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.
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.
The relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by ...whole-genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.
•AML subclones are discrete, genetically distinct entities in AML samples•AML subclones often have unique functional and morphological properties•Engraftment of AML cells in mice is not defined by evolutionary hierarchy•The AML founding clone is not equivalent to the AML-initiating cell in mice
Klco et al. track acute myeloid leukemia (AML) subclones identified by whole-genome sequencing and find that subclones of AML can correspond to different cellular populations within a single AML sample and can have different functional properties in vitro and in immunodeficient mice.
Hematopoietic clones harboring specific mutations may expand over time. However, it remains unclear how different cellular stressors influence this expansion. Here we characterize clonal ...hematopoiesis after two different cellular stressors: cytotoxic therapy and hematopoietic transplantation. Cytotoxic therapy results in the expansion of clones carrying mutations in DNA damage response genes, including TP53 and PPM1D. Analyses of sorted populations show that these clones are typically multilineage and myeloid-biased. Following autologous transplantation, most clones persist with stable chimerism. However, DNMT3A mutant clones often expand, while PPM1D mutant clones often decrease in size. To assess the leukemic potential of these expanded clones, we genotyped 134 t-AML/t-MDS samples. Mutations in non-TP53 DNA damage response genes are infrequent in t-AML/t-MDS despite several being commonly identified after cytotoxic therapy. These data suggest that different hematopoietic stressors promote the expansion of distinct long-lived clones, carrying specific mutations, whose leukemic potential depends partially on the mutations they harbor.
The randomized, double-blind, placebo-controlled, phase 3 COMFORT-I trial evaluated the JAK1/JAK2 inhibitor ruxolitinib in patients with intermediate-2/high-risk myelofibrosis. The primary and ...planned 3-year analyses of COMFORT-I data demonstrated that ruxolitinib-the first myelofibrosis-approved therapy-reduced splenomegaly and prolonged overall survival versus placebo. Here, we present the final 5-year results.
Patients managed in Australia, Canada, and the USA were randomized centrally (interactive voice response system) 1:1 to oral ruxolitinib twice daily (15 or 20 mg per baseline platelet counts) or placebo. Investigators and patients were blinded to treatment. The secondary endpoints evaluated in this analysis were durability of a ≥35% reduction from baseline in spleen volume (spleen response) and overall survival, evaluated in the intent-to-treat population. Safety was evaluated in patients who received study treatment.
Patients were randomized (September 2009-April 2010) to ruxolitinib (n = 155) or placebo (n = 154). At termination, 27.7% of ruxolitinib-randomized patients and 25.2% (28/111) who crossed over from placebo were on treatment; no patients remained on placebo. Patients randomized to ruxolitinib had a median spleen response duration of 168.3 weeks and prolonged median overall survival versus placebo (ruxolitinib group, not reached; placebo group, 200 weeks; HR, 0.69; 95% CI, 0.50-0.96; P = 0.025) despite the crossover to ruxolitinib. The ruxolitinib safety profile remained consistent with previous analyses. The most common new-onset all-grade nonhematologic adverse events starting <12 versus ≥48 months after ruxolitinib initiation were fatigue (29.0 vs 33.3%) and diarrhea (27.8 vs 14.6%). New-onset grade 3 or 4 anemia and thrombocytopenia both primarily occurred within the first 6 months, with no cases after 42 months. The most common treatment-emergent adverse event-related deaths in the ruxolitinib-randomized group were sepsis (2.6%), disease progression (1.9%), and pneumonia (1.9%).
The final COMFORT-I results continue to support ruxolitinib as an effective treatment for patients with intermediate-2/high-risk MF.
ClinicalTrials.gov, NCT00952289.
New therapies for patients with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) are needed. Interleukin 15 (IL-15) is a cytokine that stimulates ...CD8+ T-cell and natural killer (NK) cell antitumor responses, and we hypothesized this cytokine may augment antileukemia/antilymphoma immunity in vivo. To test this, we performed a first-in-human multicenter phase 1 trial of the IL-15 superagonist complex ALT-803 in patients who relapsed >60 days after allo-HCT. ALT-803 was administered to 33 patients via the IV or subcutaneous (SQ) routes once weekly for 4 doses (dose levels of 1, 3, 6, and 10 μg/kg). ALT-803 was well tolerated, and no dose-limiting toxicities or treatment-emergent graft-versus-host disease requiring systemic therapy was observed in this clinical setting. Adverse events following IV administration included constitutional symptoms temporally related to increased serum IL-6 and interferon-γ. To mitigate these effects, the SQ route was tested. SQ delivery resulted in self-limited injection site rashes infiltrated with lymphocytes without acute constitutional symptoms. Pharmacokinetic analysis revealed prolonged (>96 hour) serum concentrations following SQ, but not IV, injection. ALT-803 stimulated the activation, proliferation, and expansion of NK cells and CD8+ T cells without increasing regulatory T cells. Responses were observed in 19% of evaluable patients, including 1 complete remission lasting 7 months. Thus, ALT-803 is a safe, well-tolerated agent that significantly increased NK and CD8+ T cell numbers and function. This immunostimulatory IL-15 superagonist warrants further investigation to augment antitumor immunity alone and combined with other immunotherapies. This trial was registered at www.clinicaltrials.gov as #NCT01885897.
•Single-agent IL-15/IL-15Rα-Fc (ALT-803) therapy was well tolerated and resulted in clinical responses in patients who relapsed post-HCT.•First-in-human use of ALT-803 promoted NK and CD8+ T-cell expansion and activation in vivo without stimulating regulatory T cells.
Display omitted
Older patients with acute myeloid leukemia (AML) have limited treatment options because of the lack of effectiveness and the toxicity of available therapies. We investigated the efficacy and toxicity ...of the hypomethylating agent decitabine as initial therapy in older patients with AML.
In this multicenter, phase II study, patients older than 60 years who had AML (ie, > 20% bone marrow blasts) and no prior therapy for AML were treated with decitabine 20 mg/m(2) intravenously for 5 consecutive days of a 4-week cycle. Response was assessed by weekly CBC and bone marrow biopsy after cycle 2 and after each subsequent cycle. Patients continued to receive decitabine until disease progression or an unacceptable adverse event occurred.
Fifty-five patients (mean age, 74 years) were enrolled and were treated with a median of three cycles (range, one to 25 cycles) of decitabine. The expert-reviewed overall response rate was 25% (complete response rate, 24%). The response rate was consistent across subgroups, including in patients with poor-risk cytogenetics and in those with a history of myelodysplastic syndrome. The overall median survival was 7.7 months, and the 30-day mortality rate was 7%. The most common toxicities were myelosuppression, febrile neutropenia, and fatigue.
Decitabine given in a low-dose, 5-day regimen has activity as upfront therapy in older patients with AML, and it has acceptable toxicity and 30-day mortality.
Diabetic stem-cell "mobilopathy" DiPersio, John F
New England journal of medicine/The New England journal of medicine,
2011-Dec-29, Letnik:
365, Številka:
26
Journal Article
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.
Allogeneic hematopoietic cell transplantation is often complicated by graft versus host disease (GvHD), primarily mediated through allo-reactive donor T cells in the donor stem cell graft. Enhancer ...of Zeste Homolog 2 (EZH2), a histone-lysine N-methyltransferase and a component of the Polycomb Repressive Complex 2, has been shown to play a role in GvHD pathology. Although not yet clear, one proposed mechanism is through selective tri-methylation of lysine 27 in histone 3 (H3K27me3) that marks the promoter region of multiple pro-apoptotic genes, leading to repression of these genes in allo-reactive T cells. We found that selective pharmacologic inhibition of H3K27me3 with EPZ6438 or GSK126 did not prevent murine GvHD. This suggests the GvHD mitigating properties of DZNep are independent from H3K27me3 inhibition. Furthermore, while pharmacologic inhibition of EZH2 by DZNep has been shown to be effective in abrogating mouse GvHD, we found that DZNep was not effective in preventing GvHD in a human T cell xenograft mouse model. Although EZH2 is an attractive target to harness donor allo-reactive T cells in the post-transplant setting to modulate GvHD and the anti-leukemia effect, our results suggest that more selective and effective ways to inhibit EZH2 in human T cells are required.
PDF
