Author Correction: BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.

Impact of trisomy 19 on outcome according to genetic makeup in patients with acute myeloid leukemia.

We retrospectively studied 97 acute myeloid leukemia patients with trisomy 19 (median age at diagnosis 57 years; range, 17- 83 years) treated between 2001 and 2019 within two multicenter study groups. Trisomy 19 occurred alone in ten (10.5%) patients, with additional abnormalities being present in non-complex karyotypes in eight (8%) patients and in complex karyotypes in 79 (82%) patients. Altogether, karyotypes characterized by trisomies only were present in 27 (28%) patients. Data on response and outcome of intensively treated patients were available for 92 cases. The median follow-up was 6.4 years (95% confidence interval [95% CI]: 2.9-9.0 years). The complete remission (CR) rate after induction therapy was 52% (48 patients); the early death rate was 10% (n=9). Notably, patients with trisomy 19 as the sole abnormality had a CR rate of 89%. Allogeneic hematopoietic stem cell transplantation (allo-HCT) was performed in 34 (35%) patients (CR, n=19; active disease, n=15). Five-year relapse-free and overall survival rates were 26% (95% CI: 16-43%) and 20% (95% CI: 13-31%), respectively. Overall survival rates were significantly higher in patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time-dependent covariable on overall survival revealed that trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only was a favorable factor (hazard ratio [HR]=0.47; P=0.021); higher age at diagnosis had an adverse impact (10 years difference; HR=1.29; P=0.002), whereas allo-HCT did not have a beneficial impact (odds ratio=1.45; P=0.21). In our cohort, patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only had a high CR rate and better clinical outcome.

Characteristics and outcome of patients with acute myeloid leukemia and trisomy 4.

We retrospectively studied 125 patients with acute myeloid leukemia and trisomy 4 (median age at diagnosis, 58 years; range, 16-77 years) treated between 2000 and 2019 within a multicenter study. Trisomy 4 was the sole abnormality in 28 (22%) patients and additional abnormalities were present in 97 (78%) patients. Twenty-two (22%) and 15 (15%) of 101 tested patients harbored NPM1 and FLT3-ITD mutations. Two (3%) of 72 tested patients had double CEBPA mutations. Data on response to intensive anthracycline-based induction therapy were available for 119 patients. Complete remission was achieved in 67% (n=80) and the early death rate was 5% (n=6). Notably, patients with trisomy 4 as sole abnormality had a complete remission rate of 89%. Allogeneic hematopoietic cell transplantation was performed in 40 (34%) patients, of whom 19 were transplanted in first complete remission. The median follow-up of the intensively treated cohort was 5.76 years (95% confidence interval [95% CI]: 2.99-7.61 years). The 5-year overall survival and relapse-free survival rates were 30% (95% CI: 22-41%) and 27% (95% CI: 18-41%), respectively. An Andersen-Gill regression model on overall survival revealed that favorable-risk according to the European LeukemiaNet classification (hazard ratio [HR]=0.34; P=0.006) and trisomy 4 as sole abnormality (HR=0.41; P=0.01) were favorable factors, whereas age with a difference of 10 years (HR=1.15; P=0.11), female gender (HR=0.74; P=0.20) and allogeneic hematopoietic cell transplantation (HR=0.64; P=0.14) did not have an significant impact. In our cohort, patients with trisomy 4 as their sole abnormality had a high complete remission rate and favorable clinical outcome. Allogeneic hematopoietic cell transplantation did not seem to improve overall survival.

BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells.

Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Outcome of relapsed or refractory acute B-lymphoblastic leukemia patients and BCR-ABL-positive blast cell crisis of B-lymphoid lineage with extramedullary disease receiving inotuzumab ozogamicin.

Acute lymphoblastic leukemia (ALL) can relapse in the extramedullary compartment, with or without medullary involvement. Response to treatment may be individual. We evaluated response to inotuzumab ozogamicin in 31 patients with relapsed/refractory B-ALL with extramedullary disease. Median age was 31 years (range, 19-81). All patients were heavily pretreated, including allogeneic hematopoietic stem cell transplantation (HSCT; n=18). Overall response rate after two cycles of inotuzumab ozogamicin was 84% (complete remission, 55%; partial remission, 29%; resistant disease, 13%; early death, 3%). The median follow-up was 29 months and median overall survival was 12.8 months. One-year and 2-year overall survival rates were 53% (95% CI: 37-76%) and 18% (95% CI: 8-43%), respectively. Age had no impact on overall survival when assessed as a continuous variable or dichotomized at 60 years. Twelve patients proceeded to allogeneic HSCT (complete remission, n=6; partial remission, n=3; resistant disease, n=3). Prior to allogeneic HSCT, eight patients received two or fewer cycles and four patients received three or four cycles of inotuzumab ozogamicin. Sinusoidal obstruction syndrome was reported in three patients, including one after transplantation. Allogeneic HSCT, evaluated as a time-dependent variable, had no impact on overall survival. Inotuzumab ozogamicin seems to be effective as a debulking strategy in relapsed/refractory ALL with extramedullary disease. However, inotuzumab ozogamicin followed by allogeneic HSCT seems not to be effective in maintaining long-term disease control.

Characteristics and outcome of patients with core-binding factor acute myeloid leukemia and FLT3-ITD: results from an international collaborative study.

The aim of this study was to evaluate the prognostic impact of FLT3-ITD in core-binding factor acute myeloid leukemia (CBFAML) in an international, multicenter survey of 97 patients of whom 52% had t(8;21)(q22;q22) and 48% had inv(16)(p13q22)/t(16;16)(p13;q22). The median age of the patients was 53 years (range, 19-81). Complete remission after anthracycline-based induction (n=86) and non-intensive therapy (n=11) was achieved in 97% and 36% of the patients, respectively. The median follow-up was 4.43 years (95% confidence interval [95% CI]: 3.35-7.39 years). The median survival after intensive and non-intensive treatment was not reached and 0.96 years, respectively. Among intensively treated patients, inv(16) with trisomy 22 (n=11) was associated with a favorable 4-year relapse-free survival rate of 80% (95% CI: 59-100%) as compared to 38% (95% CI: 27-54%; P=0.02) in all other patients with CBFAML/ FLT3-ITD (n=75). Overall, 24 patients underwent allogeneic hematopoietic cell transplantation (HCT), 12 in first complete remission and 12 after relapse. Allogeneic HCT in first complete remission was not beneficial (P=0.60); however, allogeneic HCT seemed to improve median survival in relapsed patients compared to that of patients treated with chemotherapy (not reached vs. 0.6 years, respectively; P=0.002). Excluding patients with inv(16) with trisomy 22, our data indicate that compathe outcome of CBF-AML patients with FLT3-ITD may be inferior to that of patients without FLT3-ITD (based on previously published data), suggesting that prognostically CBF-AML patients with FLT3-ITD should not be classified as favorable-risk. FLT3-inhibitors may improve the outcome of these patients.

The extracellular matrix proteins type I collagen, type III collagen, fibronectin, and laminin 421 stimulate migration of cancer cells.

For metastasis formation, individual cells from a primary tumor must migrate toward other tissues. The aim of this study was to determine if mesenchymal stromal cells (MSCs) from human bone marrow are able to emit signals that induce this migratory activity in cancer cells. We separated the supernatant of MSCs derived from human bone marrow by size-exclusion and ion-exchange chromatography and have subsequently studied the migratory behavior of the prostate cancer cell line PC3 and the breast cancer cell line MDA-MB-231 toward the respective fractions in a transwell migration assay. We identified the extracellular matrix (ECM) proteins type I collagen, type III collagen, fibronectin, and laminin 421 as potential drivers of cancer cell migration. These results could be reproduced using the corresponding isolated or recombinant ECM proteins. Knockdown of the gene encoding beta 1 integrin, an important cell surface receptor for fibronectin, has led to inhibition of cancer cell migration. This supports the hypothesis that beta 1 integrin signaling represents an initial event that leads to metastasis, and that signaling is triggered by binding of integrin heterodimers to ECM molecules. Further characterization of signaling factors and their respective receptors will have implications for anticancer drug development.

Glucose Metabolism and Aging of Hematopoietic Stem and Progenitor Cells.

Comprehensive proteomics studies of human hematopoietic stem and progenitor cells (HSPC) have revealed that aging of the HSPC compartment is characterized by elevated glycolysis. This is in addition to deregulations found in murine transcriptomics studies, such as an increased differentiation bias towards the myeloid lineage, alterations in DNA repair, and a decrease in lymphoid development. The increase in glycolytic enzyme activity is caused by the expansion of a more glycolytic HSPC subset. We therefore developed a method to isolate HSPC into three distinct categories according to their glucose uptake (GU) levels, namely the GUhigh, GUinter and GUlow subsets. Single-cell transcriptomics studies showed that the GUhigh subset is highly enriched for HSPC with a differentiation bias towards myeloid lineages. Gene set enrichment analysis (GSEA) demonstrated that the gene sets for cell cycle arrest, senescence-associated secretory phenotype, and the anti-apoptosis and P53 pathways are significantly upregulated in the GUhigh population. With this series of studies, we have produced a comprehensive proteomics and single-cell transcriptomics atlas of molecular changes in human HSPC upon aging. Although many of the molecular deregulations are similar to those found in mice, there are significant differences. The most unique finding is the association of elevated central carbon metabolism with senescence. Due to the lack of specific markers, the isolation and collection of senescent cells have yet to be developed, especially for human HSPC. The GUhigh subset from the human HSPC compartment possesses all the transcriptome characteristics of senescence. This property may be exploited to accurately enrich, visualize, and trace senescence development in human bone marrow.

Analysis of nonleukemic cellular subcompartments reconstructs clonal evolution of acute myeloid leukemia and identifies therapy-resistant preleukemic clones.

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+ /CD38- /ALDH+ cells for AML with rare-ALDH+ blasts (<1.9% ALDH+ cells) were defined as the nonleukemia compartments. WES identified 62 point-mutations in the leukemia compartment derived from 12 AML-patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point-mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point-mutations that have not yet been described in AML. Some leukemia-specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T-lymphocytes, B-lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow-up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia-specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long-term cure.

Characteristics and outcome of patients with acute myeloid leukaemia and t(8;16)(p11;p13): results from an International Collaborative Study.

In acute myeloid leukaemia (AML) t(8;16)(p11;p13)/MYST3-CREBBP is a very rare abnormality. Previous small series suggested poor outcome. We report on 59 patients with t(8;16) within an international, collaborative study. Median age was 52 (range: 16-75) years. AML was de novo in 58%, therapy-related (t-AML) in 37% and secondary after myelodysplastic syndrome (s-AML) in 5%. Cytogenetics revealed a complex karyotype in 43%. Besides MYST3-CREBBP, whole-genome sequencing on a subset of 10 patients revealed recurrent mutations in ASXL1, BRD3, FLT3, MLH1, POLG, TP53, SAMD4B (n = 3, each), EYS, KRTAP9-1 SPTBN5 (n = 4, each), RUNX1 and TET2 (n = 2, each). Complete remission after intensive chemotherapy was achieved in 84%. Median follow-up was 5·48 years; five-year survival rate was 17%. Patients with s-/t-AML (P = 0·01) and those with complex karyotype (P = 0·04) had an inferior prognosis. Allogeneic haematopoietic cell transplantation (allo-HCT) was performed in 21 (36%) patients, including 15 in first complete remission (CR1). Allo-HCT in CR1 significantly improved survival (P = 0·04); multivariable analysis revealed that allo-HCT in CR1 was effective in de novo AML but not in patients with s-AML/t-AML and less in patients exhibiting a complex karyotype. In summary, outcomes of patients with t(8;16) are dismal with chemotherapy, and may be substantially improved with allo-HCT performed in CR1.

Feasibility and Safety of CD19 Chimeric Antigen Receptor T Cell Treatment for B Cell Lymphoma Relapse after Allogeneic Hematopoietic Stem Cell Transplantation.

Although CD19-directed chimeric antigen receptor (CAR) T cells have been successfully used after a preceding allogeneic stem cell transplant (alloHCT) in patients with acute lymphoblastic leukemia, little is known about the feasibility and outcome of CAR T cell treatment in patients who have been previously allotransplanted for lymphoma. In a single-center retrospective analysis, course and outcome of all allografted patients treated with CD19 CAR constructs for B cell lymphoma between October 2018 and November 2019 were studied. CAR therapy consisted either of a third-generation CAR (HD-CAR-1) or of commercially manufactured axicabtagene ciloleucel (axi-cel; Gilead, Santa Monica, U.S.). Altogether, 10 CAR T cell dosings using recipient leukapheresis products were performed in 8 patients: 4 patients (2 mantle cell lymphoma, 2 chronic lymphocytic leukemia) received 6 dosings with HD-CAR-1 and 4 patients (all with diffuse large B cell lymphoma) received 4 dosings with axi-cel. Overall, 6 of 8 patients (75%) responded. CAR treatment was well tolerated with grade ≥ 3 cytokine release syndrome and neurotoxicity each being observed after 1 of 10 dosings. A single patient had moderate chronic graft-versus-host disease. Of note, 3 of 4 patients who received axi-cel had ongoing grade ≥ 3 cytopenia 3 months postdosing, whereas prolonged cytopenia was not observed in 9 alloHCT-naive patients who received axi-cel during the same time period. In conclusion, CAR T cell treatment from recipient-derived leukapheresis products after a prior alloHCT appears to be feasible, effective, and safe in patients with B cell lymphoma. Protracted cytopenia after axi-cel treatment is a matter of concern and requires further exploration.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Allogeneic hematopoietic cell transplantation improves outcome of adults with t(6;9) acute myeloid leukemia: results from an international collaborative study.

Acute myeloid leukemia (AML) with t(6;9)(p22;q34) is a distinct entity accounting for 1-2% of AML cases. A substantial proportion of these patients have a concomitant FLT3-ITD. While outcomes are dismal with intensive chemotherapy, limited evidence suggests allogeneic hematopoietic cell transplantation (allo-HCT) may improve survival if performed early during first complete remission. We report on a cohort of 178 patients with t(6;9)(p22;q34) within an international, multicenter collaboration. Median age was 46 years (range: 16-76), AML was de novo in 88%, FLT3-ITD was present in 62%, and additional cytogenetic abnormalities in 21%. Complete remission was achieved in 81% (n=144), including 14 patients who received high-dose cytarabine after initial induction failure. With a median follow up of 5.43 years, estimated overall survival at five years was 38% (95%CI: 31-47%). Allo-HCT was performed in 117 (66%) patients, including 89 in first complete remission. Allo-HCT in first complete remission was associated with higher 5-year relapse-free and overall survival as compared to consolidation chemotherapy: 45% (95%CI: 35-59%) and 53% (95%CI: 42-66%) versus 7% (95%CI: 3-19%) and 23% (95%CI: 13-38%), respectively. For patients undergoing allo-HCT, there was no difference in overall survival rates at five years according to whether it was performed in first [53% (95%CI: 42-66%)], or second [58% (95%CI: 31-100%); n=10] complete remission or with active disease/relapse [54% (95%CI: 34-84%); n=18] (P=0.67). Neither FLT3-ITD nor additional chromosomal abnormalities impacted survival. In conclusion, outcomes of t(6;9)(p22;q34) AML are poor with chemotherapy, and can be substantially improved with allo-HCT.

AL amyloidosis patients with low amyloidogenic free light chain levels at first diagnosis have an excellent prognosis.

The difference between involved minus uninvolved serum free light chains (dFLC) has been established as an invaluable hematologic parameter in systemic amyloid light chain (AL) amyloidosis. However, patients with an initial dFLC level <50 mg/L are currently deemed not evaluable for response to therapy. Therefore, we aimed to characterize this subgroup of patients and to define novel hematologic response parameters. We retrospectively analyzed 783 AL patients newly diagnosed at our center between 2002 and 2016. Patients with a dFLC level <50 mg/L showed smaller bone marrow plasmacytosis compared to patients with a dFLC level ≥50 mg/L (7% vs 10%, P < .001), but no significant differences in all analyzed chromosomal aberrations. Cardiac involvement was less frequent (45% vs 80%, P < .001) and less severe (Mayo 2004 stage III: 18% vs 51%, P < .001), whereas kidney involvement was more prevalent (83% vs 53%, P < .001) and proteinuria was higher (7.3 g/L vs 5.0 g/L, P < .001). In multivariate analyses, a dFLC level <50 mg/L appeared to be an independent prognostic factor with respect to overall survival (hazard ratio [HR] = 0.50, P = .003) and renal survival (HR = 0.56, P = .020). Patients with a dFLC level <50 mg/L showed a higher proportion of complete hematologic response after first-line therapy compared to patients with a dFLC level ≥50 mg/L (39% vs 9%, P < .001). Receiver-operating characteristics analysis identified a low-dFLC partial response (dFLC <10 mg/L for patients with a dFLC between 20 and 50 mg/L), which predicted overall and renal survival already at 3 months after the start of therapy. Importantly, a parallel Italian study validated this new hematologic remission parameter. The outcome of prospective clinical trials might be adversely influenced by exclusion of the favorable clinical subgroup with an initial dFLC <50 mg/L. We propose the appreciation of dFLC in hematologic response assessment for all patients with a baseline dFLC >20 mg/L.

Marker chromosomes can arise from chromothripsis and predict adverse prognosis in acute myeloid leukemia.

Metaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non-core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P < .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53 mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features.

Consensus guidelines for the diagnosis and management of patients with classic hairy cell leukemia.

Hairy cell leukemia is an uncommon hematologic malignancy characterized by pancytopenia and marked susceptibility to infection. Tremendous progress in the management of patients with this disease has resulted in high response rates and improved survival, yet relapse and an appropriate approach to re-treatment present continuing areas for research. The disease and its effective treatment are associated with immunosuppression. Because more patients are being treated with alternative programs, comparison of results will require general agreement on definitions of response, relapse, and methods of determining minimal residual disease. The development of internationally accepted, reproducible criteria is of paramount importance in evaluating and comparing clinical trials to provide optimal care. Despite the success achieved in managing these patients, continued participation in available clinical trials in the first-line and particularly in the relapse setting is highly recommended. The Hairy Cell Leukemia Foundation convened an international conference to provide common definitions and structure to guide current management. There is substantial opportunity for continued research in this disease. In addition to the importance of optimizing the prevention and management of the serious risk of infection, organized evaluations of minimal residual disease and treatment at relapse offer ample opportunities for clinical research. Finally, a scholarly evaluation of quality of life in the increasing number of survivors of this now manageable chronic illness merits further study. The development of consensus guidelines for this disease offers a framework for continued enhancement of the outcome for patients.

Protein abundance of AKT and ERK pathway components governs cell type-specific regulation of proliferation.

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro-proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type-specific proliferation. First, cell type-specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate-limiting for faster cycling cells while slower cell cycles are controlled at the G1-S progression. The integrated mathematical model of Epo-driven proliferation explains cell type-specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti-proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Prognostic impact of cytogenetic aberrations in AL amyloidosis patients after high-dose melphalan: a long-term follow-up study.

Cytogenetic aberrations detected by interphase fluorescence in situ hybridization (iFISH) of plasma cells are routinely evaluated as prognostic markers in multiple myeloma. This long-term follow-up study aimed to assess the prognosis of systemic light chain amyloidosis (AL) patients treated with high-dose melphalan (HDM) chemotherapy and autologous stem cell transplantation, depending on iFISH results. Therefore, we analyzed a consecutive cohort of 123 AL patients recruited from 2003 to 2014. HDM was safe, with only 1 of 123 patients dying as a result of treatment-related mortality, and effective, with a complete remission (CR) rate of 34%. Translocation t(11;14) as the most prevalent aberration (59%) led to an improved CR rate after high-dose therapy (41.2% vs 20.0%; P = .02), translating into a prolonged hematologic event-free survival (hemEFS; median, 46.1 vs 28.1 months; P = .05) and a trend for better overall survival (median, not reached vs 93.7 months; P = .07). In multivariate analysis, t(11;14) was confirmed as a favorable prognostic factor regarding hemEFS along with lower values for the difference between involved and uninvolved free light chains. Conversely, deletion 13q14, gain of 1q21, and hyperdiploidy had no significant prognostic impact. The high-risk cytogenetic aberrations t(4;14), t(14;16), and del(17p13) conferred an unfavorable prognosis, although statistical significance was reached only for univariate CR analysis in this small group of 9 patients. Thus, t(11;14) positivity in HDM-treated AL patients conferred superior CR rates and hemEFS. In view of the reduced response of t(11;14) to bortezomib, this highlights the impact of therapy on the prognostic role of cytogenetic aberrations.

BRAF inhibition in hairy cell leukemia with low-dose vemurafenib.

The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care.