Showing papers in "Leukemia in 2009"

Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma

Abstract: New systems have emerged for diagnosis, staging and response assessment in multiple myeloma (MM). The diagnostic and response criteria recommended are primarily derived from the International Myeloma Working Group, with certain updates and clarifications. The International Staging System is the current standard for staging of myeloma. A new risk stratification model is provided to specifically define high-risk patients who may benefit from novel therapeutic strategies. This paper provides the current criteria for diagnosis, staging, risk stratification and response assessment of MM.

Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia.

Abstract: Imatinib mesylate is considered standard of care for first-line treatment of chronic phase chronic myeloid leukemia (CML-CP). In the phase III, randomized, open-label International Randomized Study of Interferon vs STI571 (IRIS) trial, previously untreated CML-CP patients were randomized to imatinib (n=553) or interferon-alpha (IFN) plus cytarabine (n=553). This 6-year update focuses on patients randomized to receive imatinib as first-line therapy for newly diagnosed CML-CP. During the sixth year of study treatment, there were no reports of disease progression to accelerated phase (AP) or blast crisis (BC). The toxicity profile was unchanged. The cumulative best complete cytogenetic response (CCyR) rate was 82%; 63% of all patients randomized to receive imatinib and still on study treatment showed CCyR at last assessment. The estimated event-free survival at 6 years was 83%, and the estimated rate of freedom from progression to AP and BC was 93%. The estimated overall survival was 88% -- or 95% when only CML-related deaths were considered. This 6-year update of IRIS underscores the efficacy and safety of imatinib as first-line therapy for patients with CML.

International Myeloma Working Group molecular classification of multiple myeloma: spotlight review.

Abstract: Myeloma is a malignant proliferation of monoclonal plasma cells. Although morphologically similar, several subtypes of the disease have been identified at the genetic and molecular level. These genetic subtypes are associated with unique clinicopathological features and dissimilar outcome. At the top hierarchical level, myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. The latter is mainly composed of cases harboring IgH translocations, generally associated with more aggressive clinical features and shorter survival. The three main IgH translocations in myeloma are the t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Trisomies and a more indolent form of the disease characterize hyperdiploid myeloma. A number of genetic progression factors have been identified including deletions of chromosomes 13 and 17 and abnormalities of chromosome 1 (1p deletion and 1q amplification). Other key drivers of cell survival and proliferation have also been identified such as nuclear factor- B-activating mutations and other deregulation factors for the cyclin-dependent pathways regulators. Further understanding of the biological subtypes of the disease has come from the application of novel techniques such as gene expression profiling and array-based comparative genomic hybridization. The combination of data arising from these studies and that previously elucidated through other mechanisms allows for most myeloma cases to be classified under one of several genetic subtypes. This paper proposes a framework for the classification of myeloma subtypes and provides recommendations for genetic testing. This group proposes that genetic testing needs to be incorporated into daily clinical practice and also as an essential component of all ongoing and future clinical trials.

International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders

Abstract: The serum immunoglobulin-free light chain (FLC) assay measures levels of free κ and λ immunoglobulin light chains. There are three major indications for the FLC assay in the evaluation and management of multiple myeloma and related plasma cell disorders (PCD). In the context of screening, the serum FLC assay in combination with serum protein electrophoresis (PEL) and immunofixation yields high sensitivity, and negates the need for 24-h urine studies for diagnoses other than light chain amyloidosis (AL). Second, the baseline FLC measurement is of major prognostic value in virtually every PCD. Third, the FLC assay allows for quantitative monitoring of patients with oligosecretory PCD, including AL, oligosecretory myeloma and nearly two-thirds of patients who had previously been deemed to have non-secretory myeloma. In AL patients, serial FLC measurements outperform PEL and immunofixation. In oligosecretory myeloma patients, although not formally validated, serial FLC measurements reduce the need for frequent bone marrow biopsies. In contrast, there are no data to support using FLC assay in place of 24-h urine PEL for monitoring or for serial measurements in PCD with measurable disease by serum or urine PEL. This paper provides consensus guidelines for the use of this important assay, in the diagnosis and management of clonal PCD.

CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers

Abstract: Hematopoietic and epithelial cancer cells express CXCR4, a seven-transmembrane G-protein-coupled chemokine receptor. Stromal cells within the bone marrow microenvironment constitutively secrete stromal cell-derived factor-1 (SDF-1/CXCL12), the ligand for CXCR4. Activation of CXCR4 induces leukemia cell trafficking and homing to the marrow microenvironment, where CXCL12 retains leukemia cells in close contact with marrow stromal cells that provide growth and drug resistance signals. CXCR4 antagonists, such as Plerixafor (AMD3100) and T140 analogs, can disrupt adhesive tumor-stroma interactions and mobilize leukemia cells from their protective stromal microenvironment, making them more accessible to conventional drugs. Therefore, targeting the CXCR4-CXCL12 axis is a novel, attractive therapeutic approach that is explored in ongoing clinical trials in leukemia patients. Initially, CXCR4 antagonists were developed for the treatment of HIV, where CXCR4 functions as a co-receptor for virus entry into T cells. Subsequently, CXCR4 antagonists were noticed to induce leukocytosis, and are currently used clinically for mobilization of hematopoietic stem cells. However, because CXCR4 plays a key role in cross-talk between leukemia cells (and a variety of other tumor cells) and their microenvironment, cancer treatment may become the ultimate application of CXCR4 antagonists. Here, we summarize the development of CXCR4 antagonists and their preclinical and clinical activities, focusing on leukemia and other cancers.

International myeloma working group consensus statement and guidelines regarding the current role of imaging techniques in the diagnosis and monitoring of multiple Myeloma.

Abstract: Several imaging technologies are used for the diagnosis and management of patients with multiple myeloma (MM). Conventional radiography, computed tomography (CT), magnetic resonance imaging (MRI) and nuclear medicine imaging are all used in an attempt to better clarify the extent of bone disease and soft tissue disease in MM. This review summarizes all available data in the literature and provides recommendations for the use of each of the technologies. Conventional radiography still remains the 'gold standard' of the staging procedure of newly diagnosed and relapsed myeloma patients. MRI gives information complementary to skeletal survey and is recommended in MM patients with normal conventional radiography and in all patients with an apparently solitary plasmacytoma of bone. Urgent MRI or CT (if MRI is not available) is the diagnostic procedure of choice to assess suspected cord compression. Bone scintigraphy has no place in the routine staging of myeloma, whereas sequential dual-energy X-ray absorptiometry scans are not recommended. Positron emission tomography/CT or MIBI imaging are also not recommended for routine use in the management of myeloma patients, although both techniques may be useful in selected cases that warrant clarification of previous imaging findings, but such an approach should ideally be made within the context of a clinical trial.

Pathogenesis of myeloma bone disease.

Abstract: Bone disease in multiple myeloma (MM) is characterized by lytic bone lesions, which can cause severe bone pain, pathologic fractures and hypercalcemia. However, the lytic bone disease in MM differs from that in other cancer patients who have lytic bone metastases. Although increased osteoclastic bone destruction is involved in MM and other tumors involving bone, in contrast to other tumors, once the MM tumor burden exceeds 50% in a local area, osteoblast activity is either suppressed or absent.1 The basis for this severe imbalance between increased osteoclastic bone resorption and decreased bone formation has been a topic of intensive investigation over the last several years and will be reviewed in this article.

TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis

Abstract: TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis

Long-term follow-up on overall survival from the MM-009 and MM-010 phase III trials of lenalidomide plus dexamethasone in patients with relapsed or refractory multiple myeloma.

Abstract: We present a pooled update of two large, multicenter MM-009 and MM-010 placebo-controlled randomized phase III trials that included 704 patients and assessed lenalidomide plus dexamethasone versus dexamethasone plus placebo in patients with relapsed/refractory multiple myeloma (MM). Patients in both studies were randomized to receive 25 mg daily oral lenalidomide or identical placebo, plus 40 mg oral dexamethasone. In this pooled analysis, using data up to unblinding (June 2005 for MM-009 and August 2005 for MM-010), treatment with lenalidomide plus dexamethasone significantly improved overall response (60.6 vs 21.9%, P<0.001), complete response rate (15.0 vs 2.0%, P<0.001), time to progression (median of 13.4 vs 4.6 months, P<0.001) and duration of response (median of 15.8 months vs 7 months, P<0.001) compared with dexamethasone-placebo. At a median follow-up of 48 months for surviving patients, using data up to July 2008, a significant benefit in overall survival (median of 38.0 vs 31.6 months, P=0.045) was retained despite 47.6% of patients who were randomized to dexamethasone-placebo receiving lenalidomide-based treatment after disease progression or study unblinding. Low β2-microglobulin and low bone marrow plasmacytosis were associated with longer survival. In conclusion, these data confirm the significant response and survival benefit with lenalidomide and dexamethasone.

New insights to the MLL recombinome of acute leukemias

Abstract: Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Cyclophosphamide, bortezomib and dexamethasone induction for newly diagnosed multiple myeloma: high response rates in a phase II clinical trial.

Abstract: We have studied a three-drug combination with cyclophosphamide, bortezomib and dexamethasone (CyBorD) on a 28-day cycle in the treatment of newly diagnosed multiple myeloma (MM) patients to assess response and toxicity. The primary endpoint of response was evaluated after four cycles. Thirty-three newly diagnosed, symptomatic patients with MM received bortezomib 1.3 mg/m2 intravenously on days 1, 4, 8 and 11, cyclophosphamide 300 mg/m2 orally on days 1, 8, 15 and 22 and dexamethasone 40 mg orally on days 1–4, 9–12 and 17–20 on a 28-day cycle for four cycles. Responses were rapid with a mean 80% decline in the sentinel monoclonal protein at the end of two cycles. The overall intent to treat response rate (⩾ partial response) was 88%, with 61% of very good partial response or better (⩾VGPR) and 39% of complete/near complete response (CR/nCR). For the 28 patients who completed all four cycles of therapy, the CR/nCR rate was 46% and VGPR rate was 71%. All patients undergoing stem cell harvest had a successful collection. Twenty-three patients underwent stem cell transplantation (SCT) and are evaluable through day 100 with CR/nCR documented in 70% and ⩾VGPR in 74%. In conclusion, CyBorD produces a rapid and profound response in patients with newly diagnosed MM with manageable toxicity.

Bone marrow microenvironment and the identification of new targets for myeloma therapy.

Abstract: The development of multiple myeloma (MM) is a complex multi-step process involving both early and late genetic changes in the tumor cell as well as selective supportive conditions by the bone marrow (BM) microenvironment. Indeed, it is now well established that MM cell-induced disruption of the BM homeostasis between the highly organized cellular and extracellular compartments supports MM cell proliferation, survival, migration and drug resistance through activation of various signaling (for example, PI3K/Akt, JAK/Stat-, Raf/MEK/MAPK-, NFκB- and Wnt-) pathways. Based on our enhanced understanding of the functional importance of the MM BM microenvironment and its inter-relation with the MM cell resulting in homing, seeding, proliferation and survival, new molecular targets have been identified and derived treatment regimens in MM have already changed fundamentally during recent years. These agents include thalidomide, its immunomodulatory derivative lenalidomide and the proteasome inhibitor bortezomib, which mediate tumor cytotoxicity in the BM milieu. Ongoing studies are further delineating MM pathogenesis in the BM to enhance cytotoxicity, avoid drug resistance and improve patient outcome.

Mutations of ASXL1 gene in myeloproliferative neoplasms

Abstract: measure epoxomicin uptake we utilized another P-gP substrate to determine if drug efflux is altered in KMS11R cells. Doxorubicin is an established substrate for P-gP and it can be easily measured in the cell because it fluoresces red. We compared the fluorescence of KMS11 and KMS11R cells treated with various concentrations of doxorubicin for 24 h. No differences in background fluorescence were observed and both cell lines could take up doxorubicin in a dose-dependent fashion (Figure 2b). However, the uptake observed was 3–5 fold lower in the resistant line. In addition, KMS11R cells are significantly resistant to doxorubicin-induced cell death (not shown). Finally, we determined the role of P-gP in the acquired resistance to epoxomicin by observing the effects of inhibiting PgP function with verapamil. Verapamil had no effect on the viability of either cell line nor did it alter the sensitivity of KMS11 cells to epoxomicin (Figure 3a) or doxorubicin (not shown). In contrast verapamil resensitized KMS11R to both drugs (Figure 3a and not shown). FACS analysis of cells treated with doxorubicin and verapamil revealed that the addition of verapamil to the KMS11R cells resulted in an increase in red fluorescence in doxorubicin-treated cells (Figure 3b). Given the promise of bortezomib it is not surprising that other proteasome inhibitors that are either irreversible and/or inhibit the other activities of the proteasome are currently being tested in the clinic. As proteasome inhibitors become more commonly used in newly diagnosed diseases it will be important to understand mechanisms of acquired resistance to this class of agents. In addition, we will need to know how acquired resistance to proteasome inhibitors alter the response to other therapeutics used to treat myeloma patients. Acquired expression of P-gP has been observed in myeloma patients and cell lines. P-gP is rarely seen in newly diagnosed patients, however, increased expression was observed in cells from approximately 75% of patients treated with vincristine, doxorubicin and dexamethasone. We observed that P-gP is expressed and the P-gP inhibitor verapimil sensitizes KMS11R to epoxomicin. These data suggest that the acquired resistance observed was the result of P-gP expression. This is the first report to demonstrate the acquisition of P-gP with a proteasome inhibitor. Although it is possible that the induction of P-gP could be the result of inhibition of protein turnover, this does not appear to be a likely mechanism. First epoxomicin does not acutely induce P-gP (Figure 2). More importantly we also observe increased expression of the MDR mRNA in these cells (not shown). Thus the mechanism is likely to be due to increased gene expression possibly because of gene amplification. This remains to be determined. Finally, our data suggest that carfilzomib could be ineffective in the treatment of P-gP-positive myeloma. Consistent with this possibility, a recent study demonstrated that the P-gP-positive cell line, RPMI8226-Dox40, is resistant to carfilzomib; however, they can be sensitized by co-treatment with verapamil. Unfortunately such resensitization of cells to P-gP substrates has not proven to be effective clinically. Several agents that inhibit drug efflux have been tested and either proven to be too toxic or had little effect on efficacy. In addition to confirming that epoxomicin-based compounds may be subject to this type of resistance mechanism we demonstrated that they can initiate resistance in this fashion. Therefore careful consideration of the use of these compounds both as single agents and in combination therapies will be needed to assure both efficacy of this therapy as well as subsequent treatment regimens.

Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy

Abstract: Dasatinib, a broad-spectrum tyrosine kinase inhibitor (TKI), predominantly targets BCR-ABL and SRC oncoproteins and also inhibits off-target kinases, which may result in unexpected drug responses. We identified 22 patients with marked lymphoproliferation in blood while on dasatinib therapy. Clonality and immunophenotype were analyzed and related clinical information was collected. An abrupt lymphocytosis (peak count range 4-20 x 10(9)/l) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy and it persisted throughout the therapy. Fifteen patients had a cytotoxic T-cell and seven patients had an NK-cell phenotype. All T-cell expansions were clonal. Adverse effects, such as colitis and pleuritis, were common (18 of 22 patients) and were preceded by LGL lymphocytosis. Accumulation of identical cytotoxic T cells was also detected in pleural effusion and colon biopsy samples. Responses to dasatinib were good and included complete, unexpectedly long-lasting remissions in patients with advanced leukemia. In a phase II clinical study on 46 Philadelphia chromosome-positive acute lymphoblastic leukemia, patients with lymphocytosis had superior survival compared with patients without lymphocytosis. By inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity associated with good clinical responses and a distinct adverse effect profile.

The DNA methyltransferase inhibitors azacitidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells.

Abstract: The three DNA methyltransferase (DNMT)-inhibiting cytosine nucleoside analogues, azacitidine, decitabine and zebularine, which are currently studied as nonintensive therapy for myelodysplastic syndromes and acute myeloid leukemia (AML), differ in structure and metabolism, suggesting that they may have differential molecular activity. We investigated cellular and molecular effects of the three substances relative to cytarabine in Kasumi-1 AML blasts. Under in vitro conditions mimicking those used in clinical trials, the DNMT inhibitors inhibited proliferation and triggered apoptosis but did not induce myeloid differentiation. The DNMT inhibitors showed no interference with cell-cycle progression whereas cytarabine treatment resulted in an S-phase arrest. Quantitative methylation analysis of hypermethylated gene promoters and of genome-wide LINE1 fragments using bisulfite sequencing and MassARRAY suggested that the hypomethylating potency of decitabine was stronger than that of azacitidine; zebularine showed no hypomethylating activity. In a comparative gene expression analysis, we found that the effects of each DNMT inhibitor on gene transcription were surprisingly different, involving several genes relevant to leukemogenesis. In addition, the gene methylation and expression analyses suggested that the effects of DNMT-inhibiting cytosine nucleoside analogues on the cellular transcriptome may, in part, be unrelated to direct promoter DNA hypomethylation, as previously shown by others.

Human mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1.

Abstract: Mesenchymal stem cells (MSCs) have an inhibitory effect on tumor proliferation, but the precise mechanisms are not fully understood. Here, we identified DKK-1 (dickkopf-1), secreted by MSCs and acting as a negative regulator of WNT signaling pathway, to be one of the molecules responsible for the inhibitory effect. When DKK-1 was neutralized by anti-DKK-1 antibodies, or when the expression of DKK-1 was downregulated by RNA interference (RNAi), the inhibitory effects of MSCs on K562 cell proliferation were attenuated. We also provide evidence that the expression of DKK-1 by MSCs is regulated by NANOG, a transcriptional factor ubiquitously expressed in some stem cells. Using the Cellmax artificial capillary modules that eliminate the immunosuppressive properties of MSCs, we further showed that MSCs were able to inhibit proliferation of K562 cells in a humoral microenvironment. Meanwhile, we recapture this effect of MSCs on primary leukemic hematopoietic progenitors from patients. MSCs probably have a general inhibitory effect on their neighboring cells, including malignant cells, en route to achieving tissue homeostasis.

Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

Abstract: Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

Global target profile of the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells

Abstract: The detailed molecular mechanism of action of second-generation BCR-ABL tyrosine kinase inhibitors, including perturbed targets and pathways, should contribute to rationalized therapy in chronic myeloid leukemia (CML) or in other affected diseases. Here, we characterized the target profile of the dual SRC/ABL inhibitor bosutinib employing a two-tiered approach using chemical proteomics to identify natural binders in whole cell lysates of primary CML and K562 cells in parallel to in vitro kinase assays against a large recombinant kinase panel. The combined strategy resulted in a global survey of bosutinib targets comprised of over 45 novel tyrosine and serine/threonine kinases. We have found clear differences in the target patterns of bosutinib in primary CML cells versus the K562 cell line. A comparison of bosutinib with dasatinib across the whole kinase panel revealed overlapping, but distinct, inhibition profiles. Common among those were the SRC, ABL and TEC family kinases. Bosutinib did not inhibit KIT or platelet-derived growth factor receptor, but prominently targeted the apoptosis-linked STE20 kinases. Although in vivo bosutinib is inactive against ABL T315I, we found this clinically important mutant to be enzymatically inhibited in the mid-nanomolar range. Finally, bosutinib is the first kinase inhibitor shown to target CAMK2G, recently implicated in myeloid leukemia cell proliferation.

Detection of mutant TET2 in myeloid malignancies other than myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML.

Abstract: Detection of mutant TET2 in myeloid malignancies other than myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML

Frequent TET2 mutations in systemic mastocytosis: clinical, KITD816V and FIP1L1-PDGFRA correlates

Abstract: TET2 (TET oncogene family member 2) is a candidate tumor suppressor gene located at chromosome 4q24, and was recently reported to be mutated in ∼14% of patients with JAK2V617F-positive myeloproliferative neoplasms. We used high-throughput DNA sequence analysis to screen for TET2 mutations in bone marrow-derived DNA from 48 patients with systemic mastocytosis (SM), including 42 who met the 2008 WHO (World Health Organization) diagnostic criteria for SM and 6 with FIP1L1-PDGFRA. Twelve (29%) SM, but no FIP1L1-PDGFRA patients, had TET2 mutations. A total of 17 mutations (13 frameshift, 2 nonsense and 2 missense) were documented in 2 (15%) of 13 indolent SM patients, 2 (40%) of 5 aggressive SM, and 8 (35%) of 23 SM associated with a clonal non-mast cell-lineage hematopoietic disease (P=0.52). KITD816V was detected by PCR sequencing in 50 or 20% of patients with or without TET2 mutation (P=0.05), respectively. Multivariable analysis showed a significant association between the presence of TET2 mutation and monocytosis (P=0.0003) or female sex (P=0.05). The association with monocytosis was also observed in non-indolent SM (n=29), in which the presence of mutant TET2 did not affect survival (P=0.98). We conclude that TET2 mutations are frequent in SM, segregate with KITD816V and influence phenotype without necessarily altering prognosis.

Contribution of bone microenvironment to leukemogenesis and leukemia progression.

Abstract: Tumor microenvironment has a major role in cancer progression and resistance to treatment. The bone marrow (BM) is a dynamic network of growth factors, cytokines and stromal cells, providing a permissive environment for leukemogenesis and progression. Both BM stroma and leukemic blasts promote angiogenesis, which is increased in acute lymphoblastic leukemia and acute myeloid leukemia. Growth factors like vascular endothelial growth factor (VEGF), basic fibroblast growth factor and angiopoietins are the main proangiogenic mediators in acute leukemia. Autocrine proleukemic loops have been described for VEGF and angiopoietin in hematopoietic cells. Interactions of stromal cells and extracellular matrix with leukemic blasts can also generate antiapoptotic signals that contribute to neoplastic progression and persistence of treatment-resistant minimal residual disease. High expression of CXC chemokine ligand 4 (CXCR4) by leukemic blasts and activation of the CXCR4–CXCL12 axis is involved in leukemia progression and disruption of normal hematopoiesis. Leukemia-associated bone microenvironment markers could be used as prognostic or predictive indicators of disease progression and/or treatment outcome. Studies related to bone microenvironment would likely provide a better understanding of the treatment resistance associated with leukemia therapy and design of new treatments.

KIR-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia.

Abstract: Donor killer cell immunoglobulin-like receptor (KIR)-ligand incompatibility is associated with decreased relapse incidence (RI) and improved leukemia-free survival (LFS) after haploidentical and HLA-mismatched unrelated hematopoietic stem cell transplantation. We assessed outcomes of 218 patients with acute myeloid leukemia (AML n=94) or acute lymphoblastic leukemia (n=124) in complete remission (CR) who had received a single-unit unrelated cord blood transplant (UCBT) from a KIR-ligand-compatible or -incompatible donor. Grafts were HLA-A, -B or -DRB1 matched (n=21) or mismatched (n=197). Patients and donors were categorized according to their degree of KIR-ligand compatibility in the graft-versus-host direction by determining whether or not they expressed HLA-C group 1 or 2, HLA-Bw4 or HLA-A3/-A11. Both HLA-C/-B KIR-ligand- and HLA-A-A3/-A11 KIR-ligand-incompatible UCBT showed a trend to improved LFS (P=0.09 and P=0.13, respectively). Sixty-nine donor-patient pairs were HLA-A, -B or -C KIR-ligand incompatible and 149 compatible. KIR-ligand-incompatible UCBT showed improved LFS (hazards ratio=2.05, P=0.0016) and overall survival (OS) (hazards ratio=2.0, P=0.004) and decreased RI (hazards ratio=0.53, P=0.05). These results were more evident for AML transplant recipients (2-year LFS and RI with or without KIR-ligand incompatibility 73 versus 38% (P=0.012), and 5 versus 36% (P=0.005), respectively). UCBT for acute leukemia in CR from KIR-ligand-incompatible donors is associated with decreased RI and improved LFS and OS.

Molecular recognition of acute myeloid leukemia using aptamers.

Abstract: Cell surface proteins can play important roles in cancer pathogenesis. Comprehensive understanding of the surface protein expression patterns of tumor cells and, consequently, the pathogenesis of tumor cells depends on molecular probes against these proteins. To be used effectively for tumor diagnosis, classification and therapy, such probes would be capable of specific binding to targeted tumor cells. Molecular aptamers, designer DNA-RNA probes, can address this challenge by recognizing proteins, peptides and other small molecules with high affinity and specificity. Through a process known as cell-based SELEX, we used live acute myeloid leukemia (AML) cells to select a group of DNA aptamers, which can recognize AML cells with dissociation constants (Kd's) in the nanomolar range. Interestingly, one aptamer (KH1C12) compared with two control cell lines (K562 and NB4) showed significant selectivity to the target AML cell line (HL60) and could recognize the target cells within a complex mixture of normal bone marrow aspirates. The other two aptamers KK1B10 and KK1D04 recognize targets associated with monocytic differentiation. Our studies show that the selected aptamers can be used as a molecular tool for further understanding surface protein expression patterns on tumor cells and thus providing a foundation for effective molecular analysis of leukemia and its subcategories.

The detection of TP53 mutations in chronic lymphocytic leukemia independently predicts rapid disease progression and is highly correlated with a complex aberrant karyotype.

Abstract: The poor prognosis of chronic lymphocytic leukemia (CLL) patients with del (17p) is well established. We analyzed whether mutation of TP53 on the remaining allele adds to the poor prognosis or whether even TP53 mutation alone may be an adverse prognostic factor. We analyzed TP53 mutations in 193 CLL patients by denaturing high performance liquid chromatography in combination with direct DNA sequencing and a TP53 resequencing research microarray. Mutations were correlated to chromosomal aberrations defined by interphase fluorescent in situ hybridization and chromosome banding analyses and to the clinical course of patients. TP53 mutations were detected in 13.5% (26 of 193) of samples, whereas the incidence of del (17p) was 9.3% (18 of 193). TP53 mutations were significantly associated with del (17p) (concordance 94%, P<0.001) and complex cytogenetic abnormalities (concordance 50%, P<0.001). Among 147 patients whose clinical data were available, patients with TP53 abnormalities (n=20) had a significantly decreased time to treatment compared to patients without TP53 aberration (P<0.001). Median time to treatment was short in patients with isolated TP53 mutation (n=6, 2.0 months) and in those with del (17p) (n=14, 21.3 months) as compared to patients without TP53 aberration (n=127, 64.9 months, P<0.001). In multivariate Cox regression analysis, VH status, TP53 mutations and also isolated TP53 mutations independently predicted rapid disease progression.

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

Abstract: Nucleophosmin (NPM1) is a highly conserved nucleo-cytoplasmic shuttling protein that shows a restricted nucleolar localization. Mutations of NPM1 gene leading to aberrant cytoplasmic dislocation of nucleophosmin (NPMc+) occurs in about one third of acute myeloid leukaemia (AML) patients that exhibit distinctive biological and clinical features. We discuss the latest advances in the molecular basis of nucleophosmin traffic under physiological conditions, describe the molecular abnormalities underlying altered transport of nucleophosmin in NPM1-mutated AML and present evidences supporting the view that cytoplasmic nucleophosmin is a critical event for leukaemogenesis. We then outline how a highly specific immunohistochemical assay can be exploited to diagnose NPM1-mutated AML and myeloid sarcoma in paraffin-embedded samples by looking at aberrant nucleophosmin accumulation in cytoplasm of leukaemic cells. This procedure is also suitable for detection of haemopoietic multilineage involvement in bone marrow trephines. Moreover, use of immunohistochemistry as surrogate for molecular analysis can serve as first-line screening in AML and should facilitate implementation of the 2008 World Health Organization classification of myeloid neoplasms that now incorporates AML with mutated NPM1 (synonym: NPMc+ AML) as a new provisional entity. Finally, we discuss the future therapeutic perspectives aimed at reversing the altered nucleophosmin transport in AML with mutated NPM1.

Clinical features and prognosis of de novo diffuse large B-cell lymphoma with t(14;18) and 8q24 /c-MYC translocations

Abstract: Diffuse large B-cell lymphoma (DLBCL) having both t(14;18) and 8q24 translocations is rare. We evaluated the clinical characteristics and prognoses of patients with DLBCL carrying both t(14;18) and 8q24 translocations. A total of 1972 patients with non-Hodgkin's lymphoma were treated in the Adult Lymphoma Treatment Study Group (ALTSG) from 1998 to 2007. Nineteen cases of de novo DLBCL with the dual translocation were identified. The dual translocation was observed in 19 of 394 patients with DLBCL (10 males and 9 females, with a median age of 61 years). The dual translocation was observed significantly more frequently among patients with high lactate dehydrogenase levels, B symptoms, bone marrow involvement and advanced stage. Immunophenotyping was performed and showed DLBCL with a germinal center type in the majority of cases. Progression-free survival and overall survival rates were significantly lower in patients with the dual translocation than in those with other translocation. DLBCL patients with concurrent t(14;18) and 8q24 translocations have very poor prognosis. Even if patients had a complete response to chemotherapy, they subsequently suffered early relapse. In this study, only a few patients received rituximab, and its usefulness could not be assessed. Future studies with larger numbers of patients are required.

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients

Abstract: Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

International myeloma working group (IMWG) consensus statement and guidelines regarding the current status of stem cell collection and high-dose therapy for multiple myeloma and the role of plerixafor (AMD 3100).

Abstract: Multiple myeloma is the most common indication for high-dose chemotherapy with autologous stem cell support (ASCT) in North America today. Stem cell procurement for ASCT has most commonly been performed with stem cell mobilization using colony-stimulating factors with or without prior chemotherapy. The target CD34+ cell dose to be collected as well as the number of apheresis performed varies throughout the country, but a minimum of 2 million CD34+ cells/kg has been traditionally used for the support of one cycle of high-dose therapy. With the advent of plerixafor (AMD3100) (a novel stem cell mobilization agent), it is pertinent to review the current status of stem cell mobilization for myeloma as well as the role of autologous stem cell transplantation in this disease. On June 1, 2008, a panel of experts was convened by the International Myeloma Foundation to address issues regarding stem cell mobilization and autologous transplantation in myeloma in the context of new therapies. The panel was asked to discuss a variety of issues regarding stem cell collection and transplantation in myeloma especially with the arrival of plerixafor. Herein, is a summary of their deliberations and conclusions.

Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells.

Abstract: The protein kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation, Syk is recruited to the BCR and becomes activated by phosphorylation at Y352. Recently, Syk was found to be constitutively phosphorylated in several common B-cell lymphoma subtypes, indicating a role for antigen-independent Syk activation in the pathogenesis of these diseases. We now report that Syk is constitutively phosphorylated on the activating Y352 residue in chronic lymphocytic leukemia (CLL) B cells. To examine the effects of constitutive Syk activity on intracellular signaling and leukemic cell survival, we performed in vitro studies with the Syk inhibitor R406. Treatment with R406 induced leukemic cell apoptosis in the majority of investigated cases and affected the basal activity or expression of several pro-survival molecules regulated by Syk, including the Akt and extracellular signal-regulated (ERK) kinases, and the anti-apoptotic protein Mcl-1. In addition, R406 prevented the increase in leukemic cell viability induced by sustained BCR engagement and inhibited BCR-induced Akt activation and Mcl-1 upregulation. Collectively, these data identify Syk as a potential target for CLL treatment and suggest that inhibition of this kinase could provide a double therapeutic benefit by disrupting both antigen-dependent and antigen-independent signaling pathways that regulate leukemic cell survival.

Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia.

Abstract: MicroRNAs (miRNAs) control the expression of protein-coding genes in normal hematopoietic cells and, consequently, aberrant expression may contribute to leukemogenesis. To identify miRNAs relevant to pediatric acute lymphoblastic leukemia (ALL), we cloned 105 known and 8 new miRNA genes expressed in patients' leukemia cells. Instead of known miRNA genes, new miRNA genes were not evolutionarily conserved. Quantification of 19 selected miRNA genes revealed an aberrant expression in ALL as compared with normal CD34+ cells (P