New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution and comprehensiveness. To help translate these methods to ...routine research and clinical practice, we developed a sequence-resolved benchmark set for identification of both false-negative and false-positive germline large insertions and deletions. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle Consortium integrated 19 sequence-resolved variant calling methods from diverse technologies. The final benchmark set contains 12,745 isolated, sequence-resolved insertion (7,281) and deletion (5,464) calls ≥50 base pairs (bp). The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.51 Gbp and 5,262 insertions and 4,095 deletions supported by ≥1 diploid assembly. We demonstrate that the benchmark set reliably identifies false negatives and false positives in high-quality SV callsets from short-, linked- and long-read sequencing and optical mapping.
Mutations within the BCR-ABL kinase domain in imatinib-treated chronic myeloid leukemia (CML) are the main mechanism of acquired resistance. The early detection of mutations should provide clinical ...benefit by allowing early intervention. Quantitative polymerase chain reaction (RQ-PCR) results of BCR-ABL mRNA were correlated with mutation analysis in 214 patients treated with imatinib. We determined whether there was a difference in the incidence of mutations between the patients with a more than 2-fold rise in BCR-ABL and patients with stable or decreasing levels. Of the 56 patients with a more than 2-fold rise, 34 (61%) had detectable mutations (median rise, 3.0-fold; 25th-75th percentiles, 2.3-5.2). In 31 (91%) of these 34 patients, the mutation was present at the time of the rise and became detectable within 3 months in the remaining patients. Only 1 (0.6%) of 158 patients with stable or decreasing BCR-ABL levels had a detectable mutation, P less than .0001. Thus, a more than 2-fold rise identified 34 (97%) of 35 patients with a mutation. We conclude that a rise in BCR-ABL of more than 2-fold can be used as a primary indicator to test patients for BCR-ABL kinase domain mutations.
Complex microbial communities play a critical role in a wide variety of biological systems in the environment and throughout the human body. Characterization of these communities has historically ...been limited to one or a small number of known genetic markers for species such as 16S rRNA genes. While the advent of inexpensive shotgun sequencing has enabled a more accurate measure of biodiversity than marker typing, short read lengths prevent accurate analysis of related strains within a mixture, as well as consistent characterization of large-scale structural variation that can distinguish highly related strains and significantly impact pathogenicity. To address these issues, we have applied the Nabsys HD-MappingTMplatform to strain-level identification of microbial strains in the context of complex mixtures. HD-Mapping employs electronic detection of tagged single DNA molecules, hundreds of kilobases in length, at a resolution superior to existing mapping approaches. The combination of long read lengths and high information density means that individual HD-Mapping reads tend to be much more specific to the genomes from which they derive than do NGS reads. As a result, differences between closely related strains of the same species become clear with minimal bioinformatics analysis. Here we describe strain-level characterization of the ZymoBIOMICS Microbial Community Standard using Nabsys HD-Mapping. DNA was extracted using a standard solution phase, kit-based isolation procedure. Single-molecule reads derived from the mixture were mapped to the NCBI database of all ~10,500 completed bacterial references, including ~1,700 references for species present in the mixture. Through analysis of unique read mapping characteristics, the correct reference was identified for each of the 8 bacterial strains present in the mixture as well as relative strain quantitation.
Bacterial genetic diversity is often described solely using base-pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplication/amplifications are ...thought to be widespread among bacteria but due to their often-intractable size and instability, comprehensive studies of these mutations are rare. We define a methodology to investigate amplifications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with
, whose insertion sequence element-rich genome provides extensive scope for amplifications to occur. Analysis of data for 2430
isolates identified 272 putative amplifications, of which 94 % were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of amplifications, suggesting unstable and sporadic characteristics. Genome instability was further described
using long-read sequencing via the Nanopore platform, which revealed that clonally derived laboratory cultures produced heterogenous populations rapidly. We extended this research to analyse a population of 1000 isolates of another important pathogen,
. We found 590 amplifications in
, and like
, these occurred primarily at hotspots. Genes amplified in
include those involved in motility and respiration, whilst in
, functions included intracellular growth and regulation of virulence. Using publicly available short-read data we predicted previously unrecognized, large amplifications in
and
. This reveals the unrecognized and dynamic genetic diversity of
and
, highlighting the need for a more holistic understanding of bacterial genetics.
The value of administering sequential courses of chemotherapy containing high-dose cytarabine in both induction and consolidation therapy for acute myeloid leukemia (AML) has not been assessed in a ...prospective randomized trial. Two hundred ninety-two AML patients aged 15 to 60 years were enrolled in the Australasian Leukaemia and Lymphoma Group (ALLG) AML trial number 7 (M7) protocol to evaluate this question. All received induction therapy with the ICE protocol (idarubicin 9 mg/m2 × 3; cytarabine 3 g/m2 twice a day on days 1, 3, 5, 7; etoposide 75 mg/m2 × 7). Complete remission was achieved in 234 (80%) patients. Two hundred two patients in remission were then randomized to either a further identical cycle of ICE or 2 attenuated courses (cytarabine 100 mg/m2 daily × 5, idarubicin × 2, etoposide × 5 IcE). ICE consolidation therapy was more toxic than IcE, however, the treatment-related death rate was not significantly different. There was no difference between the 2 consolidation arms for relapse-free survival at 3 years (49% for ICE vs 46% for IcE; P = .66), survival following randomization (61% vs 62%; P = .91), or the cumulative incidence of relapse (43% vs 51%; P = .31), and there was no difference within cytogenetic risk groups. Intensive induction chemotherapy incorporating high-dose cytarabine results in high complete remission rates, but further intensive consolidation treatment does not appear to confer additional benefit.
Abstract 2599
FLT3-ITD is a major risk factor for relapse and poor clinical outcome in AML. Markedly elevated levels of FLT3 ligand (FLT3L) occur after intensive chemotherapy and in patients with ...relapsed AML. In addition, elevated circulating FLT3L in relapsed/refractory FLT3-ITD+ AML has been proposed to limit the response to FLT3 inhibitors (Sato T, et al. Blood 2011; 3286). Novel agents including hypomethylating agents and mTOR inhibitors are being investigated as salvage options in AML but their impact on circulating FLT3 ligand is unknown.
To investigate the effect of azacitidine in combination with mTOR inhibitors on FLT3 ligand levels in relation to clinical outcome in relapsed and refractory AML.
A phase Ib/II open label dose escalation study using azacitidine 75 mg/m2 sc daily on days 1–5 and 8–9 of each 28-day cycle with 2.5, 5 or 10 mg everolimus orally on days 5–21. Serum was sampled at baseline and on days 5, 12 19 and 25 of cycle 1 and FLT3 ligand measured quantitated by ELISA.
37 patients, median age 65 years (range 17–78), with relapsed (73%) or refractory (27%) AML, after failing 1 (n=16), 2 (n=13) or 3 (n=8) previous lines of chemotherapy received azacitidine in combination with 2.5mg (n=6), 5mg (n=12) or 10mg (n=19) everolimus. Poor risk karyotype was present in 10/34 (29%) and FLT3-ITD in 4/16 (25%) of those evaluable. Clinical response was 32% (2 CR, 10 PR). At a median follow up of 252 days, median OS is 211 days (194d in primary refractory and 211d in relapsed AML) and median PFS 178 days. 3/5 patients treated for relapsed AML after allo-SCT had clinical responses and remain alive at 245, 252 and 525 days. In comparison to the typically large increase in FLT3L in a patient given intensive HiDAC-based induction chemotherapy (Figure 1), only 4/26 patients given azacitidine + everolimus had FLT3L levels above 1000 pg/ml within the first month of therapy (Figure 2). Furthermore, patients A, B and C (Figure 2) achieved CR, PR or had SD on therapy, suggesting that elevated FLT3L was unlikely to affect the clinical response to this treatment regimen. Finally, of 4 patients with FLT3-ITD+ AML failing prior intensive chemotherapy, absolute changes in the number of bone marrow blasts in those given azacitidine + everolimus were −80%, −85%, +5% and +7%. Display omitted
Azacitidine in combination with the mTOR inhibitor everolimus has notable activity in chemoresistant AML, including those with FLT3-ITD and does not trigger clinically significant changes in circulating FLT3L that may impact on the efficacy of other therapeutic options.
Wei:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Schwarer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria; Hospira: Membership on an entity's Board of Directors or advisory committees. Patil:Celgene: Research Funding.
Abstract 3301
Although the demethylating agent azacitidine has an established role in myelodysplastic syndromes and encouraging activity in oligoblastic acute myeloid leukemia (AML), information ...regarding its role in relapsed and refractory AML is still emerging. The French ATU reported an overall response rate (ORR; CR/CRi+PR) in relapsed and refractory AML of 11% (Itzykson et al. ASH 2009 #1054). In a similar population, azacitidine salvage therapy produced a CR/CRi rate of 19% (Ayari S, et al. ASH 2009 #2044). Rapamycin, an inhibitor of the AKT downstream target mammalian Target Of Rapamycin (mTOR), is reported to specifically target leukemic stem cells and orally bioavailable rapamycin derivatives, such as everolimus (RAD001), are in active clinical development. Clinical responses with single agent everolimus in relapsed, refractory AML, however, have been modest (Yee et al, Clin Cancer Research 2006 and Boehm et al, European Journal of Internal Medicine 2009).
Building on our experience combining everolimus with low dose cytarabine (submitted to ASH, 2010), we have sought to investigate the feasibility and preliminary efficacy of combining everolimus with azacitidine in relapsed and refractory AML.
Phase Ib/II open label dose escalation study. Patients were treated with azacitidine 75 mg/m2 s.c. daily on days 1–5 and 8–9 of each 28-day cycle with either 2.5, 5 or 10 mg everolimus orally on days 5–21 for a maximum of 12 cycles.
This preliminary analysis includes 20 patients (M 14, F 6), median age 64 years (range 17–76) receiving 2.5 mg (n=6), 5 mg (n=12) or 10 mg (n=2) everolimus. 9 (45%) had chemotherapy refractory and 11 (55%) relapsed AML after 1 (n=8), 2 (n=10) or 3 (n=2) previous lines of therapy. 7/17 (41%) had poor risk and 10/17 (59%) intermediate risk cytogenetics. 6/19 (32%) had secondary AML. The overall response rate (ORR) in 14 evaluable patients was 36% (2 CR, 3 PR). Stable disease (SD) was observed in 7 (50%) patients and 2 (14%) had progressive disease. Absolute bone marrow blast reductions from baseline in the 5 responders ranged from 9 to 88% (Figure 1). Grade 3/4 non-hematologic toxicities are summarized as follows: 2.5 mg everolimus cohort- septicemia (n=1) and mucositis (n=1, dose limiting toxicity; DLT), 5 mg everolimus cohort- septic arthritis (n=1, DLT). Febrile neutropenia during the first cycle of therapy was reported in 5/20 (25%). Safety analysis in the 10 mg everolimus cohort is ongoing. With a median follow up of 82 days, 30 day mortality was 0%. Enrolment continues to a planned 40 patients. Of interest, 2 out of 3 patients with FLT3-ITD+ AML refractory to high-dose cytarabine and antracyclines, had a striking reduction in bone marrow blasts after commencing azacitidine + everolimus (2.5 mg) therapy, with the absolute blast count falling from 95% to 16% and 92% to 5%, respectively, and lasting for at least 5 months in both. One of these patients has so far proceeded to allogeneic stem cell transplant (allo-SCT). Another patient with 3rd relapse of AML after failing allo-SCT, achieved CR after 3 cycles of treatment with azacitidine + everolimus (2.5 mg) and remains in CR after 157 days.
Display omitted
In relapsed and refractory AML, azacitidine in combination with the mTOR inhibitor everolimus was well tolerated and demonstrates substantial clinical activity in this advanced AML population. Further evaluation of this promising combination is ongoing.
Wei:Novartis: Advisory board, Research Funding; Celgene: Research Funding. Off Label Use: AML therapy. Catalano:Celgene: Research Funding; Roche: Honoraria, Research Funding, Travel Grants.
The vast majority of acute promyelocytic leukemia (APL) cases are characterized by the formation of a PML/RARA fusion gene. Disruptions of retinoic acid receptor α (RARα) function have also been ...described in four types of variant APL in which an alternative partner gene (PLZF, NPM, NUMA, or STAT5B) is fused to RARA. We describe a novel variant APL with a RARA fusion formed by a complex gene rearrangement which is undetectable by conventional cytogenetics. A 66 yr old male with a history of mild thrombocytopenia was diagnosed with APL based on the blood and marrow morphology, the coagulopathy, and a microspeckled PML immunofluorescence pattern. The bone marrow immunophenotype was negative for CD2, CD19, CD34, CD56, CD117 and HLA-DR, and with weak expression of CD13, CD33 and CD11b, a pattern atypical for APL. The diagnostic bone marrow karyotype was 47,XY,+225/46,XY30 with no t(15;17)(q22;q21). FISH with the Vysis LSI PML/RARA dual fusion translocation probe did not show any fusion signals but there was splitting of an RARA signal on one 17q. A second probe, the Vysis LSI RARA break apart probe, showed deletion of the 5′ RARA probe and the 3′ RARA probe appeared to localize more distally than normal. The Cytocell PML/RARA ES probe also showed no fusion signals but one RARA signal appeared smaller. The diagnostic marrow was negative for PML/RARA transcripts by RT-PCR using PML and RARA specific primers, but an atypical product was observed. Sequencing of this product showed partial homology to the PRKAR1A gene that maps to 17q24 and encodes the regulatory subunit type I-alpha (RIα) of cyclic AMP-dependent protein kinase A. RT-PCR using PRKAR1A and RARA specific primers amplified two transcript splice variants of a PRKAR1A/RARA fusion gene. The shorter out-of-frame fusion transcript lacked PRKAR1A exon 3 and encoded a carboxy-truncated RIα protein. The longer in-frame fusion transcript resulted from cryptic splicing of the first 100 bases of PRKAR1A exon 3 to RARA exon 3, and encoded a chimeric RIα-RARα fusion protein that contained the dimerization domain of RIα and the same carboxy terminal domains of RARα that are found in all other known RARA rearrangements in APL. FISH using a BAC probe (RP11–120M18) encompassing the PRKAR1A gene identified signals on both copies of 17q; a strong signal on the normal 17 and a weaker signal on der(17). Before cytogenetic, FISH and molecular results were available, the patient was registered on the Australasian Leukaemia and Lymphoma Group's APML4 treatment protocol which includes ATRA, age-adjusted idarubicin and arsenic trioxide. Arsenic was ceased on day 22 due to toxicity. Morphological and cytogenetic FISH complete remission was documented on day 35. A bone marrow biopsy eleven months from original diagnosis showed no evidence of leukemia and PRKAR1A/RARA RT-PCR was indicative of molecular remission. This novel PRKAR1A/RARA gene rearrangement identified in a variant APL is the fifth variant APL in which the RARA partner gene has been identified and the second known rearrangement of PRKAR1A in a malignant disease.
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