Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol.

Prognostic stratification is critical for making therapeutic decisions and maximizing survival of patients with acute myeloid leukemia. Advances in the genomics of acute myeloid leukemia have identified several recurrent gene mutations whose prognostic impact is being deciphered. We used HaloPlex target enrichment and Illumina-based next generation sequencing to study 24 recurrently mutated genes in 42 samples of acute myeloid leukemia with a normal karyotype. Read depth varied between and within genes for the same sample, but was predictable and highly consistent across samples. Consequently, we were able to detect copy number changes, such as an interstitial deletion of BCOR, three MLL partial tandem duplications, and a novel KRAS amplification. With regards to coding mutations, we identified likely oncogenic variants in 41 of 42 samples. NPM1 mutations were the most frequent, followed by FLT3, DNMT3A and TET2. NPM1 and FLT3 indels were reported with good efficiency. We also showed that DNMT3A mutations can persist post-chemotherapy and in 2 cases studied at diagnosis and relapse, we were able to delineate the dynamics of tumor evolution and give insights into order of acquisition of variants. HaloPlex is a quick and reliable target enrichment method that can aid diagnosis and prognostic stratification of acute myeloid leukemia patients.

Ibrutinib inhibits BTK-driven NF-κB p65 activity to overcome bortezomib-resistance in multiple myeloma.

Multiple Myeloma (MM) is a haematologic malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Over the last 10-15 y the introduction of the proteasome-inhibitor bortezomib has improved MM prognosis, however relapse due to bortezomib-resistance is inevitable and the disease, at present, remains incurable. To model bortezomib-resistant MM we generated bortezomib-resistant MM cell lines (n = 4 ) and utilised primary malignant plasma cells from patients relapsing after bortezomib treatment (n = 6 ). We identified enhanced Bruton's tyrosine kinase (BTK) activity in bortezomib-resistant MM cells and found that inhibition of BTK, either pharmacologically with ibrutinib (0.5 µM) or via lenti-viral miRNA-targeted BTK interference, re-sensitized previously bortezomib-resistant MM cells to further bortezomib therapy at a physiologically relevant concentration (5 nM). Further analysis of pro-survival signaling revealed a role for the NF-κB p65 subunit in MM bortezomib-resistance, thus a combination of BTK and NF-κB p65 inhibition, either pharmacologically or via further lenti-viral miRNA NF-κB p65 interference, also restored sensitivity to bortezomib, significantly reducing cell viability (37.5 ± 6 .9 %, ANOVA P ≤ 0 .001). Accordingly, we propose the clinical evaluation of a bortezomib/ibrutinib combination therapy, including in patients resistant to single-agent bortezomib.

Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis.

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age.

Fludarabine and cytosine are less effective than standard ADE chemotherapy in high-risk acute myeloid leukemia, and addition of G-CSF and ATRA are not beneficial: results of the MRC AML-HR randomized trial.

The optimum chemotherapy schedule for reinduction of patients with high-risk acute myeloid leukemia (relapsed, resistant/refractory, or adverse genetic disease) is uncertain. The MRC AML (Medical Research Council Acute Myeloid Leukemia) Working Group designed a trial comparing fludarabine and high-dose cytosine (FLA) with standard chemotherapy comprising cytosine arabinoside, daunorubicin, and etoposide (ADE). Patients were also randomly assigned to receive filgrastim (G-CSF) from day 0 until neutrophil count was greater than 0.5 x 10(9)/L (or for a maximum of 28 days) and all-trans retinoic acid (ATRA) for 90 days. Between 1998 and 2003, 405 patients were entered: 250 were randomly assigned between FLA and ADE; 356 to G-CSF versus no G-CSF; 362 to ATRA versus no ATRA. The complete remission rate was 61% with 4-year disease-free survival of 29%. There were no significant differences in the CR rate, deaths in CR, relapse rate, or DFS between ADE and FLA, although survival at 4 years was worse with FLA (16% versus 27%, P = .05). Neither the addition of ATRA nor G-CSF demonstrated any differences in the CR rate, relapse rate, DFS, or overall survival between the groups. In conclusion these findings indicate that FLA may be inferior to standard chemotherapy in high-risk AML and that the outcome is not improved with the addition of either G-CSF or ATRA.

Long-term outcomes of myeloablation and autologous transplantation of relapsed acute myeloid leukemia in second remission: a British Society of Blood and Marrow Transplantation registry study.

Relapsed acute myeloid leukemia (AML) in adults has a poor prognosis if treated with chemotherapy alone. Case series have previously supported the role of myeloablation and autologous transplantation as a potentially curative treatment. This study aimed to use the large numbers and extended follow-up data in the British Society of Blood and Marrow Transplantation (BSBMT) registry database to establish long-term outcomes and relate these to biological and procedural factors. The BSBMT registry database was used to retrospectively identify 152 adult patients (age, 16-69 years) with AML in second remission treated with autologous transplantation in 1982-2003. Cytogenetic data were available for 68% of the patients; of these, at diagnosis, 42% had good risk features, 57% had standard risk features, and 1% had poor risk features. Conditioning regimens varied; autologous rescue was provided with bone marrow (BM) (71%), peripheral blood stem cells (PBSCs) (18%), or both (11%), which were harvested during first complete remission (CR1) and/or second CR (CR2). Median follow-up was 84 months (range, 2-200 months). At 10 years, actuarial overall survival (OS) was 32%, progression-free survival (PFS) was 28%, and relapse rate (RR) was 57%. The 100-day nonrelapse mortality (NRM) was 7%, rising to 11% at 1 year and to 14% at 10 years. OS was significantly related to M3 subtype (5-year OS, 66%; P = .005), patient age at diagnosis (P = .005) and transplantation (P = .026), and length of CR1, with greatest significance if the patient was dichotomized at CR1 duration of < 8 months or > or = 8 months (P = .0001). There was no difference in OS between regimens containing total body irradiation (TBI) and chemotherapy alone (P = .7). In relation to the nature of autologous graft material, there was improved OS (P = .025) and PFS (P = .009) with the use of cells harvested entirely in CR1 compared with cells harvested in CR2 or in both CR1 and CR2. Engraftment times were significantly shortened with the use of PBSCs alone or in combination with BM compared with BM alone (P = .0001), but there was no significant long-term impact on OS, PFS, RR, or NRM. This study provides long-term follow-up data in one of the largest series of patients with standard-risk and good-risk AML in CR2 treated with autologous transplantation and supports earlier observations that long-term survival is achievable in about 1/3 of patients overall and in about 2/3 of patients with M3 with a relatively low NRM. Outcomes are better in patients with CR1 > or = 8 months by use of grafts obtained entirely in CR1 and use of PBSCs. TBI conditioning did not confer an advantage. Randomized studies against unrelated donor transplantation are warranted.

Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors alpha and beta.

Estrogen has multifunctional effects influencing growth, differentiation, and function in many tissues. High-dose estrogen has been shown to produce anabolic skeletal effects in the skeleton of postmenopausal women with increased megakaryocyte (MK) population in the bone marrow, suggesting a possible role for these cells in bone remodelling. To investigate if estrogen stimulates megakaryocytopoiesis and affects on estrogen receptor (ER) expression, CD34(+) cells were cultured for 6, 9, and 14 days plus or minus low-dose or high-dose 17 beta estradiol (E). Cells were immunolocalised for CD61, CD41, ER alpha and beta. ER mRNA expression was assessed by RT-PCR. Cells formed more CD61 positive MK colonies with low- and high-dose E treatment (P < 0.001) at 6 and 9 days. CD41 expression was increased dose-dependently in MK (3- and 5-fold P < 0.001) at 9 days. E-stimulated ER alpha expression at 6 days (P < 0.001) whilst ER beta was dose-dependently increased only at 9 days (P < 0.01). ER alpha mRNA was increased at 6 days but not at 14 days whilst ER beta mRNA expression was only increased at 14 days with E treatment. These results demonstrate that E stimulates the colony forming potential of CD34(+) cells to a more megakaryocytic phenotype in vitro. This finding together with the stimulation of ER protein and mRNA expression adds to the increasing evidence for a role for MKs in estrogen-induced bone formation.