thrombotic thrombocytopenic purpura; ticlopidine; ADAMTS13; ADAMTS13 inhibitor; Japan
MLL rearrangements were analysed in the blood of a patient receiving chemotherapy for diffuse large B-cell lymphoma using inverse polymerase chain reaction targeting exon 12, parallel sequencing and a custom algorithm design. Of thirteen MLL rearrangements detected, five were capable of generating MLL fusion genes, including MLL-MLLT3, the most common fusion in acute myeloid leukaemia (AML). Other fusions, all previously clinically unobserved, included MLL-NKD1, a fusion to the negative regulator of Wnt/β-catenin signaling, a pathway linked to leukaemic cell proliferation. The majority of the fusions exhibited clonal persistence from before treatment until six months post-chemotherapy, suggesting the fusions may confer a survival advantage to the mutant clone. MLL breakpoints were partly clustered at a specific location, indicating commonality in the process of their formation. Further, the same MLL breakpoint location exhibited a 50- to 100-fold increase in C to T transitions, consistent with attack by activation-induced cytidine deaminase (AICDA). As is also observed in AML and acute lymphoblastic leukaemia, in this single patient setting, MLL is capable of interacting with multiple fusion partners. This finding defines a discrete site of MLL susceptibility to fragmentation, linked to possible deregulation of AICDA function.
AICDA; tAML; Clonal MLL fusion; C to T transition; MLL rearrangement
lymphoma; stem cells; drug resistance
We report results of a pilot study of high-dose vitamin D in sickle cell disease (SCD). Subjects were followed for 6 months after receiving a six-week course of oral high-dose cholecalciferol or placebo. Vitamin D insufficiency and deficiency was present at baseline in 82.5% and 52.5% of subjects, respectively. Subjects who received high-dose vitamin D achieved higher serum 25-hydroxyvitamin D, experienced fewer pain days per week, and had higher physical activity quality-of-life scores. These findings suggest a potential benefit of vitamin D in reducing the number of pain days in SCD. Larger prospective studies with longer duration are needed to confirm these effects.
Vitamin D; Chronic Pain; Sickle Cell; Paediatrics; Quality of Life
The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide-specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA-A2-specific CD138 epitope against MM cells. A novel immunogenic HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide was identified from the full-length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138+ MM cells. The peptide-induced CD138-CTL contained a high percentage of CD8+ activated/memory T cells with a low percentage of CD4+ T cell and naive CD8+ T cell subsets. The CTL displayed HLA-A2-restricted and CD138 antigen-specific cytotoxicity against MM cell lines. In addition, CD138-CTL demonstrated increased degranulation, proliferation and γ–interferon secretion to HLA-A2+/CD138+ myeloma cells, but not HLA-A2−/CD138+ or HLA-A2+/CD138− cells. The immune functional properties of the CD138-CTL were also demonstrated using primary HLA-A2+/CD138+ cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138260-268 (GLVGLIFAV) peptide can induce antigen-specific CTL, which might be useful for the treatment of MM patients with peptide-based vaccine or cellular immunotherapy strategies.
CD138 peptide; multiple myeloma; peptide vaccine; cytotoxic T lymphocytes
Clinical management of severe pain associated with sickle cell disease (SCD) remains challenging. Development of optimal therapy would be facilitated by use of murine model(s) with varying degrees of sickling and pain tests that are most sensitive to vasoocclusion. We found that young (≤3 month old) NY1DD and S+SAntilles mice (having modest and moderate sickle phenotype, respectively) exhibit evidence of deep tissue/musculoskeletal pain. Deep tissue pain and cold sensitivity in S+SAntilles mice increased significantly with both age and incitement of hypoxia/reoxygenation (H/R). C57/BL6 mice (genetic background strain of NY1DD and S+SAntilles) were hypersensitive to mechanical and heat stimuli, even without the sickle transgene. H/R treatment of HbSS-BERK mice with severe sickle phenotype resulted in significantly decreased withdrawal thresholds and enhanced mechanical, thermal and deep tissue hyperalgesia. Deep hyperalgesia incited by H/R in HbSS-BERK was ameliorated by CP 55940, a cannabinoid receptor agonist. Thus, assessment of deep tissue pain appears to be the most sensitive measure for studying pain mechanisms across mouse models of SCD, and HbSS-BERK mice may best model vaso-occlusive and chronic pain of SCD.
Pain; sickle cell disease; hypoxia; ischemia; reperfusion injury
Aurora kinases play an important role in the control of the cell cycle and have been implicated in tumourigenesis in a number of cancers. Among the haematological malignancies, overexpression of Aurora kinases has been reported in acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, multiple myeloma, aggressive non-Hodgkin lymphoma and Hodgkin lymphoma. A large number of Aurora kinase inhibitors are currently in different stages of clinical development. In addition to varying in their selectivity for the different Aurora kinases, some also have activity directed at other cellular kinases involved in important molecular pathways in cancer cells. This review summarizes the biology of Aurora kinases and discusses why they may be good therapeutic targets in different haematological cancers. We describe preclinical data that has served as the rationale for investigating Aurora kinase inhibitors in different haematological malignancies, and summarize published results from early phase clinical trials. While the anti-tumour effects of Aurora kinase inhibitors appear promising, we highlight important issues for future clinical research and suggest that the optimal use of these inhibitors is likely to be in combination with cytotoxic agents already in use for the treatment of various haematological cancers.
cell cycle; myeloid leukaemia; lymphoma; myeloma
Myelodysplasia; acute myeloid leukaemia; azacitidine; relapse; outcome
Prior to the introduction of the International Network for Cancer Treatment and Research (INCTR) protocol INCTR 03-06, survival of patients with Burkitt lymphoma at 4 tertiary care centres in equatorial Africa was probably no more than 10–20%. The results reported here for 356 patients have demonstrated marked improvement in survival through the use of a uniform treatment protocol consisting of cyclophosphamide, methotrexate, vincristine, and intrathecal therapy, and the introduction of non-cross resistant second-line (salvage) therapy, consisting of ifosfamide, mesna, etoposide and cytarabine, when patients failed to achieve a complete response to first-line therapy or relapsed early. Overall survival rates of 67% and 62% were observed at 1 and 2 years (relapse is rare after one year). Of interest was the small impact of cerebrospinal fluid (CSF) and bone marrow involvement on outcome. However, the presence or absence of abdominal involvement clearly defined two prognostic groups. An additional finding was the association between CSF pleocytosis and orbital tumours, suggesting that spread of tumour cells to the central nervous system may occur via direct involvement of cranial nerves in the orbit. Survival rates may be increased in patients with abdominal involvement by combining first- and second-line therapy, but verification will require a further clinical study.
Childhood haematological malignancies; clinical research; chemotherapy
To assess the associations between a doctor diagnosis of asthma and wheezing (independent of a diagnosis of asthma) with sickle cell disease (SCD) morbidity, we conducted a retrospective review of Emergency Department (ED) visits to the Mount Sinai Medical Center for SCD between 1 January 2007 and 1 January 2011. Outcomes were ED visits for pain and acute chest syndrome. The cohort included 262 individuals, median age 23·8 years, (range: 6 months to 67·5 years). At least one episode of wheezing recorded on a physical examination was present in 18·7% (49 of 262). Asthma and wheezing did not overlap completely, 53·1% of patients with wheezing did not carry a diagnosis of asthma. Wheezing was associated with a 118% increase in ED visits for pain (95% confidence interval [CI]: 56–205%) and a 158% increase in ED visits for acute chest syndrome (95% CI: 11–498%). A diagnosis of asthma was associated with a 44% increase in ED utilization for pain (95% CI: 2–104%) and no increase in ED utilization for acute chest syndrome (rate ratio 1·00, 95%CI 0·41–2·47). In conclusion, asthma and wheezing are independent risk factors for increased painful episodes in individuals with SCD. Only wheezing was associated with more acute chest syndrome.
sickle cell; asthma; wheeze; pain; acute chest syndrome
Gene amplification is defined as a copy number (CN) increase in a restricted region of a chromosome arm, and is a mechanism for acquired drug resistance and oncogene activation. In multiple myeloma (MM), high CNs of genes in a 1q12~23 amplicon have been associated with disease progression and poor prognosis. To investigate the mechanisms for gene amplification in this region in MM, we performed a comprehensive metaphase analysis combining G-banding, fluorescence in situ hybridization, and spectral karyotyping in 67 patients with gain of 1q. In six patients (9%), evidence for at least one breakage-fusion-bridge (BFB) cycle was found. In three patients (4%), extended ladders of 1q12~23 amplicons were identified. Several key structures that are predicted intermediates in BFB cycles were observed, including: equal-spaced organization of amplicons, inverted repeat organization of amplicons along the same chromosome arm, and deletion of sequences distal to the amplified region. The 1q12 pericentromeric heterochromatin region served as both a recurrent breakpoint as well as a fusion point for sister chromatids, and ultimately bracketed both the proximal and distal boundaries of the amplicon. Our findings provide evidence for a novel BFB mechanism involving 1q12 pericentromeric breakage in the amplification of a large number of genes within a 1q12~23 amplicon.
Multiple myeloma; cytogenetics; gene amplification; pericentromeric heterochromatin; breakage-fusion-bridge cycles
Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line-only control. Furthermore, the combined co-culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.
mesenchymal cells; macrophages; multiple myeloma; marrow stroma; myeloma
Childhood obesity is rapidly increasing in prevalence. We compared circulating membrane-bound tissue factor (FIII, F3) procoagulant activity (TF-PCA) and plasma markers of coagulation, fibrinolysis and endothelial dysfunction in 21 obese (10.1±1.5 years, mean ± standard deviation) and 22 healthy weight children (9.9±1.6 years), classified by Body Mass Index (BMI). TF-PCA and factor VII coagulant activity (FVII:C), plasminogen activator inhibitor (PAI-1, SERPINE1) and soluble vascular cell adhesion molecule 1 (sVCAM1) were higher in obese children. BMI correlated positively with TF-PCA, FVII:C, and PAI-1. Childhood obesity is associated with a procoagulant state and endothelial dysfunction. Studies are needed to assess whether weight reduction reverses these abnormalities.
Childhood Obesity; Coagulation; Tissue Factor; Fibrinolysis; Endothelium
Complex karyotype (CK) on metaphase cytogenetics discriminates poor outcome in chronic lymphocytic leukaemia (CLL) patients undergoing salvage treatment; we hypothesized that it might provide prognostic information for patients undergoing allogeneic stem cell transplant. Fifty-one CLL patients were analysed following transplant; 18-month overall survival (OS), event-free survival (EFS) and cumulative incidence of progression estimates were 35%, 14% and 63%, respectively, in patients with CK (n = 19) versus 83%, 68% and 29% in patients without (n = 32) (P ≤ 0.0001, P ≤ 0.0001, and P = 0.02). In patients with high-risk interphase cytogenetics, CK remained predictive of worse OS (P = 0.02) and EFS (P = 0.009). These findings support further evaluation of metaphase karyotype in transplant risk assessment.
chronic lymphocytic leukaemia; transplant; fluorescent insitu hybridization; metaphase cytogenetics; conditioning
In the last decade, the novel agents lenalidomide, bortezomib, and thalidomide have dramatically improved outcomes for patients with multiple myeloma (MM). A number of new therapies with precise targets involved in MM cell growth and replication are now in development and have the potential for further improvements. Second-generation proteasome inhibitors and thalidomide derivatives may offer increased efficacy and safety. Investigational therapies with rationally selected targets in MM include inhibitors of histone deacetylase, heat shock protein 90, mammalian target of rapamycin, BCL2, Akt, mitogen-activated protein kinase, and telomerase. In addition, monoclonal antibodies directed against several targets have been developed and many are showing promise in initial clinical trials in MM. Interest in the ancient remedy of arsenic trioxide has been revived because of its proapoptotic effects on mitochondria, despite its established toxicities. In general, combination regimens are proving the most efficacious, which is to be expected given the multiple overlapping pathways responsible for MM growth and progression.
multiple myeloma; tanespimycin; lenalidomide; bortezomib; thalidomide
Lenalidomide has demonstrated impressive antileukaemic effects in patients with chronic lymphocytic leukaemia (CLL). The mechanism(s) by which it mediates these effects remain unclear. Clinically, CLL patients treated with lenalidomide demonstrate an acute inflammatory reaction, the tumour flare reaction that is suggestive of an immune activation phenomenon. Samples from CLL patients treated with lenalidomide were used to evaluate its effect on the tumour cell and components of its microenvironment (immune cellular and cytokine). Lenalidomide was unable to directly induce apoptosis in CLL cells in vitro, however it modulated costimulatory (CD80, CD83, CD86) surface molecules on CLL cells in vitro and in vivo. Concurrently, we demonstrated that NK cell proliferation was induced by lenalidomide treatment in patients and correlated with clinical response. Cytokine analysis showed increase in levels of TNF-α post-lenalidomide treatment, consistent with acute inflammatory reaction. Furthermore, the basal cytokine profile (high IL-8, MIG, IP-10 and IL-4 levels and low IL-5, MIP1a, MIP1b, IL12/p70) was predictive of clinical response to lenalidomide. Collectively, our correlative studies provide further evidence that the antileukaemic effect of lenalidomide in CLL is mediated not only through modulation of the leukaemic clone but also through elements of the tumour microenvironment.
lenalidomide; chronic lymphocytic leukaemia; immune cells; microenvironment; immune activation
Follicular lymphoma (FL) comprises nearly 25% of non-Hodgkin lymphoma cases and is clinically characterized by initial sensitivity to chemotherapy followed by relapse. FL stroma contains a special type of stromal cell found in germinal centre of lymph nodes—the follicular dendritic cell (FDC). We first isolated tumourigenic cells from the FL cell line FLK-1 by side population (SP) technique, and found that SP cells, which express ABCG2, were enriched by chemotherapy and radiation treatments. In vitro, SP cells were attracted by and adhered to FDCs through chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4) signalling. In vivo, limiting dilution assays showed SP cells were highly enriched in cancer stem cell (CSC), but required FDC for tumour formation in non-obese diabetic/severe combined immunodeficiency mice. Treatment with AMD3100, a specific CXCL12/CXCR4 inhibitor, eliminated tumour growth. These findings were then verified with FL cells isolated from an FL patient’s ascitic fluid (FLA-1). Finally, we detected the ABCG2 expressing lymphoma cells in FL clinical specimens. Thus, we found that the highly tumourigenic FL cells having CSC-like activities (FL-SC) interact with FDCs in a CXCL12/CXCR4 dependent manner to resist chemotherapy. Our results indicate the importance of FL-SC and niche cell signalling in maintaining tumourigenicity. These signals represent novel targets for CSC eradication.
Follicular lymphoma; Tumour/stromal cell interactions; Side population; Follicular dendritic cells; CXCL12 (SDF-1α)
The prognosis of patients with relapsed and refractory acute leukaemia (RRAL) is very poor. Forty patients with RRAL were enrolled (28 acute myeloid leukaemia [AML], 12 acute lymphoblastic leukaemia [ALL]) in this Phase 1 dose-escalation trial of daily-infused clofarabine (CLO) followed by cyclophosphamide (CY) for 4 consecutive days (CLO-CYx4). The median age was 48.5 years. The median number of prior regimens was 2 (range 1–5), and 6/40 patients (15%) had prior allogeneic haematopoietic stem cell transplant. 28/40 patients (70%) had adverse genetic features. 6/40 patients (15%) died within 60 days of induction (2 infections, 4 progressive disease). The average time to neutrophil recovery (absolute neutrophil count ≥ 0.5 × 109/l was 34 days, (range, 17–78). The overall response rate (ORR) was 33% (13/40), with 7 complete remissions (18%), 4 complete remissions with incomplete recovery of blood counts (10%), and 2 partial remissions (5%). ORR was 25% (7/28), and 50% (6/12), for AML and ALL, respectively. Notably, the clinical responses were independent of dose level. 7/17 patients (41%) exhibited CLO-mediated enhancement of CY-induced DNA, which was associated with, but not necessary for, improved clinical outcomes. In summary, the CLO-CYx4 regimen was well tolerated and had activity in patients with RRAL, especially relapsed ALL. Therefore, CLO-CYx4 can be considered a salvage therapy for adults with RRALs, and warrants further investigations.
refractory leukaemia; clofarabine; cyclophosphamide; acute lymphoblastic leukaemia; acute myeloid leukaemia
Proper splicing of pre-mRNA is required for protein synthesis and therefore is a fundamental cellular function. The discovery of a variety of somatic spliceosomal mutations in hematologic malignancies, including myeloid neoplasms and chronic lymphocytic leukemia has pointed to a new leukemogenic pathway involving spliceosomal dysfunction. Theoretically, spliceosomal mutations can lead to activation of incorrect splice sites, intron retention or aberrant alternative splicing occurring in patterns generated by mutations of individual spliceosomal proteins. Such events can produce a defective balance between protein isoforms leading to functional consequences including defective regulation of proliferation and differentiation. The observed pattern of occurrence of highly specific missense mutations coupled with the lack of nonsense mutations and deletions, implies a gain-of-function or better gain-of-dysfunction mechanism. Incorrect splicing of downstream genes such as tumor suppressor genes may result in haploinsufficient expression through nonsense mediated mRNA decay. Thus sliceosomal mutations may, depending on the pattern of affected proteins, lead to similar functional effects on tumor suppressor genes as chromosomal deletions, epigenetic silencing or inactivating/hypomorphic mutations. The prognostic value of the most common mutations and their phenotypic association in the clinical setting is currently being investigated. It is likely that spliceosomal mutations may indicate sensitivity to spliceosome inhibitors applied in the form of a synthetic lethal approach. This manuscript discusses the most current aspects of spliceosomal research in the context of hematologic malignancies.
Allogeneic marrow transplantation offers curative therapy for children with severe aplastic anaemia (SAA). We report the outcomes of 148 children with SAA who received human leucocyte antigen (HLA)-matched related marrow grafts between 1971 and 2010. Patients were divided into 3 groups, reflecting changes in conditioning and graft-versus-host disease (GVHD) prophylaxis regimens that occurred over time. Patients in Group 1 were conditioned with cyclophosphamide (CY; 200 mg/kg) followed by "long" (102 days) methotrexate (MTX). Patients in Groups 2 and 3 received CY alone (Group 2) or combined with anti-thymocyte globulin (Group 3) followed by "short" (days 1, 3, 6, and 11) MTX and ciclosporin (until day 180). With a median follow-up of 25 years, the 5-year survivals were 66%, 95%, and 100% for Groups 1, 2, and 3, respectively (overall p<0.0001). The 3-year estimates of graft rejection were 22%, 32%, and 7%, respectively. The probabilities of grades III-IV acute and 2-year chronic GVHD were 15%, 0%, and 3%, and 21%, 21%, and 10%, respectively. Advances in preparative and GVHD prophylaxis regimens, and supportive care during the past 40 years have led to improved outcomes for children with SAA. These results confirm the use of allogeneic marrow transplantation for children with SAA who have HLA-matched related donors.
Bone marrow transplantation; Paediatric aplastic anaemia; Haematopoietic cell transplantation
Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.
D-tyrosinol; chronic lymphocytic leukaemia; p38 mitogen-activated protein kinase (p38 MAPK); apoptosis; BIRC5
We recently showed that increasing Wnt/β-catenin signalling in the bone marrow microenvironment or in multiple myeloma (MM) cells clearly suppresses osteoclastogenesis in SCID-hu mice; however, this regulation of osteoclastogenesis could result directly from activation of Wnt/β-catenin signalling in osteoclasts or indirectly from effects on osteoblasts. The present studies characterized Wnt/β-catenin signalling and its potential role in osteoclasts. Systematic analysis of expression of WNT, FZD, LRP and TCF gene families demonstrated that numerous Wnt-signalling components were expressed in human osteoclasts from patients with MM. Functional Wnt/β-catenin signalling was identified by accumulation of total and active β-catenin and increases in Dvl-3 protein in response to Wnt3a or LiCl. Furthermore, Wnt-induced increases in β-catenin and Dvl-3 were attenuated by Wnt antagonists Dkk1 and sFRP1. Finally, Wnt3a-induced TCF/LEF transcriptional activity suggests that canonical Wnt signalling is active in osteoclasts. Supernatants from dominant-negative-β-catenin–expressing osteoblast clones significantly stimulated tartrate-resistant acid phosphatase–positive osteoclast formation from primary MM-derived osteoclasts, compared with supernatants from control cells. These results suggested that Wnt/β-catenin signalling is active in osteoclasts in MM and is involved in osteoclastogenesis in bone marrow, where it acts as a negative regulator of osteoclast formation in an osteoblast-dependent manner in MM.
Wnt3a; β-catenin; osteoclast; bone disease; multiple myeloma
Contrary to Total Therapy 2 (TT2), FGFR3-translocation bore no adverse effects on outcome in Total Therapy 3 (TT3) with added bortezomib. DelTP53, another poor-risk feature in TT2, was examined for its prognostic consequences in TT3 present in 10% of 441 patients treated. Not affecting rate or duration of complete response, TP53 haplo-insufficiency also did not compromise, in the 83% with genomically defined low-risk myeloma, survival or event-free survival. FGFR+ and FGFR3− molecular subgroups fared worse in the presence of delTP53 when applying TT2 but not TT3. Thus, delTP53’s prognostic implications were protocol- as well as genome-defined risk- and molecular subgroup-dependent.
Myeloma; TP53; Bortezomib; Prognosis; Genomics