Prognostic impact of CEBPA mutational subgroups in adult AML.

Despite recent refinements in the diagnostic and prognostic assessment of CEBPA mutations in AML, several questions remain open, i.e. implications of different types of basic region leucin zipper (bZIP) mutations, the role of co-mutations and the allelic state. Using pooled primary data analysis on 1010 CEBPA-mutant adult AML patients, a comparison was performed taking into account the type of mutation (bZIP: either typical in-frame insertion/deletion (InDel) mutations (bZIPInDel), frameshift InDel or nonsense mutations inducing translational stop (bZIPSTOP) or single base-pair missense alterations (bZIPms), and transcription activation domain (TAD) mutations) and the allelic state (single (smCEBPA) vs. double mutant (dmCEBPA)). Only bZIPInDel patients had significantly higher rates of complete remission and longer relapse free and overall survival (OS) compared with all other CEBPA-mutant subgroups. Moreover, co-mutations in bZIPInDel patients (e.g. GATA2, FLT3, WT1 as well as ELN2022 adverse risk aberrations) had no independent impact on OS, whereas in non-bZIPInDel patients, grouping according to ELN2022 recommendations added significant prognostic information. In conclusion, these results demonstrate bZIPInDel mutations to be the major independent determinant of outcome in CEBPA-mutant AML, thereby refining current classifications according to WHO (including all dmCEBPA and smCEBPA bZIP) as well as ELN2022 and ICC recommendations (including CEBPA bZIPms).

Sex-associated differences in frequencies and prognostic impact of recurrent genetic alterations in adult acute myeloid leukemia (Alliance, AMLCG).

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients' sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3-ITD, DNMT3A, NPM1 and WT1 mutations and less often complex karyotype, ASXL1, SRSF2, U2AF1, RUNX1, or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients' sex in analyses of AML biology and prognostication.

Validation and refinement of the 2022 European LeukemiaNet genetic risk stratification of acute myeloid leukemia.

The revised 2022 European LeukemiaNet (ELN) AML risk stratification system requires validation in large, homogeneously treated cohorts. We studied 1118 newly diagnosed AML patients (median age, 58 years; range, 18-86 years) who received cytarabine-based induction chemotherapy between 1999 and 2012 and compared ELN-2022 to the previous ELN-2017 risk classification. Key findings were validated in a cohort of 1160 mostly younger patients. ELN-2022 reclassified 15% of patients, 3% into more favorable, and 12% into more adverse risk groups. This was mainly driven by patients reclassified from intermediate- to adverse-risk based on additional myelodysplasia-related mutations being included as adverse-risk markers. These patients (n = 79) had significantly better outcomes than patients with other adverse-risk genotypes (5-year OS, 26% vs. 12%) and resembled the remaining intermediate-risk group. Overall, time-dependent ROC curves and Harrel's C-index controlling for age, sex, and AML type (de novo vs. sAML/tAML) show slightly worse prognostic discrimination of ELN-2022 compared to ELN-2017 for OS. Further refinement of ELN-2022 without including additional genetic markers is possible, in particular by recognizing TP53-mutated patients with complex karyotypes as "very adverse". In summary, the ELN-2022 risk classification identifies a larger group of adverse-risk patients at the cost of slightly reduced prognostic accuracy compared to ELN-2017.

A clinically applicable gene expression-based score predicts resistance to induction treatment in acute myeloid leukemia.

Prediction of resistant disease at initial diagnosis of acute myeloid leukemia (AML) can be achieved with high accuracy using cytogenetic data and 29 gene expression markers (Predictive Score 29 Medical Research Council; PS29MRC). Our aim was to establish PS29MRC as a clinically usable assay by using the widely implemented NanoString platform and further validate the classifier in a more recently treated patient cohort. Analyses were performed on 351 patients with newly diagnosed AML intensively treated within the German AML Cooperative Group registry. As a continuous variable, PS29MRC performed best in predicting induction failure in comparison with previously published risk models. The classifier was strongly associated with overall survival. We were able to establish a previously defined cutoff that allows classifier dichotomization (PS29MRCdic). PS29MRCdic significantly identified induction failure with 59% sensitivity, 77% specificity, and 72% overall accuracy (odds ratio, 4.81; P = 4.15 × 10-10). PS29MRCdic was able to improve the European Leukemia Network 2017 (ELN-2017) risk classification within every category. The median overall survival with high PS29MRCdic was 1.8 years compared with 4.3 years for low-risk patients. In multivariate analysis including ELN-2017 and clinical and genetic markers, only age and PS29MRCdic were independent predictors of refractory disease. In patients aged ≥60 years, only PS29MRCdic remained as a significant variable. In summary, we confirmed PS29MRC as a valuable classifier to identify high-risk patients with AML. Risk classification can still be refined beyond ELN-2017, and predictive classifiers might facilitate clinical trials focusing on these high-risk patients with AML.

Double Drop-Off Droplet Digital PCR: A Novel, Versatile Tool for Mutation Screening and Residual Disease Monitoring in Acute Myeloid Leukemia Using Cellular or Cell-Free DNA.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

Validation and refinement of the revised 2017 European LeukemiaNet genetic risk stratification of acute myeloid leukemia.

The revised 2017 European LeukemiaNet (ELN) recommendations for genetic risk stratification of acute myeloid leukemia have been widely adopted, but have not yet been validated in large cohorts of AML patients. We studied 1116 newly diagnosed AML patients (age range, 18-86 years) who had received induction chemotherapy. Among 771 patients not selected by genetics, the ELN-2017 classification re-assigned 26.5% of patients into a more favorable or, more commonly, a more adverse-risk group compared with the ELN-2010 recommendations. Forty percent of the cohort, and 51% of patients ≥60 years, were classified as adverse-risk by ELN-2017. In 599 patients <60 years, estimated 5-year overall survival (OS) was 64% for ELN-2017 favorable, 42% for intermediate-risk and 20% for adverse-risk patients. Among 517 patients aged ≥60 years, corresponding 5-year OS rates were 37, 16, and 6%. Patients with biallelic CEBPA mutations or inv(16) had particularly favorable outcomes, while patients with mutated TP53 and a complex karyotype had especially poor prognosis. DNMT3A mutations associated with inferior OS within each ELN-2017 risk group. Our results validate the prognostic significance of the revised ELN-2017 risk classification in AML patients receiving induction chemotherapy across a broad age range. Further refinement of the ELN-2017 risk classification is possible.

Genetic heterogeneity of cytogenetically normal AML with mutations of CEBPA.

Biallelic mutations of the CCAAT/enhancer binding protein α (CEBPA) gene define a distinct genetic entity of acute myeloid leukemia (AML) with favorable prognosis. The presence of GATA2 and CSF3R mutations that are specifically associated with this subgroup but not mutated in all samples suggests a genetic heterogeneity of biCEBPA-mutated AML. We characterized the mutational landscape of CEBPA-mutated cytogenetically normal AML by targeted amplicon resequencing. We analyzed 48 biallelically mutated CEBPA (biCEBPA), 32 monoallelically mutated CEBPA (moCEBPA), and 287 wild-type CEBPA (wtCEBPA) patient samples from German AML Cooperative Group studies or registry. Targeted sequencing of 42 genes revealed that moCEBPA patients had significantly more additional mutations and additional mutated genes than biCEBPA patients. Within the group of biCEBPA patients, we identified 2 genetic subgroups defined by the presence or absence of mutations in chromatin/DNA modifiers (C), cohesin complex (C), and splicing (S) genes: biCEBPA CCSpos (25/48 [52%]) and biCEBPA CCSneg (23/48 [48%]). Equivalent subgroups were identified in 51 biCEBPA patients from the Cancer Genome Project. Patients in the biCEBPA CCSpos group were significantly older and had poorer overall survival and lower complete remission rates following intensive chemotherapy regimens compared with patients in the biCEBPA CCSneg group. Patients with available remission samples from the biCEBPA CCSpos group cleared the biCEBPA mutations, but most had persisting CCS mutations in complete remission, suggesting the presence of a preleukemic clone. In conclusion, CCS mutations define a distinct biological subgroup of biCEBPA AML that might refine prognostic classification of AML. This trial was registered at www.clinicaltrials.gov as #NCT00266136 and NCT01382147.

Sequential high-dose cytarabine and mitoxantrone (S-HAM) versus standard double induction in acute myeloid leukemia-a phase 3 study.

Dose-dense induction with the S-HAM regimen was compared to standard double induction therapy in adult patients with newly diagnosed acute myeloid leukemia. Patients were centrally randomized (1:1) between S-HAM (2nd chemotherapy cycle starting on day 8 = "dose-dense") and double induction with TAD-HAM or HAM(-HAM) (2nd cycle starting on day 21 = "standard"). 387 evaluable patients were randomly assigned to S-HAM (N = 203) and to standard double induction (N = 184). The primary endpoint overall response rate (ORR) consisting of complete remission (CR) and incomplete remission (CRi) was not significantly different (P = 0.202) between S-HAM (77%) and double induction (72%). The median overall survival was 35 months after S-HAM and 25 months after double induction (P = 0.323). Duration of critical leukopenia was significantly reduced after S-HAM (median 29 days) versus double induction (median 44 days)-P < 0.001. This translated into a significantly shortened duration of hospitalization after S-HAM (median 37 days) as compared to standard induction (median 49 days)-P < 0.001. In conclusion, dose-dense induction therapy with the S-HAM regimen shows favorable trends but no significant differences in ORR and OS compared to standard double induction. S-HAM significantly shortens critical leukopenia and the duration of hospitalization by 2 weeks.

Genetic analysis of adults heterozygous for ALPL mutations.

Hypophosphatasia (HPP) is a rare inherited metabolic bone disease due to a deficiency of the tissue nonspecific alkaline phosphatase isoenzyme (TNSALP) encoded by the ALPL gene. Patients have consistently low serum alkaline phosphatase (AP), so that this parameter is a good hallmark of the disease. Adult HPP is heterogeneous, and some patients present only mild nonpathognomonic symptoms which are also common in the general population such as joint pain, osteomalacia and osteopenia, chondrocalcinosis, arthropathy and musculoskeletal pain. Adult HPP may be recessively or dominantly inherited; the latter case is assumed to be due to the dominant negative effect (DNE) of missense mutations derived from the functional homodimeric structure of TNSALP. However, there is no biological argument excluding the possibility of other causes of dominant HPP. Rheumatologists and endocrinologists are increasingly solicited for patients with low AP and nonpathognomonic symptoms of HPP. Many of these patients are heterozygous for an ALPL mutation and a challenging question is to determine if these symptoms, which are also common in the general population, are attributable to their heterozygous ALPL mutation or not. In an attempt to address this question, we reviewed a cohort of 61 adult patients heterozygous for an ALPL mutation. Mutations were distinguished according to their statistical likelihood to show a DNE. One-half of the patients carried mutations predicted with no DNE and were slightly less severely affected by the age of onset, serum AP activity and history of fractures. We hypothesized that these mutations result in another mechanism of dominance or are recessive alleles. To identify other genetic factors that could trigger the disease phenotype in heterozygotes for potential recessive mutations, we examined the next-generation sequencing results of 32 of these patients for a panel of 12 genes involved in the differential diagnosis of HPP or candidate modifier genes of HPP. The heterozygous genotype G/C of the COL1A2 coding SNP rs42524 c.1645C > G (p.Pro549Ala) was associated with the severity of the phenotype in patients carrying mutations with a DNE whereas the homozygous genotype G/G was over-represented in patients carrying mutations without a DNE, suggesting a possible role of this variant in the disease phenotype. These preliminary results support COL1A2 as a modifier gene of HPP and suggest that a significant proportion of adult heterozygotes for ALPL mutations may have unspecific symptoms not attributable to their heterozygosity.

Combination of t(4;14), del(17p13), del(1p32) and 1q21 gain FISH probes identifies clonal heterogeneity and enhances the detection of adverse cytogenetic profiles in 233 newly diagnosed multiple myeloma.

BACKGROUND: Our aim was to set the FISH combination of del(17p13), t(4;14), 1q21 gain and del(1p32), four adverse cytogenetic factors rarely evaluated together, and compare our technical thresholds with those defined in the literature. METHODS: Two hundred thirty-three patients with MM at diagnosis were studied using FISH to target 4 unfavorable cytogenetic abnormalities: 17p13 deletion, t(4;14) translocation, 1p32 deletion and 1q21 gain. Technical thresholds were determined for each probe using isolated CD138-expressing PC from patients without MM. RESULTS: The FISH analysis identified abnormalities in 79.0% of patients. Del(17p13) was detected in 15.0% of cases, t(4;14) in 11.5%, 1q21 gain in 37.8% and del(1p32) in 8.7%. Adding 1p32/1q21 FISH probes has enabled us to identify adverse cytogenetic profiles in 39.0% of patients without del(17p13) or t(4;14). Clonal heterogeneity was observed in 51.1% of patients as well as an increase in the number of adverse abnormalities when related clones were greater than or equal to 2 (85.1% against 45.6%). CONCLUSION: FISH allowed detecting accumulation of adverse abnormalities and clonal heterogeneity in MM with a combination of 4 probes. The impacts of these two parameters need to be evaluated, and could be included in future cytogenetic classifications.

Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia.

The clinical and prognostic relevance of many recently identified driver gene mutations in adult acute myeloid leukemia (AML) is poorly defined. We sequenced the coding regions or hotspot areas of 68 recurrently mutated genes in a cohort of 664 patients aged 18 to 86 years treated on 2 phase 3 trials of the German AML Cooperative Group (AMLCG). The median number of 4 mutations per patient varied according to cytogenetic subgroup, age, and history of previous hematologic disorder or antineoplastic therapy. We found patterns of significantly comutated driver genes suggesting functional synergism. Conversely, we identified 8 virtually nonoverlapping patient subgroups, jointly comprising 78% of AML patients, that are defined by mutually exclusive genetic alterations. These subgroups, likely representing distinct underlying pathways of leukemogenesis, show widely divergent outcomes. Furthermore, we provide detailed information on associations between gene mutations, clinical patient characteristics, and therapeutic outcomes in this large cohort of uniformly treated AML patients. In multivariate analyses including a comprehensive set of molecular and clinical variables, we identified DNMT3A and RUNX1 mutations as important predictors of shorter overall survival (OS) in AML patients <60 years, and particularly in those with intermediate-risk cytogenetics. NPM1 mutations in the absence of FLT3-ITD, mutated TP53, and biallelic CEBPA mutations were identified as important molecular prognosticators of OS irrespective of patient age. In summary, our study provides a comprehensive overview of the spectrum, clinical associations, and prognostic relevance of recurrent driver gene mutations in a large cohort representing a broad spectrum and age range of intensively treated AML patients.

The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor.

In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor.

Young woman with mild bone marrow dysplasia, GATA2 and ASXL1 mutation treated with allogeneic hematopoietic stem cell transplantation.

Heterozygous mutations in GATA2 underlie different syndromes, previously described as monocytopenia and mycobacterial avium complex infection (MonoMAC); dendritic cell, monocytes, B- and NK lymphocytes deficiency (DCML); lymphedema, deafness and myelodysplasia (Emberger syndrome) and familiar myelodysplastic syndrome/acute myeloid leukemia (MDS / AML). Onset and severity of clinical symptoms vary and preceding cytopenias are not always present. We describe a case of symptomatic DCML deficiency and rather discrete bone marrow findings due to GATA2 mutation. Exome sequencing revealed a somatic ASXL1 mutation and the patient underwent allogeneic stem cell transplantation successfully.

Long-term follow-up of cytogenetically normal CEBPA-mutated AML.

BACKGROUND: The aim of this study was to analyze the long-term survival of AML patients with CEBPA mutations. PATIENTS AND METHODS: We investigated 88 AML patients with a median age of 61 years and (1) cytogenetically normal AML (CN-AML), (2) monoallelic (moCEBPA) or biallelic (biCEBPA) CEBPA mutation, and (3) intensive induction treatment. 60/88 patients have been described previously with a shorter follow-up. RESULTS: Median follow-up time was 9.8 years (95% CI: 9.4-10.1 years) compared to 3.2 and 5.2 years in our former analyses. Patients with biCEBPA mutations survived significantly longer compared to those with moCEBPA (median overall survival (OS) 9.6 years vs. 1.7 years, p = 0.008). Patients ≤ 60 years and biCEBPA mutations showed a favorable prognosis with a 10-year OS rate of 81%. CONCLUSIONS: In this long-term observation we confirmed the favorable prognostic outcome of patients with biCEBPA mutations compared to moCEBPA-mutated CN-AML. The high probability of OS (81%) in younger patients is helpful to guide intensity of postremission therapy.

PTK2 expression and immunochemotherapy outcome in chronic lymphocytic leukemia.

Addition of rituximab (R) to fludarabine and cyclophosphamide (FC) has significantly improved patient outcomes in chronic lymphocytic leukemia (CLL). Whether baseline gene expression can identify patients who will benefit from immunochemotherapy over chemotherapy alone has not been determined. We assessed genome-wide expression of 300 pretreatment specimens from a subset of 552 patients in REACH, a study of FC or R-FC in relapsed CLL. An independent test set was derived from 282 pretreatment specimens from CLL8, a study of FC or R-FC in treatment-naïve patients. Genes specific for benefit from R-FC were determined by assessing treatment-gene interactions in Cox proportional hazards models. REACH patients with higher pretreatment protein tyrosine kinase 2 (PTK2) messenger RNA levels derived greater benefit from R-FC, with significant improvements in progression-free survival, independent of known prognostic factors in a multivariate model. Examination of PTK2 gene expression in CLL8 patients yielded similar results. Furthermore, PTK2 inhibition blunted R-dependent cell death in vitro. This retrospective analysis from 2 independent trials revealed that increased PTK2 expression is associated with improved outcomes for CLL patients treated with R-FC vs FC. PTK2 expression may be a useful biomarker for patient selection in future trials. These trials were registered at www.clinicaltrials.gov as #NCT00090051 (REACH) and #NCT00281918 (CLL8).

Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis.

In acute myeloid leukemia (AML), isolated trisomy 13 (AML+13) is a rare chromosomal abnormality whose prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and performed exome sequencing, targeted candidate gene sequencing and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, P = .006; median OS 9.3 vs. 14.8 months, P = .004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%. Recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogeneous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogeneous subgroup with alterations in a few critical cellular pathways. Clinicaltrials.gov identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.

Molecular response assessment by quantitative real-time polymerase chain reaction after induction therapy in NPM1-mutated patients identifies those at high risk of relapse.

Monitoring minimal residual disease is an important way to identify patients with acute myeloid leukemia at high risk of relapse. In this study we investigated the prognostic potential of minimal residual disease monitoring by quantitative real-time polymerase chain reaction analysis of NPM1 mutations in patients treated in the AMLCG 1999, 2004 and 2008 trials. Minimal residual disease was monitored - in aplasia, after induction therapy, after consolidation therapy, and during follow-up - in 588 samples from 158 patients positive for NPM1 mutations A, B and D (with a sensitivity of 10(-6)). One hundred and twenty-seven patients (80.4%) achieved complete remission after induction therapy and, of these, 56 patients (44.1%) relapsed. At each checkpoint, minimal residual disease cut-offs were calculated. After induction therapy a cut-off NPM1 mutation ratio of 0.01 was associated with a high hazard ratio of 4.26 and the highest sensitivity of 76% for the prediction of relapse. This was reflected in a cumulative incidence of relapse after 2 years of 77.8% for patients with ratios above the cut-off versus 26.4% for those with ratios below the cut-off. In the favorable subgroup according to European LeukemiaNet, the cut-off after induction therapy also separated the cohort into two prognostic groups with a cumulative incidence of relapse of 76% versus 6% after 2 years. Our data demonstrate that in addition to pre-therapeutic factors, the course of minimal residual disease in an individual is an important prognostic factor and could be included in clinical trials for the guidance of post-remission therapy. The trials from which data were obtained were registered at www.clinicaltrials.gov (#NCT01382147, #NCT00266136) and at the European Leukemia Trial Registry (#LN_AMLINT2004_230).