Use of granulocyte colony stimulating factor (G-CSF)–mobilized peripheral blood hematopoietic progenitor cells (HPC) has largely replaced bone marrow (BM) as a source of stem cells for both autologous and allogeneic cell transplantation. With G-CSF alone, up to 35% of patients are unable to mobilize sufficient numbers of CD34 cells/kg to ensure successful and consistent multi-lineage engraftment and sustained hematopoietic recovery. To this end, research is ongoing to identify new agents or combinations which will lead to the most effective and efficient stem cell mobilization strategies, especially in those patients who are at risk for mobilization failure. We describe both established agents and novel strategies at various stages of development. The latter include but are not limited to drugs that target the SDF-1/CXCR4 axis, S1P agonists, VCAM/VLA-4 inhibitors, parathyroid hormone, proteosome inhibitors, Groβ, and agents that stabilize HIF. While none of the novel agents have yet gained an established role in HPC mobilization in clinical practice, many early studies exploring these new pathways show promising results and warrant further investigation.
Stem cell mobilization; G-CSF; Plerixafor; SDF-1; Parathyroid hormone
Many patients with acute myeloid leukemia (AML) will eventually develop refractory or relapsed disease. In the absence of standard therapy for this population, there is currently an urgent unmet need for novel therapeutic agents. Targeted therapy with small molecule inhibitors (SMIs) represents a new therapeutic intervention that has been successful for the treatment of multiple tumors (e.g., gastrointestinal stromal tumors, chronic myelogenous leukemia). Hence, there has been great interest in generating selective small molecule inhibitors targeting critical pathways of proliferation and survival in AML. This review highlights a selective group of intriguing therapeutic agents and their presumed targets in both preclinical models and in early human clinical trials.
acute myeloid leukemia; small molecule inhibitors; therapeutic agents
Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used 18F-9-(4-fluoro-3-hydroxymethylbutyl)guanine (18FHBG) and positron emission tomography (PET)/CT scans to follow T cells transduced with herpes simplex virus thymidine kinase after administration to patients. Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of graft vs. host disease. Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans vs. animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered.
suicide gene; gene therapy; thymidine kinase; PET-imaging; transplant biology; regenerative medicine
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often complicated by alloreactive donor T cell-mediated graft-versus-host disease (GvHD). The major polyphenol of green tea, epigallocatechin-3-gallate (EGCG), is an inhibitor of both DNA methyltransferase 1 (DNMT1) and signal transducer and activator of transcription 1 (STAT1), which are essential for induction of GvHD. Thus, in this report, we examine if in vivo administration of EGCG mitigates GvHD in several different animal models. While we concede that refinement of EGCG treatment might result in GvHD prevention, our results suggest that EGCG treatment might not be an effective therapy against GvHD in the clinic.
GvHD; allogeneic hematopoietic stem cell transplantation; EGCG; DNMT1; interferon gamma receptor signaling; STAT1
Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders that are more common in patients aged ≥ 60 years and are incurable with conventional therapies. Reduced-intensity conditioning (RIC) allogeneic hematopoietic stem-cell transplantation is potentially curative but has additional mortality risk. We evaluated RIC transplantation versus nontransplantation therapies in older patients with MDS stratified by International Prognostic Scoring System (IPSS) risk.
Patients and Methods
A Markov decision model with quality-of-life utility estimates for different MDS and transplantation states was assessed. Outcomes were life expectancy (LE) and quality-adjusted life expectancy (QALE). A total of 514 patients with de novo MDS aged 60 to 70 years were evaluated. Chronic myelomonocytic leukemia, isolated 5q– syndrome, unclassifiable, and therapy-related MDS were excluded. Transplantation using T-cell depletion or HLA-mismatched or umbilical cord donors was also excluded. RIC transplantation (n = 132) stratified by IPSS risk was compared with best supportive care for patients with nonanemic low/intermediate-1 IPSS (n = 123), hematopoietic growth factors for patients with anemic low/intermediate-1 IPSS (n = 94), and hypomethylating agents for patients with intermediate-2/high IPSS (n = 165).
For patients with low/intermediate-1 IPSS MDS, RIC transplantation LE was 38 months versus 77 months with nontransplantation approaches. QALE and sensitivity analysis did not favor RIC transplantation across plausible utility estimates. For intermediate-2/high IPSS MDS, RIC transplantation LE was 36 months versus 28 months for nontransplantation therapies. QALE and sensitivity analysis favored RIC transplantation across plausible utility estimates.
For patients with de novo MDS aged 60 to 70 years, favored treatments vary with IPSS risk. For low/intermediate-1 IPSS, nontransplantation approaches are preferred. For intermediate-2/high IPSS, RIC transplantation offers overall and quality-adjusted survival benefit.
Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplant (allo-HCT) has been associated with reduced risk of relapse in patients with acute myeloid leukemia (AML). However the influence of the conditioning regimen on this protective effect of CMV reactivation after allo-HCT is relatively unexplored. To address this, we evaluated the risk of relapse in 264 AML patients who received T cell replete, 6/6 HLA matched sibling or 10/10 HLA matched unrelated donor transplantation at a single institution between 2006 and 2011. Out of these 264 patients, 206 received myeloablative (MA) and 58 received reduced intensity conditioning (RIC) regimens. CMV reactivation was observed in 88 patients with MA conditioning and 37 patients with RIC. At a median follow up of 299 days, CMV reactivation was associated with significantly lower risk of relapse in patients who received MA conditioning both in univariate (P= .01) and multivariate analyses (hazard ratio of 0.5246, P= .006), however CMV reactivation did not significantly affect the risk of relapse in our RIC cohort. These results confirm the protective effect of CMV reactivation on relapse in AML patients after allo-HCT reported by previous studies, however they suggest that this protective effect of CMV reactivation on relapse is influenced by the conditioning regimen used with the transplant.
Acute graft-versus-host disease (GVHD) remains a significant barrier to a more widespread application of allogeneic hematopoietic stem cell transplantation (HSCT). Vorinostat (suberoylanilide hydroxamic acid) is a histone deacetylases (HDAC) inhibitor that has been shown to attenuate GVHD in pre-clinical models. We aimed to study the safety and activity of vorinostat in combination with standard immunoprophylaxis for GVHD prevention in patients undergoing related donor reduced intensity conditioning HSCT.
In this prospective, single-arm phase 1/2 study of vorinostat, we recruited patients with high-risk hematologic malignances at two centers in the USA. We enrolled patients aged 18 years or older who were candidates for a reduced intensity conditioning HSCT and had an available 8/8- or 7/8-Human Leukocyte Antigen (HLA) matched related donor. Disease status had to be adequately controlled at the time of transplant. All patients received a conditioning regimen consisting of fludarabine 40 mg/m2 daily for four days (total dose 160 mg/m2) and busulfan 3·2 mg/kg daily for two days (total dose 6·4 mg/kg). GVHD prophylaxis consisted of mycophenolate mofetil 1 gram three times daily from day 0 and through day 28 and tacrolimus beginning on day −3 pre-HSCT and tapered beginning on day 56 and discontinued by day 180 post-HSCT in the absence of GVHD. The investigational agent, vorinostat, was initiated on day −10 through day 100 post-HSCT. The primary endpoint of the study was grade 2–4 acute GVHD by day 100. We expected to reduce the incidence to 25% from 42% based on similarly treated patients from the study centers and published literature. Patients were assessed for both toxicity and the primary endpoint if at least 21 days of vorinostat were administered. Patients who received less than 21 days of therapy were still assessed for toxicity and were replaced in accordance to the protocol. The trial is registered with ClinicalTrials.gov, NCT00810602.
Between March 2008 and February 2013, we enrolled 50 patients evaluable for both toxicity and response. All patients engrafted neutrophils and platelets at expected times post-HSCT. The median percentages of chimerism in whole-blood at day 100 and 1-year were 98% (interquartile range [IQR], 98–100) and 100% (IQR, 100–100), respectively. The primary endpoint of the study was met with a day 100 cumulative incidence of grade 2–4 acute GVHD of 22% (95% cumulative incidence: 13%, 36%). Eight additional patients enrolled were assessed for toxicity only, in accordance with the protocol, because they received less than 21 days of study drug. The most common non-hematologic adverse events were all grade 3 and included electrolyte disturbances (N=15), hyperglycemia (N=10), infections (N=4), mucositis (N=4), and elevated liver enzymes (N=3). There was one grade 4 hypokalemia event and two grade 4 infections. Non-symptomatic thrombocytopenia which occurred after engraftment was the most common hematologic grade 3 or 4 adverse event (N=9), but was transient and all cases resolved swiftly.
Administration of vorinostat in combination with standard GVHD prophylaxis after related donor reduced intensity conditioning HSCT is safe and appears to reduce severe GVHD. Future studies are needed to assess the effect of vorinostat in the prevention of GVHD in broader HSCT settings.
GVHD; hematopoietic stem cell transplantation; HDAC inhibitor; vorinostat
We have recently reported that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells result in significantly less graft-versus-host disease (GvHD) than wild-type (WT) T cells, while maintaining an anti-leukemia or graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that IFNγR signaling regulates alloreactive T cell trafficking to GvHD target organs through expression of the chemokine receptor CXCR3 in alloreactive T cells. Since IFNγR signaling is mediated via JAK1/JAK2, we tested the effect of JAK1/JAK2 inhibition on GvHD. While we demonstrated that pharmacologic blockade of JAK1/JAK2 in WT T cells using the JAK1/JAK2 inhibitor, INCB018424 (Ruxolitinib), resulted in a similar effect to IFNγR−/− T cells both in vitro (reduction of CXCR3 expression in T cells) and in vivo (mitigation of GvHD after allo-HSCT), it remains to be determined if in vivo administration of INCB018424 will result in preservation of GvL while reducing GvHD. Here, we report that INCB018424 reduces GvHD and preserves the beneficial GvL effect in two different murine MHC-mismatched allo-HSCT models and using two different murine leukemia models (lymphoid leukemia and myeloid leukemia). In addition, prolonged administration of INCB018424 further improves survival after allo-HSCT and is superior to other JAK1/JAK2 inhibitors, such as TG101348 or AZD1480. These data suggest that pharmacologic inhibition of JAK1/JAK2 might be a promising therapeutic approach to achieve the beneficial anti-leukemia effect and overcome HLA-barriers in allo-HSCT. It might also be exploited in other diseases besides GvHD, such as organ transplant rejection, chronic inflammatory diseases and autoimmune diseases.
This investigation of the association between body mass index at the time of multiple myeloma diagnosis and overall survival in a cohort of patients within the Veterans Health Administration system showed that disease-related weight loss may be an important and heretofore unknown indicator of poor prognosis in multiple myeloma. Weight loss ≥10% of baseline in the year before diagnosis was associated with significantly increased mortality.
Describe the association between baseline BMI, weight loss, and survival in MM.Explain the importance of BMI and baseline weight loss as part of the standard history obtained in patients with MM.
We investigated the association between body mass index (BMI) at the time of multiple myeloma (MM) diagnosis and overall survival in a cohort of patients within the Veterans Health Administration system. We also evaluated the association between weight loss in the year prior to diagnosis and survival.
Patients and Methods.
Prospective analysis was performed on a retrospectively assembled cohort of 2,968 U.S. veterans diagnosed and treated for MM between September 1, 1999, and September 30, 2009, with follow-up information through October 22, 2011. Cox modeling controlling for patient- and disease-related prognostic variables was used to analyze the data.
Underweight patients (BMI <18.5 kg/m2) had increased mortality, whereas patients who were overweight (BMI 25–29.9 kg/m2) and obese (BMI ≥30 kg/m2) had lower mortality compared with healthy-weight patients (BMI 18.5–24.9 kg/m2). Weight loss ≥10% of baseline in the year before diagnosis was also associated with increased mortality and made the association between increased BMI and survival nonsignificant.
Disease-related weight loss may be an important and heretofore unknown indicator of poor prognosis in MM. Assessment of weight loss prior to MM diagnosis should become a standard component of the clinical history in patients with newly diagnosed MM. Further research may identify relationships between disease-related weight loss and currently used prognostic factors in MM, further defining the role of this clinical factor in prognostic stratification.
Multiple myeloma; Survival; Body mass index; Obesity; Overweight; Mortality
Successful hematopoietic stem cell transplant (HSCT) requires the infusion of a sufficient number of hematopoietic stem/progenitor cells (HSPCs) that are capable of homing to the bone marrow cavity and regenerating durable trilineage hematopoiesis in a timely fashion. Stem cells harvested from peripheral blood are the most commonly used graft source in HSCT. While granulocyte colony-stimulating factor (G-CSF) is the most frequently used agent for stem cell mobilization, the use of G-CSF alone results in suboptimal stem cell yields in a significant proportion of patients. Both the chemokine receptor CXCR4 and the integrin α4β1 (VLA-4) play important roles in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid and reversible mobilization of hematopoietic stem cells into the peripheral circulation and is synergistic when combined with G-CSF. In this review we discuss the development of small molecule CXCR4 and VLA-4 inhibitors and how they may improve the utility and convenience of peripheral blood stem cell transplantation.
Hematopoietic stem cell transplantation; hematopoietic stem cell mobilization; CXCR4; VLA-4; plerixafor
The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.
Sequencing the genomic DNA of cancers has revealed that tumors are not homogeneous. As a tumor grows, new mutations accumulate in individual cells, and as these cells replicate, the mutations are passed on to their offspring, which comprise only a portion of the tumor when it is sampled. We present a method for identifying the fraction of cells containing specific mutations, clustering them into subclonal populations, and tracking the changes in these subclones. This allows us to follow the clonal evolution of cancers as they respond to chemotherapy or develop therapy resistance, processes which may radically alter the subclonal composition of a tumor. It also gives us insight into the spatial organization of tumors, and we show that multiple biopsies from a single breast cancer may harbor different subclones that respond differently to treatment. Finally, we show that sequencing multiple samples from a patient's tumor is often critical, as it reveals cryptic subclones that cannot be discerned from only one sample. This is the first tool that can efficiently leverage multiple samples to identify these as distinct subpopulations of cells, thus contributing to understanding the biology of the tumor and influencing clinical decisions about therapy.
Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions—the population frequency of individual clones, their genetic composition, and their evolutionary relationships—which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.
Human cancers are genetically diverse populations of cells that evolve over the course of their natural history or in response to the selective pressure of therapy. In theory, it is possible to infer how this variation is structured into related populations of cells based on the frequency of individual mutations in bulk samples, but the accuracy of these models has not been evaluated across a large number of variants in individual cells. Here, we report a strategy for analyzing hundreds of variants within a single cell, and we apply this method to assess models of tumor clonality derived from bulk samples in three cases of leukemia. The data largely support the predicted population structure, though they suggest specific refinements. This type of approach not only illustrates the biological complexity of human cancer, but it also has the potential to inform patient management. That is, precise knowledge of which variants are present in which populations of cells may allow physicians to more effectively target combinations of mutations and predict how patients will respond to therapy.
Myelofibrosis (MF) patients can present with a wide spectrum of disease characteristics. We analysed the consistency of ruxolitinib efficacy across patient subgroups in the COntrolled MyeloFibrosis Study With ORal JAK Inhibitor Treatment (COMFORT-I,) a double-blind trial, where patients with intermediate-2 or high-risk MF were randomized to twice-daily oral ruxolitinib (n = 155) or placebo (n = 154). Subgroups analysed included MF subtype (primary, post-polycythaemia vera, post-essential thrombocythaemia), age (≤65, > 65 years), International Prognostic Scoring System risk group, baseline Eastern Cooperative Oncology Group performance status (0, 1, ≥2), JAK2 V617F mutation (positive, negative), baseline haemoglobin level (≥100, <100 g/l), baseline platelet count (100–200 × 109/l, >200 × 109/l), baseline palpable spleen size (≤10, >10 cm), and baseline quartile of spleen volume and Total Symptom Score (TSS; Q1 = lowest, Q4 = highest). Mean percentage change from baseline to week 24 in spleen volume and TSS were calculated for ruxolitinib and placebo in each subgroup. Overall survival was estimated by Kaplan–Meier method according to original randomization group. In ruxolitinib-treated patients, reductions in spleen volume and TSS and evidence of improved survival relative to placebo across subgroups were consistent with those seen in the COMFORT-I population, confirming that ruxolitinib is an effective therapy for the spectrum of MF patients studied in COMFORT-I.
Myelofibrosis; ruxolitinib; subgroups; spleen volume; symptoms
Acute graft-versus-host disease (aGVHD) results from a robust response of donor T cells transferred during hematopoietic stem cell transplantation (HSCT) to allogeneic peptide–major histocompatibility complex antigens. Previous investigations have not identified T cell subsets that selectively mediate either protective immunity or pathogenic alloreactivity. We demonstrate that the small subset of peripheral T cells that naturally express two T cell receptors (TCRs) on the cell surface contributes disproportionately to aGVHD in patients after allogeneic HSCT. Dual TCR T cells from patients with aGVHD demonstrate an activated phenotype and produce pathogenic cytokines ex vivo. Dual receptor clones from a patient with symptomatic aGVHD responded specifically to mismatched recipient human leukocyte antigens (HLAs), demonstrating pathologic alloreactivity. Human dual TCR T cells are strongly activated and expanded by allogeneic stimulation in vitro, and disproportionately contribute to the repertoire of T cells recognizing both major (HLA) and minor histocompatibility antigens, providing a mechanism for their observed activity in vivo in patients with aGVHD. These results identify dual TCR T cells as a target for focused analysis of a T cell subset mediating GVHD and as a potential prognostic indicator.
Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome.
High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM.
We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype.
These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.
Multiple myeloma (MM) is an incurable, B-cell malignancy, characterized by the clonal proliferation and accumulation of malignant plasma cells in bone marrow. Despite recent advances in understanding of genomic aberrations, a comprehensive catalogue of clinically actionable mutations in MM is just beginning to emerge. The tyrosine kinase (TK) and RAS oncogenes, which encode important regulators of various signaling pathways, are among the most frequently altered gene families in cancer. To clarify the role of TK and RAS genes in pathogenesis of MM, we performed a systematic, targeted screening of mutations on prioritized RAS and TK genes, in CD138 sorted bone marrow specimens from 42 untreated patients. We identified a total of 24 mutations in KRAS, PIK3CA, INSR, LTK and MERTK genes. In particular, seven novel mutations in addition to known KRAS mutations were observed. Prediction analysis tools, PolyPhen and SIFT were used to assess the functional significance of these novel mutations. Our analysis predicted that these mutations may have a deleterious effect, resulting in functional alteration of proteins involved in the pathogenesis of myeloma. While further investigation is needed to determine the functional consequences of these proteins, mutational testing of the RAS and TK genes in larger myeloma cohorts might be also useful to establish the recurrent nature of these mutations.
Multiple Myeloma; Tyrosine Kinase; RAS; Resequencing; Mutation analysis; Cancer
Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability, driving clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of AML samples with a known initiating event (PML-RARA) vs. normal karyotype AML samples, and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is “captured” as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.
Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Models of immunodeficient mice that consistently and efficiently reconstitute with xenoreactive human T cells would be a valuable tool for the in vivo study of GVHD, as well as other human immune responses.
Materials and Methods
We developed a consistent and sensitive model of human GVHD by retro-orbitally injecting purified human T cells into sublethally irradiated NOD/SCID-β2mnull recipients. In addition, we characterized for the first time the trafficking patterns and expansion profiles of xenoreactive human T cells in NOD/SCID-β2mnull recipients using in vivo bioluminescence imaging.
All NOD/SCID-β2mnull mice conditioned with 300 cGy of total body irradiation and injected with 1 × 107 human T cells exhibited human T cell engraftment, activation, and expansion, with infiltration of multiple target tissues and a subsequent greater than 20% loss of pretransplant body weight. Importantly, histological examination of the GVHD target tissues revealed changes consistent with human GVHD. Furthermore, we also showed by in vivo bioluminescence imaging that the development of lethal GVHD in the NOD/SCID-β2mnull recipients was dependent upon the initial retention and early expansion of human T cells in the retroorbital sinus cavity.
Our NOD/SCID-β2mnull mouse model provides a system to study the pathophysiology of acute GVHD induced by human T cells and aids in the development of more effective therapies for human GVHD.
Positron emission tomography using [18F]fluorodeoxyglucose (FDG-PET) has emerged as the standard response assessment tool in front-line therapy for classical Hodgkin lymphoma (cHL). The ability of FDG-PET to predict outcomes in patients with relapsed cHL treated with modern standard salvage chemotherapy and autologous stem cell transplantation (ASCT) remains uncertain. Forty-six patients with relapsed/refractory cHL treated from 2001–2007 with standard salvage/ASCT therapy had FDG-PET available for blinded review. The results of pre-ASCT FDG-PET interpreted by the international harmonization project (IHP) criteria were compared to published prognostic models for prediction of event free survival (EFS) and overall survival (OS). Overall, 3 year EFS was 62% and OS was 78% with a median follow-up of 38 months. Pre-ASCT FDG-PET response significantly predicted 3 year EFS in FDG-PET negative (82%) versus FDG-PET positive (41%) patients (p=0.02). A trend was observed for 3 year OS comparing FDG-PET negative (91%) versus positive (64%) patients (p=0.08). Multivariate analysis demonstrated the independent prognostic significance of pre-ASCT FDG-PET for EFS with a HR of 3.2 (CI 1.1–9.0, p=0.03). Pre-ASCT FDG-PET scans predict EFS in patients with relapsed cHL patients treated with modern salvage/ASCT therapy, and warrant prospective evaluation.
autologous stem cell transplantation; classical Hodgkin lymphoma; FDG-PET; prognosis
The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood.
We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations.
Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene.
Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.)
Most patients with acute myeloid leukemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level1,2. To determine the mutational spectrum associated with relapse, we sequenced the primary tumor and relapse genomes from 8 AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to precisely define clonality and clonal evolution patterns at relapse. Besides discovering novel, recurrently mutated genes (e.g. WAC, SMC3, DIS3, DDX41, and DAXX) in AML, we found two major clonal evolution patterns during AML relapse: 1) the founding clone in the primary tumor gained mutations and evolved into the relapse clone, or 2) a subclone of the founding clone survived initial therapy, gained additional mutations, and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific vs. primary tumor mutations in all 8 cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped in part by the chemotherapy that the patients receive to establish and maintain remissions.
Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole genome sequencing to perform an unbiased comprehensive screen to discover all the somatic mutations in a sAML sample and genotyped these loci in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (S34) in U2AF1 was recurrently mutated in 13/150 (8.7%) de novo MDS patients, with suggestive evidence of an associated increased risk of progression to sAML. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3′ end of introns and mutations are located in highly conserved zinc fingers in U2AF11,2. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This novel, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.
Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells.
Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of 3H-adenosine.
Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used.
The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.
Alterations in DNA methylation have been implicated in the pathogenesis of myelodysplastic syndromes (MDS), although the underlying mechanism remains largely unknown. Methylation of CpG dinucleotides is mediated by DNA methyltransferases, including DNMT1, DNMT3A, and DNMT3B. DNMT3A mutations have recently been reported in patients with de novo acute myeloid leukemia (AML), providing a rationale for examining the status of DNMT3A in MDS samples. Here, we report the frequency of DNMT3A mutations in patients with de novo MDS, and their association with secondary AML. We sequenced all coding exons of DNMT3A using DNA from bone marrow and paired normal cells from 150 patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12/150 patients (8.0%). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/13 mutations. DNMT3A mutations were expressed in the majority of cells in all tested mutant samples regardless of blast counts, suggesting that DNMT3A mutations occur early in the course of MDS. Patients with DNMT3A mutations had worse overall survival compared to patients without DNMT3A mutations (p=0.005) and more rapid progression to AML (p=0.007), suggesting that DNMT3A mutation status may have prognostic value in de novo MDS.
myelodysplastic syndrome; DNMT3A; mutation
The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known.
We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome.
We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis.
By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.