for evaluating clinically significant responses in MDS. These criteria included measures of alteration in the natural history of disease, hematologic improvement, cytogenetic response, and improvement in health-related quality of life. The relevance of the response criteria has now been validated prospectively in MDS clinical trials, and they have gained acceptance in research studies and in clinical practice. Because limitations of the IWG criteria have surfaced, based on practical and reported experience, some modifications were warranted. In this report, we present recommendations for revisions of some of the initial criteria.

Therapy-related Myeloid Neoplasms (tMN) comprise a poor risk subset of myelodysplastic syndromes and acute myelogenous leukemia, are increasing in incidence, and represent a serious complication following treatment for primary malignancies. In our previous study of 11 genes in 38 tMN patient samples, the data suggested that the mutational spectrum of tMN was distinct from de novo myeloid malignancies. To confirm this finding and to refine the tMN mutation profile, we investigated the mutation profile in samples from 88 patients and 28 genes using Sanger and next-generation sequencing approaches. We performed amplification using RainDance microfluidic PCR, followed by HiSeq next-generation sequencing. Mutations were identified using a modified pipeline for SNP calling employing variant detection software programs. Our study cohort included 88 patients, 71 of whom had complete clinical data for analyses. Patients had a history of epithelial and hematologic malignancies (³2 malignancies n=11; breast n=9; colorectal n=5; head and neck n=4; genital-urinary n=6; lung n=1; lymphoma n=25; melanoma n=2; ovarian n=1; sarcoma n=2; other, n=5). Treatment of primary cancers included chemotherapy alone (n=27), radiation alone (n=8), autologous stem cell transplant (n=11), or chemotherapy plus radiation (n=25). The median latency time between primary malignancy treatment and tMN diagnosis was 5.7yrs (range, 0.7 - 30.9 yrs). Median age at tMN diagnosis was 64yrs (range, 26 - 85 yrs). International Prognostic Scoring System (IPSS) risk group for MDS at tMN diagnosis were Low risk (n=8), Int-1 (n=11), Int-2 (n=30), High risk (n=9). We identified somatic mutations in 56 of 88 (64%) patients (83 patients were evaluated by next-generation sequencing and 5 by Sanger sequencing only). Mutations in TP53 were most common and were detected in 27/88 patients (30.7%), followed by mutations in TET2 in 12/88 (13.6%), DNMT3A in 9/88 (10.2%), NRAS in 8/83 (9.6%), KRAS in 5/83 (6.0%), and KIT in 5/83 (6.0%). Gene mutations detected at lower frequencies included those in ASXL1 in 5/88 (5.7%), RUNX1 in 2/83 (2.4%), EZH2 in 1/88 (1.1%), and SF3B1 in 1/88 (1.1%). Of the 58 patients with complete sequencing and FISH data, 4 patients exhibited biallelic somatic TP53 mutations and 3 patients had TP53 mutation combined with del 17p TP53 loss, demonstrating that 7 of 58 evaluable patients (12.1%) experienced biallelic loss of TP53. We also identified biallelic mutations in TET2 and DNMT3A in 2 separate patients. 25 patients had 2 or more concurrent somatic mutations. The highest number of co-occurring mutations in one patient was 5 mutations; 12 patients had 2 somatic mutations. The most common co-occurrence was TP53 and TET2, which was observed in 5 patients. All 5 ASXL1 mutations co-occurred with additional mutations. By analyzing variant allele frequencies (VAFs) in patients with multiple mutations, we observed that some tMN patients harbored multiple clones with distinct VAFs. This observation was also supported by the co-occurrence of typical class I driver mutations in the same patient, (e.g. KRAS 6% and NRAS 21% VAF; NRAS 9% and KIT 34%; NRAS 26% and KIT 9% in individual patients). The allele frequency data also suggested that ASXL1 is likely an early occurring mutation as the VAF was higher than for other co-occurring mutations (mean VAF ASXL1 50%, other co-occurring genes 23.5%, p Because of previous reports on the prognostic significance of point mutations in myeloid malignancies (e.g. TP53 in MDS and TET2 in AML), we tested the impact of individual mutations on prognosis. TP53 mutation or loss was associated with worse prognosis in tMN (OS 17.6 vs 25.2 mos, n=72, p Our data reveal that tMNs display distinct mutation profile compared to de novo disease (Fig B). TP53 mutations and loss are the most common abnormalities and predict for adverse outcome. Epigenetic modifier mutations also occur in tMNs and can serve as disease-initiating mutations. Collectively our results demonstrate that characterizing these mutation profiles can enhance our understanding of disease mechanisms in tMNs and may guide the development of future therapies for these difficult to treat disorders. Figure 1 Figure 1. Disclosures Sekeres: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective hematopoiesis leading to cytopenias and a high rate of progression to acute myeloid leukemia (AML). The NUP98-HOXD13 (NHD13) fusion has been found in patients with MDS and AML. A transgenic (Tg) mouse model, generated by Peter Aplan’s group, which utilizes Vav 1 regulatory elements to direct expression of the NHD13 transgene in hematopoietic tissues, displays the phenotypic features of MDS including a chronic phase of cytopenias followed by transformation to AML (Lin et al., 2005). We previously reported that loss of one or both alleles of p53 did not rescue the MDS phenotype in NHD13+ Tg mice, but rather exacerbated the MDS phenotype and accelerated the development of AML (Xu et al., 2012). Expression of p21WAF1/CIP1(p21) was increased in the Lin−Sca-1+c-Kit+ (LSK) cells isolated from NHD13+ Tg mice, so we generated and analyzed NHD13+p21+/– and NHD13+p21–/– mice to further investigate whether the accelerated MDS and AML that occurs in the absence of p53 relates to the defective expression of the p53 target gene. Deletion of p21 significantly altered the fate of the NHD13+ Tg mice. All of the NHD13+p21–/– mice died of AML, rather than MDS. Only 18% (4 out of total 22 mice) of the NHD13+p21+/– mice developed MDS with a median survival of 289 d; in contrast 31% (9 out of total 29 mice) of NHD13+ Tg mice died from MDS, with a median survival of 230 d (p Disclosures Armstrong: Epizyme : Consultancy.

Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

Epigenetic mechanisms underlying tumorigenesis have recently received much attention as potential therapeutic targets of human cancer. We designed a pilot study to target DNA methylation and histone deacetylation through the sequential administration of 5-azacytidine followed by sodium phenylbutyrate (PB) in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Ten evaluable patients (eight AML, two MDS) were treated with seven consecutive daily subcutaneous injections of 5-azacytidine at 75 mg/m2 followed by 5 days of sodium PB given intravenously at a dose of 200 mg/kg. Five patients (50%) were able to achieve a beneficial clinical response (partial remission or stable disease). One patient with MDS proceeded to allogeneic stem cell transplantation and is alive without evidence of disease 39 months later. The combination regimen was well tolerated with common toxicities of injection site skin reaction (90% of the patients) from 5-azacytidine, and somnolence/fatigue from the sodium PB infusion (80% of the patients). Correlative laboratory studies demonstrated the consistent reacetylation of histone H4, although no relationship with the clinical response could be demonstrated. Results from this pilot study demonstrate that a combination approach targeting different mechanisms of transcriptional modulation is clinically feasible with acceptable toxicity and measurable biologic and clinical outcomes.

Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an index AML patient, an algorithm of analyses was applied using clinical material. In vivo and in vitro investigation of primary blasts at relapse revealed persistent TK phosphorylation of FLT3 despite sufficient PKC412 serum levels. Through additional molecular analyses, we identified a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in this patient. Reconstitution experiments expressing the N676K mutant in 32D cells demonstrated that FLT3-ITD-N676K was sufficient to confer an intermediate level of resistance to PKC412 in vitro. These studies point out that a genetically complex malignancy such as AML may retain dependence on a single oncogenic signal.

OLIG2 (originally designated BHLHB1) encodes a transcription factor that contains the basic helix-loop-helix motif. Although expression of OLIG2 is normally restricted to neural tissues, overexpression of OLIG2 has been shown in patients with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL). In the current study, we found that overexpression of OLIG2 was not only found in oligodendroglioma samples and normal neural tissue but also in a wide spectrum of malignant cell lines including leukemia, non–small cell lung carcinoma, melanoma, and breast cancer cell lines. To investigate whether enforced expression of OLIG2 is oncogenic, we generated transgenic mice that overexpressed OLIG2 in the thymus. Ectopic OLIG2 expression in the thymus was only weakly oncogenic as only 2 of 85 mice developed pre-T LBL. However, almost 60% of transgenic mice that overexpressed both OLIG2 and LMO1 developed pre-T LBL with large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2/LMO1 mice revealed up-regulation of Notch1 as well as Deltex1 (Dtx1) and pre T-cell antigen receptor α (Ptcra), two genes that are considered to be downstream of Notch1. Of note, we found mutations in the Notch1 heterodimerization or proline-, glutamic acid-, serine-, and threonine-rich domain in three of six primary thymic tumors. In addition, growth of leukemic cell lines established from OLIG2/LMO1 transgenic mice was suppressed by a γ-secretase inhibitor, suggesting that Notch1 up-regulation is important for the proliferation of OLIG2-LMO1 leukemic cells.

The AML1-ETO fusion protein, generated by the t(8;21) in acute myeloid leukemia (AML), exerts dominant-negative functions and a variety of gains of function, including a positive effect on the growth of primary human CD34 + hematopoietic stem/progenitor cells. We now show that AML1-ETO expression up-regulates the level of TRKA mRNA and protein in these cells and that AML1-ETO-expressing CD34 + hematopoietic cells grown in the presence of five early-acting hematopoietic cytokines further proliferate in response to nerve growth factor (NGF). These cells also show a unique response to NGF and IL-3; namely, they expand in liquid culture. To determine the biological relevance of our findings, we analyzed 262 primary AML patient samples using real-time RT-PCR and found that t(8;21)-positive AML samples express significantly higher levels of TRKA mRNA than other subtypes of AML. NGF, which is normally expressed by bone marrow stromal cells, could provide important proliferative or survival signals to AML1-ETO-expressing leukemic or preleukemic cells, and the NGF/TRKA signaling pathway may be a suitable target for therapeutic approaches to AML.

Polycomb group proteins are epigenetic gene silencers that have been implicated in cancer development and stem cell maintenance.1 Biological and genetic studies indicate that PcG proteins exist in at least two separate protein complexes, polycomb-repressive complex 2 (PRC2) and polycomb-repressive complex 1 (PRC1), which act in concert to promote and maintain gene repression.1 EZH2, the catalytically active component of PRC2, plays an important role in many biological processes through its ability to trimethylate lysine 27 in histone H3. The core enzymatic activity of PRC1 is an E3 ubiquitin ligase activity contributed by RING1B (and enhanced by BMI1), which ubiquitinates histone H2A at lysine 119.1 PcG proteins are displaced from the promoters of one set of target genes while being recruited to the promoters of another set of target genes during lineage specification.1 How extracellular signaling regulates this process is not well-understood. In 2005, Cha and colleagues found Akt mediated phosphorylation of EZH2 results in a decrease of lysine 27 trimethylation and derepression of silenced genes.2 Recently, we reported that phosphorylation of BMI1 at serine 316 by Akt impairs its function by triggering its dissociation from the Ink4a-Arf locus, which results in decreased histone H2A ubiquitination and the inability of BMI1 to promote cell growth and tumorigenesis.3 It is very likely that other PcG proteins, including EZH1 and MEL18, are downstream targets of Akt. Thus, Akt appears to regulate the activities of both the PRC2 and PRC1 complexes in the cell (Fig. 1). Figure 1. PI3K-Akt signaling regulates polycomb group protein and hematopoietic stem cell self-renewal. PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs).1 HSCs have both the capacity to self-renew and to differentiate into many different mature blood cell types. Understanding the molecular mechanisms governing stem cell self-renewal remains the holy grail of stem cell biology and holds great promise for the development of stem cell-based therapies aimed at treating debilitating and life-threatening diseases such as cancer. Self-renewal requires the integration of survival signals and proliferation controls with the maintenance of an undifferentiated state. This demands a complex crosstalk between extrinsic signals from the microenvironment and the cell-intrinsic regulators of HSCs to maintain an undifferentiated state.4 However, how the crosstalk is coordinated has not been well-defined at the molecular level. Activation of the PI3K-Akt signaling impairs HSC self-renewal,5 and we demonstrated that BMI1 is a critical downstream target of PI3K-Akt signaling in HSCs and that Akt-mediated phophorylation of BMI1 inhibits its ability to promote hematopoietic stem/progenitor cell (HSPC) self-renewal.3 At virtually the same time, the van Lohuizen lab reported that Akt-mediated BMI1 phosphorylation enhances its oncogenic potential in an Ink4a and Arf-independent mouse model of human prostate cancer.6 They identified three sites of serine phosphorylation [Ser251, Ser253 and Ser255 (SDSGS)], none of which were consensus Akt phosphorylation sites (RXRXXS or T).6 Nonetheless, it appears that the inhibition of cell proliferation and oncogenesis by Akt-mediated phosphorylation of BMI1, occurs in a site-specific and possibly context-dependent manner. Similar to BMI1, a component of Polycomb-repressive complex 1 (PRC1), PRC2 and its components play important roles in hematopoiesis. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis.7 On the contrary, Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response, suggesting that Ezh1 is an important histone methyltransferase for adult HSC maintenance.8 Therefore, it is possible that Akt-mediated phosphorylation of EZH2 or EZH1 may limit HSC function as well. Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of diseases. The ability to increase the number of HSCs either in vivo or in vitro would provide new treatment options, but the amplification of HSCs has been difficult to achieve. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Most hematopoietic cell growth factors activate Akt in ex vivo cultures, promoting HSC differentiation. Therefore, treating HSCs with Akt inhibitors may expand HSCs ex vivo. Our understanding of the crosstalk between PI3K-Akt signaling and polycomb group proteins may facilitate the development of innovative clinical strategies that can enhance ex vivo HSC expansion.

Transforming growth factor β (TGFβ) is one of few known negative regulators of hematopoiesis, yet the mechanisms by which it affects cell cycle arrest and stem cell quiescence are poorly understood. Induction of the cyclin-dependent kinase inhibitors, p15INK4b (p15) and p21WAF1 (p21) is important for TGFβ-mediated cytostasis in epithelial cells but not in hematopoietic cells. Using primary human hematopoietic cells and microarray analysis, we identified p57KIP2 (p57) as the only cyclin-dependent kinase inhibitor induced by TGFβ. Up-regulation of p57 mRNA and protein occurs before TGFβ-induced G 1 cell cycle arrest, requires transcription, and is mediated via a highly conserved region of the proximal p57 promoter. The up-regulation of p57 is essential for TGFβ-induced cell cycle arrest in these cells, because two different small interfering RNAs that prevent p57 up-regulation block the cytostatic effects of TGFβ on human hematopoietic cells. Reduction of basal p57 expression by this approach also allows hematopoietic cells to proliferate more readily in the absence of TGFβ. p57 is a putative tumor suppressor gene whose expression is frequently silenced by promoter hypermethylation in hematologic malignancies. Our studies identify a molecular pathway by which TGFβ mediates its cytostatic effects on human hematopoietic cells and suggests an explanation for the frequent silencing of p57 expression.

Fms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated AML. Mutation screening of FLT3 exon 20 in AML patients (n = 110) revealed 2 patients with a novel mutation (Y842C) within the highly conserved activation loop of FLT3. FLT3-Y842C-transfected 32D cells showed constitutive FLT3 tyrosine phosphorylation and interleukin 3 (IL-3)-independent growth. Treatment with PKC412 led to inhibition of proliferation and apoptotic cell death. Primary AML blasts bearing FLT3-Y842C mutations showed constitutive FLT3 and signal transducer and activator of transcription 5 (STAT-5) tyrosine phosphorylation. Ex vivo PKC412 treatment of primary blasts resulted in suppression of constitutive FLT3 and STAT-5 activation and apoptotic cell death. Inspection of the FLT3 structure revealed that Y842 is the key residue in regulating the switch from the closed to the open (= active) conformation of the FLT3 activation loop. Overall, our data suggest that mutations at Y842 represent a significant new activating mutation in AML blasts. Since FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412 are currently being investigated in clinical trials in AML, extended sequence analysis of FLT3 may be helpful in defining the spectrum of TKI-sensitive FLT3 mutations in AML.

Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to < 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.

The human L3MBTL gene is located in 20q12, a region that is commonly deleted in myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). L3MBTL is highly homologous to the D-lethal(3) malignant brain tumor [D-l(3)mbt] gene, which is a putative tumor-suppressor gene (TSG) identified in Drosophila and which is closely related to the Drosophila sex combs on midleg (SCM) protein, a member of the Polycomb group (PcG) family of transcriptional repressors. To examine whether L3MBTL functions as a "classic" TSG in human hematologic malignancies, we screened a panel of 17 myeloid leukemia cell lines and peripheral blood or bone marrow samples from 29 MDS and 13 MPD patients for mutations in the entire L3MBTL coding sequence, including intron/exon splice junctions. No mutations were identified, although two single nucleotide differences were found (in intron 14 and in exon 15), which were interpreted as polymorphic changes. We used real-time RT-PCR to quantify the level of L3MBTL mRNA in various normal myeloid and lymphoid cell populations. L3MBTL is expressed in normal CD34+ bone marrow cells, and we found that the pattern of L3MBTL expression was similar to that of BMIl, a well-studied PcG gene with oncogenic activity, suggesting that L3MBTL and BMII may be co-regulated during hematopoiesis. The expression of L3MBTL mRNA in 30 of 35 cell lines and 13 of IS AML samples was comparable to the level of L3MBTL expression in the normal cell populations. However, five leukemia cell lines showed no L3MBTL expression, and two of the AML samples showed aberrant L3MBTL expression. These data suggest that L3MBTL is not mutated in MDS or MPD. However, given the known dosage effects of PcG proteins in regulating gene expression, reduced or absent L3MBTL expression may be relevant in some cases of myeloid leukemia. (C) 2004 Wiley-Liss, Inc. (Less)

We evaluated the results of high-dose chemotherapy and autologous hematopoietic stem cell transplantation in patients with diffuse aggressive non-Hodgkin lymphoma (NHL) in first relapse (Rel 1) or second complete remission (CR 2). Data were evaluated from the Autologous Blood and Marrow Transplant Registry on 429 patients with diffuse aggressive NHL who underwent transplantation in Rel 1 or CR 2. Transplantations were performed between 1989 and 1996 and were reported to the Autologous Blood and Marrow Transplant Registry by 93 centers in North and South America. The probability of 3-year survival was 44% (95% confidence interval [CI], 33%–55%). The probability at 3 years of progression-free survival was 31% (95% CI, 27%–36%). Patients who underwent transplantation in CR 2 had a 3-year probability of progression-free survival of 38% (95% CI, 30%–46%) compared with 28% (95% CI, 22%–33%) for those who were not in remission at the time of transplantation (P < .001). In multivariate analysis, chemotherapy resistance, increased lactic dehydrogenase at diagnosis, an interval of

Patients with relapsed or primary refractory diffuse large B-cell lymphoma (DLBCL) who achieve complete response (CR) before autologous stem cell transplantation (ASCT) generally have better outcomes than those who achieve only partial response (PR). We investigated whether adding rituximab to the ifosfamide-carboplatin-etoposide (ICE) chemotherapy regimen (RICE) could increase the CR rate of patients with DLBCL under consideration for ASCT. Thirty-six eligible patients were treated with RICE, and 34 received all 3 planned cycles. The CR rate was 53%, significantly better than the 27% CR rate (P = .01) achieved among 147 similar consecutive historical control patients with DLBCL treated with ICE; the PR rate was 25%. Febrile neutropenia was the most frequent grade 3 or 4 nonhematologic toxicity; it occurred in 7.5% of delivered cycles. No patient had RICE-related toxicity that precluded ASCT. The median number of CD34+ cells per kilogram mobilized was 6.3 × 106. Progression-free survival rates of patients who underwent transplantation after RICE were marginally better than those of 95 consecutive historical control patients who underwent transplantation after ICE (54% vs 43% at 2 years; P = .25). RICE appears to induce very high CR rates in patients with relapsed and refractory DLBCL; however, further studies are necessary to determine whether this treatment regimen will improve outcomes after ASCT.

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) is essential for a number of proapoptotic and growth-suppressive pathways as well as for the activity of differentiating agents such as retinoic acid (RA). In human APL, the dose of PML is reduced to heterozygosity given that one allele is involved in the chromosomal translocation while the status of the remaining PML allele is unknown. We have therefore used single-strand conformational polymorphism (SSCP) and sequencing analysis to screen DNA from APL patients for mutations at the PML locus. We identified DNA sequence variations resulting in a truncated PML protein in APL cases that displayed RA resistance and a very poor prognosis. Mutation analysis also led to the identification of aberrant PML sequence variations in other hematopoietic malignancies. Complete functional loss of PML is therefore selected by the APL phenotype and associates with poor prognosis and RA unresponsiveness. (C) 2004 by The American Society of Hematology.

Anaplastic oligodendroglioma is a chemosensitive glial neoplasm. To improve disease control and postpone cranial radiotherapy, we designed a phase II study of intensive procarbazine, lomunstine and vincristine followed by high-dose thiotepa with autologous stem cell rescue for patients with newly diagnosed anaplastic or aggressive oligodendroglioma.Sixty-nine patients with a median age of 42 (range: 18-67) and a median Karnofsky Performance Score of 90 (range: 70-100) were enrolled. Sixteen patients had a prior diagnosis of low-grade oligodendroglioma and 16 had mixed oligoastrocytoma pathology. Only patients with demonstrably chemosensitive enhancing tumors or those free of enhancing tumor after surgery and induction therapy were eligible to receive high-dose thiotepa.Thirty-nine patients (57%) completed the transplant regimen; their estimated median progression-free survival is 69 months and median overall survival has not been reached. Twelve transplanted patients (31%) relapsed. Neither histology nor prior low-grade oligodendroglioma correlated with relapse; however, persistent non-enhancing tumor at transplant conferred an increased risk of relapse (p = 0.028). The transplant regimen was well-tolerated; median hospital stay was 20 days (range: 7-43) with a median time to ANC and platelet engraftment of 10 days. Thirty patients (43%) did not receive high-dose thiotepa because of stable or progressive disease (n = 21), excessive toxicity (n = 4), refusal of further therapy (n = 2), failure to obtain insurance coverage (n = 2), or other (n = 1). No treatment-related or long-term neurotoxicity was seen in the transplanted patients.High-dose chemotherapy with stem cell rescue as initial treatment for anaplastic oligodendroglioma is feasible and associated with prolonged tumor control in some patients.

Purpose: To assess the safety and efficacy of intensive methotrexate-based chemotherapy followed by high-dose chemotherapy (HDT) with autologous stem-cell rescue in patients with newly diagnosed primary CNS lymphoma (PCNSL). Patients and Methods: Twenty-eight patients received induction chemotherapy using high-dose systemic methotrexate (3.5 g/m2) and cytarabine (3 g/m2 daily for 2 days). Fourteen patients with chemosensitive disease evident on neuroimaging then received high-dose therapy using carmustine, etoposide, cytarabine, and melphalan with autologous stem-cell rescue. Results: The objective response rate to the induction-phase chemotherapy was 57%, and median overall survival is not yet assessable, with a median follow-up time of 28 months. The overall median event-free survival time is 5.6 months for all patients and 9.3 months for 14 patients who underwent transplantation. Six of these 14 patients (43%) remained disease-free at last follow-up. Treatment was well tolerated; there was one transplantation-related death. Prospective neuropsychologic evaluations have revealed no evidence of treatment-related neurotoxicity. Conclusion: This treatment approach is feasible in patients with newly diagnosed PCNSL without evidence of significant related neurotoxicity. Although the transplantation results are similar to those achieved in patients with aggressive or poor-prognosis systemic lymphoma, the low response rate to induction chemotherapy and the significant number of patients who experienced relapse soon after HDT suggest that more aggressive induction chemotherapy may be warranted.

To determine the outcome of patients with chemosensitive relapsed or primary refractory Hodgkin's disease (HD) or aggressive non-Hodgkin's lymphoma (NHL) whose disease progresses after autologous stem cell transplantation (ASCT), we reviewed the records of 82 patients with HD and 139 patients with NHL transplanted between 1993 and 2000. Disease progression occurred in 25 patients with HD and 66 patients with NHL, with median times to progression (TTP) of 3.8 and 5.1 months, respectively. Median survival times following ASCT failure were 26 and 7.7 months for patients with HD and NHL, respectively. The second-line international prognostic index (sIPI) and the TTP (before or after 3 months from ASCT) independently were predictive of survival for NHL patients. In addition, treatment with rituximab for patients with B cell NHL was associated with improved survival (median 28.6 vs 4.1 months, P=0.003), independent of the sIPI and TTP. Prognostic factors for patients with HD were not identified. Only two patients, one of whom was among six patients who received second autologous transplants, remain disease-free. The uniformly poor outcome associated with disease progression after ASCT should prompt efforts to assess the feasibility and utility of detecting and treating post transplant residual disease during a minimal disease state, before overt progression.

Malignant plasma cells generally grow within the bone marrow microenvironment; however, they can also grow at extramedullary sites. To identify the tumour-specific alterations required for extramedullary growth, we analysed the expression profiles of a series of plasma cell neoplasms including primary multiple myeloma (MM), plasma cell leukaemia (PCL) and extramedullary plasmacytoma (EPC). Hierarchical clustering analysis segregated the EPCs from the remaining samples, and revealed an expression pattern associated with angiogenesis in the EPCs, involving higher expression of the genes TIE2, NOTCH3, CD31 and endoglin. Direct comparison of EPC samples with the MM samples identified 156 genes significantly upregulated and 85 genes significantly downregulated (P0.005, t-test) in the EPCs, including several genes involved in angiogenesis and adhesion that were upregulated (including angiopoietin 1, SPARC, Notch3 and fibronectin 1). Immunohistochemical staining demonstrated CD31 and endoglin protein expression in the EPC tumour cells, which are both angiogenesis related and could confer malignant plasma cells with the ability to grow outside the normal bone marrow environment. Defining how malignant plasma cell growth is regulated in the bone marrow versus at extramedullary sites will help to delineate the mechanisms underlying the dependence of tumour cell growth on angiogenesis and cell adhesion.

Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Valpha24+/Vbeta11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon-gamma-producing NKT cells in response to the NKT ligand, alpha-galactosyl ceramide, although influenza-virus-specific effector T-cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.

We report on the X-ray structure of three 100-amino acid mbt repeats in h-l(3)mbt, a polycomb group protein involved in transcriptional repression, whose gene is located in a region of chromosome 20 associated with hematopoietic malignancies. Interdigitation between the extended arms and cores of the mbt repeats results in a three-leaved propeller-like architecture, containing a central cavity. We have identified one ligand binding pocket per mbt repeat, which accommodates either the morphilino ring of MES or the proline ring of the C-terminal peptide segment, within a cavity lined by aromatic amino acids. Strikingly, phenotypic alterations resulting from point mutations or deletions in the mbt repeats of the related Drosophila SCM protein are clustered in and around the ligand binding pocket.

Second-line chemotherapy followed by high-dose therapy (HDT) with autologous stem cell transplantation (ASCT) cures less than half of the patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Prognostic models capable of predicting outcome are essential. In 3 sequential clinical trials, conducted from January 1993 to August 2000, we treated 150 patients with relapsed or primary refractory DLBCL with ifosfamide, carboplatin, and etoposide (ICE) chemotherapy followed by HDT/ASCT for patients with chemosensitive disease. We evaluated the age-adjusted International Prognostic Index at the initiation of second-line therapy (sAAIPI) as a predictor of progression-free survival (PFS) and overall survival (OS). At a median follow-up of 4 years, the PFS and OS are 28% and 34% by intention to treat and 39% and 45% for only those patients with chemosensitive disease. Three risk groups with different PFS and OS were identified by the sAAIPI: low risk (0 factors), 70% and 74%; intermediate risk (1 factor), 39% and 49%; and high risk (2 or 3 factors), 16% and 18% (P < .001 for both PFS and OS). The sAAIPI also predicts the PFS and OS for patients with ICEchemosensitive disease: low risk, 69% and 83%; intermediate risk, 46% and 55%; and high risk, 25% and 26% (P < .001 PFS and OS). The sAAIPI predicts outcome for patients with relapsed or primary refractory DLBCL in both intent-to-treat and chemosensitive populations. This powerful prognostic instrument should be used to evaluate new treatment approaches and to compare results of different regimens.

Optimal methods of stem cell mobilization in multiple myeloma are undefined, and contaminating clonotypic cells could contribute to disease recurrence. A phase 2 trial of intravenous melphalan (60 mg/m2) and granulocyte colony-stimulating factor (G-CSF) (10 μg/kg/d) for mobilization was performed. To enhance reliability, contamination was assessed with 2 sensitive methods, immunoglobulin light and heavy chain variable region patient-specific limiting-dilution polymerase chain reaction (PCR). We evaluated 29 stem cell components (SCCs) from 15 patients; for 9 SCCs, only VL PCR was used because of light chain disease or technical problems with VH primers. For 20 SCCs, VL and VH PCR results were highly correlated (r2 = 0.93, P < .01), with 35% (7 of 20) having identical estimates. VH PCR gave significantly higher estimates for 8—and VL PCR for 5—SCCs, supporting the utility of using 2 methods. Estimated clonotypic contamination per SCC was 0.0009% (range, 0%-0.1%) or 0.5 × 104 clonotypic cells per kilogram (range, 0-41.2 × 104/kg), and contamination correlated with CD34+ cells collected (r2 = 0.42, P < .01). Melphalan-mobilized SCCs contain minimal clonotypic contamination.

Erythroid leukemia (ERL or AML-M6) is an uncommon subtype of acute myeloid leukemia, the clinical, morphological, and genetic behavior of which needs further characterization. We analyzed a homogeneous group of 23 de novo AML-M6 patients whose bone marrow cells showed complex karyotypes. We also analyzed eight leukemia cell lines with erythroid phenotype, performing detailed molecular cytogenetic analyses, including spectral karyotyping (SKY) in all samples. The main features are: (1) A majority of patients (56%) had hypodiploidy. Loss of genetic material was the most common genetic change, especially monosomies of chromosome 7 or 18, and deletions of chromosome arm 5q. Taken together, 87% of the cases displayed aberrations involving chromosome 5 or 8. (2) We describe a novel, cryptic, and recurrent translocation, t(11;19)(p11.2;q13.1). Another translocation, t(12;21)(p11.2;q11.2), was found to be recurrent in a patient with ERL and in the K562 cell line. (3) MLL gene rearrangements were detected in 20% of cases (three translocations and three amplifications) and, overall, we defined 52 rearrangements (excluding deletions) with a mean of 2.3 translocations per patient. (4) Of the structural aberrations, 21% involved chromosomes 11 and 19. Most of the rearrangements were unbalanced; only 13 reciprocal translocations were observed. The general picture of chromosomal aberrations in cell lines did not reflect what occurred in patient samples. However, both primary samples and cell lines shared three common breakpoints at 19q13.1, 20q11.2, and 21q11.2. This is the first molecular cytogenetic description of the karyotype abnormalities present in patients with ERL. It should assist in the identification of genes involved in erythroleukemogenesis.

H-L(3)MBT, the human homolog of theDrosophila lethal(3)malignant brain tumor protein, is a member of the polycomb group (PcG) of proteins, which function as transcriptional regulators in large protein complexes. Homozygous mutations in the l(3)mbt gene cause brain tumors inDrosophila, identifying l(3)mbt as a tumor suppressor gene. The h-l(3)mbt gene maps to chromosome 20q12, within a common deleted region associated with myeloid hematopoietic malignancies. H-L(3)MBT contains three repeats of 100 residues called MBT repeats, whose function is unknown, and a C-terminal α-helical structure, the SPM (SCM,PH, MBT domain, which is structurally similar to the SAM (sterile alphamotif) protein-protein interaction domain, found in several ETS transcription factors, including TEL (translocationEts leukemia). We report that H-L(3)MBT is a transcriptional repressor and that its activity is largely dependent on the presence of a region containing the three MBT repeats. H-L(3)MBT acts as a histone deacetylase-independent transcriptional repressor, based on its lack of sensitivity to trichostatin A. We found that H-L(3)MBT binds in vivo to TEL, and we have mapped the region of interaction to their respective SPM/SAM domains. We show that the ability of TEL to repress TEL-responsive promoters is enhanced by the presence of H-L(3)MBT, an effect dependent on the H-L(3)MBT and the TEL interacting domains. These experiments suggest that histone deacetylase-independent transcriptional repression by TEL depends on the recruitment of PcG proteins. We speculate that the interaction of TEL with H-L(3)MBT can direct a PcG complex to genes repressed by TEL, stabilizing their repressed state.

Objective The AML/RUNX family of transcription factors plays important roles in hematopoiesis, neurogenesis, bone development, and segmentation in vertebrate embryos. The aim of this study was to isolate runt -related genes in a genetically and embryologically exploitable system, the zebrafish, and characterize their function during hematopoietic development. Materials and Methods Two runt -related genes were isolated by degenerate PCR and standard library screening, and a radiation hybrid panel, T51 RH, was used to resolve their chromosomal localization. In situ hybridization demonstrated their expression whereas their transcriptional activity was assessed using an AML1-responsive reporter gene in the MLA 144 T-cell line. Results We isolated the zebrafish runxa and runxb cDNAs, which encode proteins highly homologous to the human and murine Runx1 (AML1) and Runx3 (AML2) proteins. In contrast to a recent report, we detected runxa expression in both hematopoietic and neural tissues of the developing zebrafish. runxa transcripts first appear during segmentation in bilateral mesodermal cells that coexpress one of the earliest blood and endothelial cell markers, scl/tal-1 . By 24 hours postfertilization (hpf), runxa transcripts are seen in the ventral wall of the dorsal aorta. Hematopoietic runxa expression is lost in cloche mutants, which are defective in blood and endothelial cell formation. runxb transcripts are seen in nonhematopoietic domains. Both Runxa and Runxb transactivate an AML1-responsive human promoter in hematopoietic cells. Genomic localization studies demonstrate that runxa is located on linkage group 1 (LG1), and the runxb gene is located on LG13. Conclusions Our gene expression analysis strongly suggests that both the functional and spatial aorta-gonad-mesonephros (AGM) region has been conserved throughout evolution. Our runxa spatiotemporal expression data shed light on the role of vertebrate Runx1/AML1 in primitive vs definitive hematopoietic development.

The immunohistochemical analysis of lymphoid neoplasms has led to refined classification schemes based on the profile of antigen expression and correlation with morphological, cytogenetic, molecular, and clinical features. Tissue microarrays (TMAs) are a powerful tool to rapidly characterize the phenotypic profile of a large number of samples. We show that this technique can be readily applied to the study of lymphoma by examining the expression profile of a series of 193 B-cell non-Hodgkin's lymphomas (NHLs) and 29 Hodgkin's lymphomas (HLs) using immunohistochemistry and in situ hybridization (ISH). The NHL cases were studied for the expression of commonly used markers—including CD3, CD5, CD10, CD20, CD23, CD30, CD43, Bcl-2, and cyclin D1 by immunohistochemical staining of TMAs—and these results were compared with whole sections (WS) of the same cases. We found a high degree of correlation between the results achieved with TMAs or WS (86% to 100% of cases). P53 and MIB-1 staining were studied, and the results were similar to that reported in the literature. HL cases were stained for CD20, CD30, CD15 (LeuM1), and latent membrane protein 1 expression, and ISH was performed using probes for EBER-1 and-2 transcripts. The results from HL cases on TMA sections matched exactly with those of WS. We correlated cytogenetic results with immunohistochemical stains and morphology in cases of mantle cell lymphoma [t(11;14)(q13;q32)] and follicular lymphoma [t(14;18)(q32;q24)]. This extensive expression profile of B-cell NHLs and HL tissues discloses the ability of TMAs to rapidly screen a large series of cases and represents the first report of method validation for this technique in the study of lymphoma. HUM PATHOL 33:968-974. Copyright 2002, Elsevier Science (USA). All rights reserved.

The leukemia-associated fusion proteins share several structural or functional similarities, suggesting that they may impart a leukemic phenotype through common modes of transcriptional dysregulation. The fusion proteins generated by these translocations usually contain a DNA-binding domain, domains responsible for homo- or hetero-dimerization, and domains that interact with proteins involved in chromatin remodeling (e.g., co-repressor molecules or co-activator molecules). It is these shared features that constitute the 'variations on the theme' that underling the aberrant growth and differentiation that is the hallmark of acute leukemia cells.

High-dose melphalan and autologous stem cell transplant (SCT) induce hematologic and clinical remissions in AL and results in prolonged survival. In a prior phase II study, we showed that...

Salvage of patients with relapsed and refractory Hodgkin disease (HD) with high-dose chemoradiotherapy (HDT) and autologous stem cell transplantation (ASCT) results in event-free survival (EFS) rates from 30% to 50%. Unfortunately, the reduction in toxicity associated with modern supportive care has improved EFS by only 5% to 10% and has not reduced the relapse rate. Results of a comprehensive 2-step protocol encompassing dose-dense and dose-intense second-line chemotherapy, followed by HDT and ASCT, are reported. Sixty-five consecutive patients, 22 with primary refractory HD and 43 with relapsed HD, were treated with 2 biweekly cycles of ifosfamide, carboplatin, and etoposide (ICE). Peripheral blood progenitor cells from responding patients were collected, and the patients were given accelerated fractionation involved field radiotherapy (IFRT) followed by cyclophosphamide-etoposide and either intensive accelerated fractionation total lymphoid irradiation or carmustine and ASCT. The EFS rate at a median follow-up of 43 months, as analyzed by intent to treat, was 58%. The response rate to ICE was 88%, and the EFS rate for patients who underwent transplantation was 68%. Cox regression analysis identified 3 factors before the initiation of ICE that predicted for outcome: B symptoms, extranodal disease, and complete remission duration of less than 1 year. EFS rates were 83% for patients with 0 to 1 adverse factors, 27% for patients with 2 factors, and 10% for patients with 3 factors (P

While great progress has been achieved in the clinical management and molecular understanding of Ph+ chronic myeloid leukemia (CML), little is known about the optimal approach to monitor and treat patients with ETV6-ABL1+ myeloproliferative neoplasms. Nine BCR-ABL1-negative CML patients with a

The majority of patients with Hodgkin’s disease (HD) can be cured with radiation therapy (RT) and/or combination chemotherapy (CT). However, patients who relapse after attaining a complete remission (CR) from CT, and those who fail to achieve a complete response with CT (primary refractory disease), have a poor outcome using conventional-dose second-line or salvage regimens (1–3). Over the past 15 yr, numerous clinical trials using high-dose chemotherapy (HDCT), or chemoradiotherapy, with autologous stem cell transplantation (ASCT) have been reported, and 30–50% of patients appear to be cured using this approach. These results compare favorably with historical data using conventional second-line chemoradiotherapy, and, currently, ASCT is the salvage treatment of choice for many patients with relapsed and primary refractory HD (4–7).

PURPOSE: To evaluate a chemotherapy regimen that consisted of ifosfamide administered as an infusion with bolus carboplatin, and etoposide (ICE) supported by granuloctye colony-stimulating factor (G-CSF) for cytoreduction and stem-cell mobilization in transplant-eligible patients with primary refractory or relapsed non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: One hundred sixty-three transplant-eligible patients with relapsed or primary refractory NHL were treated from October 1993 to December 1997 with ICE chemotherapy at Memorial Sloan-Kettering Cancer Center. Administration of three cycles of ICE chemotherapy was planned at 2-week intervals. Peripheral-blood progenitor cells were collected after cycle 3, and all patients who achieved a partial response (PR) or complete response (CR) to ICE chemotherapy were eligible to proceed to transplantation. Event-free and overall survival, ICE-related toxicity, and the number of CD34+ cells collected after treatment with ICE and G-CSF were evaluated. RESULTS: All 163 patients were assessable for response, and there was no treatment-related mortality. A major response (CR/PR) was evident in 108 patients (66.3%); 89% of the responding patients underwent successful transplantation. Patient who underwent transplantation and achieved a CR to ICE had a superior overall survival to that of patients who achieved a PR (65% v 30%; P = .003). The median number of CD34+ cells/kg collected was 8.4 × 106. The dose-limiting toxicity of ICE was hematologic, with 29.4% of patients developing grade 3/4 thrombocytopenia. There were minimal nonhematologic side effects. CONCLUSION: ICE chemotherapy, with ifosfamide administered as a 24-hour infusion to decrease CNS side effects, and the substitution of carboplatin for cisplatin to minimize nephrotoxicity, is a very effective cytoreduction and mobilization regimen in patients with NHL. Furthermore, the quality of the clinical response to ICE predicts for posttransplant outcome.

The nuclear hormone receptor superfamily proteins are ligand modulated transcription factors that directly connect cellular transcriptional responses to extracellular lipophilic signalling compounds, such as retinoids, steroids, and perhaps fatty acid and arachidonic acid metabolites as well (1). These receptors regulate major cellular processes including reproduction, development, homeostasis, differentiation, and oncogenesis (2). Retinoic acid receptors (RARs), v-erb A, and other nuclear receptors have important transcription regulating interactions with proteins from other transcription factor families, for example fos and jun (3–5). Nuclear receptors also have significant interactions with other members of their nuclear hormone receptor superfamily, for example retinoid X receptors, (RXRs) form functionally important heterodimers with the RARs (6). RXRs, however, can also heterodimerize with a number of different nuclear hormone receptors in vitro, including the Peroxisome Proliferatior Activated Receptors, (PPARs) (7).

Although DNA methyltransferase Inhibitor (DNMTI) therapy demonstrates efficacy in patients (pts) with Myelodysplastic Syndromes (MDS), to date there are no clinical or genomic markers to select patients most likely to benefit from DNMTI therapy. Two recent retrospective studies have suggested TET2 mutations (muts) are associated with a higher response rate to 5-azacytidine (5-aza) and decitabine (DAC). We investigated the efficacy of DAC (20 mg/m2/day x 5 days) plus tretinoin (ATRA) given on days 10-19 of a 28-day schedule in MDS pts in a phase I/II study. We comprehensively analyzed pretreatment samples from 23 pts (15M,8F; median age 67, range, 44-78) treated on this study for alterations in known oncogenes to identify potential correlations between mut profile and clinical outcomes. The median follow-up was 41 mos (range, 10-66). IPSS cytogenetics (CG) risk categories included Good (n=8), Intermediate (n=5), and High risk (n=8) pts (2 karyotype failures). The overall IPSS risk categories were Int-1 (n=3), Int-2 (n=16), and High (n=4). Of 23 pts, 16 responded, including 2 CRs, 9 mCR+/-HI, 1 PR, and 4 HI. Genomic DNA and total RNA were isolated from all pt samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >584X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The median number of muts detected was 2 (range, 0-6), with 22/23 (96%) pts having at least 1 mut. The number of muts detected for 14 PB samples with ≤ 1% blasts was 2 (range, 0-6) compared to 4 muts (range, 1-5) for 8 pts with > 1% PB or BM blasts (unknown source for 1 sample). The most common mut identified in this cohort was TP53 (35%). Previous studies have shown that complex CGs and mut TP53 are strong poor prognostic indicators. Six pts with complex CGs and either mut or deleted TP53 had a median of 0 (range 0-2) additional muts, whereas pts with non-complex CGs who were wild-type (WT) TP53 (n=12) had a median of 3 (range 1-6) muts in other disease alleles (p=0.006). We identified mutually exclusive recurrent muts in RNA splicing factors including SRSF2 (26%), SF3B1 (17%), and ZRSR2 (9%), and recurrent muts in RUNX1 (22%), IDH1/2 (22%), TET2 (17%), ASXL1 (17%), PTPN11 (13%), CEBPA (13%), STAG2 (9%), and KRAS (9%). Thirteen additional muts were detected in single pts, and we identified novel candidate muts in DNA repair pathways (ATM, ATR, FANCA BRCA2), and in the recently described tumor suppressor FAT3. We also hypothesized that prognostication of pts with normal karyotype (NK) MDS may be aided by molecular stratification, as we and others have shown in AML. Of note, all 5 pts with IDH1/2 muts occurred in pts with NK, and in 4/5 cases they were associated with concomitant SRSF2 muts that were previously shown to be associated with adverse outcomes. We identified TET2 muts in 4 pts who were also ASXL1 WT. Notably, all 4 TET2 mutated/ASXL1 WT pts responded to DAC/ATRA therapy. The other 12 responders included 7 pts with TP53 mut/loss +/- other muts. No other muts were associated with a differential response to DAC/ATRA in this trial cohort. The strong association between TET2 muts and DAC/ATRA response, and more detailed mutational profiling of the 12 TET2 WT pts who responded to DAC/ATRA therapy is being investigated in additional pt samples and will be presented in more detail. Pts with TP53 muts showed a trend toward a worse survival which was not statistically significant; however, no other individual mut was associated with a differential survival in this small cohort. Conclusions The use of comprehensive mutational profiling employed in this study allowed us to identify muts in 25 genes in MDS pts, and allowed us to show that the overall mut frequency was higher in pts with a NK and without TP53 muts, thereby providing evidence for alternate disease initiation events and pathogenesis in these two genomically defined MDS subsets. All pts with mutated TET2 responded to DAC/ATRA, suggesting that pts with muts in TET2 are the best candidates for novel therapeutic trials which include DNMTI therapy. Disclosures: Wang: Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc.: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Despite past and ongoing research efforts, the pathogenetic mechanisms of MDS remain far from understood sufficiently to point to single genes or molecular pathways as putative targets for generation of better mouse models or drug development. In lack of clear targets, the manipulation of disease-associated phenotypes may at present be the best opportunity for disease study and therapeutic intervention. Furthermore, because cellular phenotypes are more proximal to molecular disease mechanisms than phenotypes seen at the level of the tissue or organism (i.e. the murine or human hematopoietic system), they may provide more sensitive and relevant readouts of the disease process. With the advent of patient-specific induced pluripotent stem cells (iPSCs), disease models based on cellular phenotypes (“disease-in-a-dish”) can now be developed and their unique properties promise to revolutionize disease study and drug development. Unlimited cell numbers of biologically relevant cells can be obtained relatively easily and cost-effectively. Here we present a new MDS model based on patient-specific iPSCs and its use in two phenotype-driven screens: (a) a focused genetic screen and (b) a high-throughput chemical screen. We have derived multiple MDS-iPSC lines with deletions of chromosomes 7q or 20q (characteristic chromosomal deletions that we are using as markers of iPSCs derived from the MDS clone) from bone marrow (BM) of 3 patients with MDS and 1 patient with sAML. We also derived isogenic karyotypically normal iPSC lines in parallel from the same BM samples. We identified two cellular phenotypes specific to the MDS-iPSCs: decreased proliferation rate and decreased potential for pan-hematopoietic differentiation and reduced clonogenic capacity of their hematopoietic progeny. These phenotypes are consistent across multiple iPSC lines from different patients and absent from their isogenic normal iPSC controls. They are reminiscent of the behavior of ex vivo cultured primary MDS BM cells and are therefore likely to be relevant to the disease process. Furthermore, they are rescued by spontaneous compensation for chr7q dosage through acquisition of an extra chr7 and recapitulated by the engineering of artificial chr7q deletions in normal iPSCs. To identify critical MDS gene(s) on chr7q, we performed a screen of 62 candidate haploinsufficient genes (with reduced expression by at least 1.5-fold in our del(7q)-iPSCs compared to their isogenic normal iPSCs) for rescue of the proliferation and/or hematopoietic differentiation phenotype. We constructed a lentiviral library of all candidate ORFs (and 14 additional alternative transcripts) linked to eGFP through a P2A peptide and each tagged with a unique 4-nt barcode sequence to its 3’ UTR. The library was packaged as a pool and transduced into two different del(7q)-iPSC lines. The cells were passaged for 16 weeks and gDNA was isolated every 2 weeks. In parallel, the cells were differentiated along the hematopoietic lineage and gDNA was isolated from CD45+ cells FACS-sorted on day 15 of differentiation. High-throughput sequencing of the barcodes identified 5 ORFs that became enriched in undifferentiated iPSCs over time (rescue of proliferation) and 4 ORFs enriched in sorted CD45+hematopoietic progenitors (rescue of hematopoietic differentiation), 2 of which overlapped. All 9 hits are being further validated in more focused screens. Second, we used the same platform for a high-throughput small molecule screen. We optimized the plating conditions and densities for a 384-well format using a luminescent cell viability assay and conducted a screen of 2000 compounds comprising known drugs, natural products, and other bioactives and chemicals, in an MDS-iPSC line (2.13) and its isogenic normal control (2.8). Primary hits were defined as compounds that enhanced the growth of the MDS-iPSC line, but not of the control normal iPSC line in a compound dose-dependent manner. 38 primary hits were retested in a dose-response survival assay in one additional MDS- (2.A1-3), one additional isogenic normal iPSC line (2.12) and one sAML iPSC line over 8 concentrations and 12 compounds were prioritized for further studies. Our data highlight the potential of this new iPSC-based model of MDS for screens to identify genes or compounds that affect cellular phenotypes by acting on previously undefined targets. Disclosures: No relevant conflicts of interest to declare.

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis and increased risk of progression to acute myeloid leukemia (AML). A hallmark of MDS is increased apoptosis, while AML undergoes decreased apoptosis and increased proliferation. BCL2, an anti-apoptotic protein, is overexpressed in high grade MDS and AML and may confer poor prognosis and increased chemotherapy resistance. In this study we examined the DNA methylation status of BCL2 in MDS and AML. Methods: We conducted DNA methylation profiling on genomic DNA extracted from bone marrows of 22 refractory anemia with ringed sideroblasts (RARS), 8 refractory anemia with excess blasts (RAEB), 21 de novo AML, 21 therapy related AML, and 7 normal volunteers. We used both Illumina 27K BeadChip and 450K BeadChip methylation arrays to assess CpG methylation. Standard student T-test was used to screen differentially methylated MDS and AML CpG sites compared to normal samples. We validated the microarray data by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Results: We identified a region in the BCL2 gene body that was among the most differentially methylated in MDS and AML versus normal bone marrow. Within that amplicon, BCL2 in both RARS, a low grade MDS, and RAEB, a high grade MDS, were hypermethylated compared to normal bone marrow (43% vs 18%, 48% vs 18%, respectively, p Conclusions: Our study identifies a region of BCL2 that is hypermethylated in both low and high risk MDS while hypomethylated in AML compared to normal bone marrows. We hypothesize aberrant epigenetic modification of BCL2 plays a key role in apoptosis and transformation from MDS to AML, and our data support the idea of combining demethylating drugs with BCL2 antagonists. We are currently investigating whether BCL2 protein expression correlates with methylation, and plan to map the histone marks with CHIP-seq to correlate chromatin accessibility and DNA methylation in BCL2. Citation Format: Sangmin Lee, Naomi Galili, Stephen Nimer, Mark D. Minden, Vundavalli V. Murty, Suresh Jhanwar, Azra Raza, Benjamin Tycko. Loss of methylation in a regulatory region of BCL2 in MDS/AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2013-2989