Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5 '-azacitidine (5 '-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5 '-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5 '-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer.

In patients with acute myeloid leukemia (AML), more common aneuploid alterations, such as complex- or monosomal karyotype (CK and MK, respectively) have been intensively studied and are known to confer an adverse prognosis. In contrast, little is known about the prognosis of clonal tetraploidy/near-tetraploidy (T/NT), another rare aneuploidy, defined by presence of 92 chromosomes in tetraploid, and 81-103 chromosomes in near-tetraploid cases. Therefore, we aimed to evaluate prevalence, clinical as well as molecular characteristics and prognostic impact of T/NT in a large cohort of AML patients. We retrospectively assessed 4259 AML patients treated within Study Alliance Leukemia (SAL) trials and/or registered in the SAL AML registry and for whom valid cytogenetic data were available from 1996 until 2020 (the cohort was subsequently enriched by 5 T/NT cases from the AML cooperative group (AMLCG) AML registry). T/NT was defined by presence of ≥ 81 chromosomes in ≥ 10% of metaphases in G-banded karyograms and/or ≥ 4% T/NT cells in interphase fluorescence in-situ hybridization (iFISH). Bone marrow mononuclear cell (BMMC) nuclear area was assessed in patients with available bone marrow smears. In 22 patients with available BMMCs were available, panel next generation sequencing (NGS) and/or whole genome sequencing (WGS) was performed. Student's t test and Kaplan-Meier estimation were used for statistical analyses. We identified 35 patients with T/NT (prevalence 0.82%). Median age was 60.1 years, 34.2% were female. Most patients were diagnosed with de-novo AML (77.2%); secondary AML and therapy-related AML was seen in 20.0% and 2.8%, respectively. Median WBC was 2.9 ×109/L, platelet count was 59 ×109/L and hemoglobin was 6.2 mmol/L; median bone marrow blast count was 64%. As per standard of care molecular studies, there was no evidence of biallelic CEBPA or NPM1 mutations, only one patient harbored an FLT3-ITD mutation; a complex karyotype was observed in 22.9%. In 24 patients considered fit/eligible for intensive chemotherapy (IC) the CR rate was 54.2%, allogeneic hematopoietic cell transplantation (HCT) was performed in 34.2% of patients. As compared to AML patients with diploid karyotype, median BMMC nuclear area was increased (209 µm2 vs. 129 µm2, p = .02). NGS/WGS revealed an accumulation of RUNX1 (15 variants in 11/22 patients), TET2 (15 alterations in 7/22 patients) and SRSF2 (7 variants in 7/22 patients) mutations. Moreover, frequent chromosome 14 aberrations, e.g. del(14q) were observed (5/22 patients). In contrast, only two patients harbored FLT3 mutations and no variants in CEBPA or NPM1 were seen; also, CBF fusions and KMT2A rearrangements were absent (Figure 1A). Median overall survival (OS) of the entire cohort was 9.6 months. Of note, when evaluating patients with sole T/NT vs. patients with T/NT and additional adverse aberrations, median OS in T/NT patients without additional adverse alterations was 26.1 months vs. 5.5 months in patients with T/NT and adverse risk (Figure 1B). Thus, T/NT per se do not seem to confer an adverse prognosis. In summary, we report the first comprehensive evaluation of AML patients with T/NT. T/NT was associated with distinct clinical (male predominance, large BMMC nuclear area) and molecular features (mutations in RUNX1, TET2, SRSF2; del(14)). Most importantly, isolated T/NT was not associated with a poor prognosis; accordingly, we suggest assigning AML patients with T/NT without additional adverse-risk alterations to the intermediate-risk category. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BACKGROUND:Flow cytometry (FCM) is a co-criterion in myelodysplastic syndromes (MDS) diagnostics according to the WHO classification. The presented study compared diagnostic power and prognostic impact of different FCM-based scores. METHODS:A total of 807 bone marrow (BM) samples of patients with cytopenia (543 MDS, 153 non-clonal cytopenias, 111 non-MDS myeloid malignancies) and 78 healthy controls have been investigated using a standardized 8-color-FCM procedure. FCSS, Ogata-score, iFS, RED-score, and ELN-NEC were analyzed for sensitivity and specificity in comparison to standard diagnostic tools. Median follow up for patients was 26 month (range: 0.2-89). RESULTS:The iFS showed the highest accuracy (80%) with the best balance between sensitivity (79%) and specificity (86%). This was also valid in MDS with very low IPSS-R and even in MDS without ring sideroblasts, with normal blast count and karyotype, where iFS could confirm diagnosis in 62% and 65% of patients. Besides the high diagnostic power, the established iFS category "consistent with MDS" was associated with inferior overall survival (OS) independent from WHO classification (median: 51 month vs. not reached, p < 0.0001). Remarkably, this iFS category redefined a subgroup of patients with worse OS within IPSS-R low-risk category (73 month vs. not reached, p = 0.0433). Finally, multivariable analysis showed that iFS added independent prognostic information regarding OS besides IPSS-R. CONCLUSIONS:The iFS separates non-clonal cytopenias and MDS with the highest accuracy, provided information in addition to standard diagnostic procedures, and refined established prognostic tools for outcome prediction.

Precision medicine can significantly improve outcomes for cancer patients, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here we describe somatic biallelic TET2 mutation (focal deletion and nonsense mutation) in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine, but acutely sensitive to 5-azacitidine (5-Aza) hypomethylating monotherapy, resulting in long-term morphological remission (overall survival (OS) 850 days). Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5-Aza compared to cells with monoallelic mutation. We subsequently identified 29 additional patients from the Study Alliance Leukemia biobank with chromosome 4 abnormalities and identified two further patients with complex biallelic TET2 mutations, including one with trisomy 4, homozygosity across the long arm and an inactivating point mutation. We also screened patients recruited to the PETHEMA FLUGAZA phase 3 clinical trial and identified three patients with biallelic TET2 mutations, two of whom had responded very well to single agent 5-Aza (OS 767 and 579 days) despite having adverse risk AML and poor performance status. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for cancer patients.

Abstract Background Lysyl oxidase (LOX) has been described as necessary for premetastatic niche formation in epithelium-derived malignancies and its expression level therefore correlates with risk of metastatic disease and overall survival. However, its role in acute myeloid leukemia (AML) has not been sufficiently analyzed. Methods We investigated LOX plasma expression in 683 AML patients (age 17–60 years) treated within the prospective AML2003 trial (NCT00180102). The optimal cut-off LOX value was determined using a minimal-p-value method dichotomizing patients into a LOX-high group (> 109 ng/mL, n = 272, 40%) and a LOX-low group (≤ 109 ng/mL, n = 411, 60%). Results Higher LOX expression was associated with lower peripheral white blood cells, lower serum LDH, and a lower frequency of FLT3-ITD and NPM1 mutations at diagnosis. Higher LOX expression was found significantly more frequently in patients with secondary AML and therapy-related AML, in patients with French-American-British M5 subtypes, and in patients with adverse-risk cytogenetics. Comparing patients in the LOX-high group and the LOX-low group revealed a 3-year overall survival (OS) of 47 and 53% (p = 0.022) and 3-year event-free survival (EFS) of 27 and 35% (p = 0.005), respectively. In the LOX-high group significantly more patients had extramedullary AML compared to the LOX-low group (p = 0.037). Combining extramedullary AML and LOX as interacting factors in a multivariate analysis resulted in an independent impact on survival for the LOX-high-extramedullary interaction for OS (HR = 2.25, p = 0.025) and EFS (HR = 2.48, p = 0.008). Furthermore, in patients with extramedullary disease (n = 59) the LOX level predicted survival. Patients within the LOX-low group had an OS of 43% and EFS of 36% as compared to the LOX-high group with an OS of 13% and EFS of 6% (p = 0.002 and p = 0.008, respectively). Conclusion We hypothesize LOX expression to be a new potential biomarker to predict outcome in AML, specifically in AML subgroups such as the prognostic heterogeneous group of AML patients with extramedullary disease. Trial registration This retrospective study was performed with patient samples registered within the prospective AML2003 trial ( NCT00180102 ). Patients were enrolled between December 2003 and November 2009.

Personalised medicine is predicted to significantly improve outcomes for cancer patients, but implementation requires comprehensive genetic characterisation of malignant cells to identify therapeutically exploitable vulnerabilities. Using an isogenic cell model system with CRISPR-inactivated TET2 in HEL acute myeloid leukemia (AML) cells and an orthotopic mouse xenograft model we demonstrate that mutant TET2 allele dosage significantly affects sensitivity to 5-azacitidine hypomethylating therapy in AML, with biallelic mutation conferring hypersensitivity relative to monoallelic mutation. In the presence of 5-azacitidine, cell clones with biallelic TET2 mutation had significantly lower cloning efficiency (P = 3 x 10-3) and proliferation in liquid culture (P < 1 x 10-4) compared to isogenic clones with monoallelic TET2 mutation. Mixed populations of monoallelic and biallelicTET2 mutated HEL AML cells were transplanted via intrafemoral injection into Rag2−/−Il2rg−/−129×Balb/c mice, and treatment with 5-azacitidine resulted in significant negative in vivo selection against TET2 null cells relative to cells with monoallelic TET2 mutation (P = 4 x 10-4). Methylation analysis revealed the acquisition of an overall hypermethylation phenotype in TET2 null cells and RNA sequencing identified significant down-regulation of ABCB1 transcript, resulting in concomitant pronounced down-regulation of the MDR1 drug efflux transporter at the protein level. RNA sequencing pathway analysis also identified a global effect on ribosome pathway (KEGG pathway ko03010) transcript levels (Padjusted = 0.002), evidenced by down-regulation of numerous RNA polymerase II components in cells with bi-allelic TET2 mutation compared to cells with monoallelic TET2 mutation. Consistent with our isogenic model data, we characterise biallelic somatic TET2 mutation in a patient with AML that was chemoresistant to anthracycline/cytarabine-based chemotherapy but acutely sensitive to 5-azacitidine, resulting in durable cytomorphological remission. Integration of next generation sequencing, interphase FISH and SNP array analysis of bone marrow at AML presentation, relapse and during remission was used to infer tumour phylogeny which indicated that disease pathogenesis was initiated by a TET2 nonsense mutation (c.2815C>T, Q939*) with subsequent deletion of the second TET2 allele and a NPM1 mutation (c.863_864ins, TCTG) that arose after the acquisition of bi-allelic TET2 mutation. Furthermore, our data demonstrate that 5-azacitidine treatment almost completely eliminated the TET2/NPM1-mutated clone. 5-azacitidine also induced a modest reduction in ancestral pre-leukemic cells carrying bi-allelic TET2 mutation but negative for the NPM1 mutation, although the majority retained viability and re-acquired the ability to differentiate and recapitulate normal haematopoiesis rendering a cytomorphological remission. These observations suggest that bi-allelic TET2 mutation confers sensitivity to the cytotoxic effects of 5-azacitidine, but that the major effect of 5-azacitidine is the induction of phenotypic re-programming. The frequency of TET2 mutation in primary AML is estimated at 10-20%, with the majority of these being monoallelic. We determined the frequency of TET2 alterations in AML patients presenting with a chromosome 4 abnormality discernible cytogenetically. TET2 copy number and mutational status were determined using high density SNP arrays and gene sequencing, respectively. In a panel of 30 AML cases with a chromosome 4 abnormality, four patients were heterozygous for TET2 mutation (all deletions resulting in reduced copy number) and three patients were homozygous for TET2 mutation (deletion plus base substitution in two cases and homozygous base substitution resulting from uniparental disomy in one case). Furthermore, all seven cases with TET2 mutation were characterised by cytogenetics that included loss or gain of material on chromosome 4. In contrast, only 1 case with a TET2 mutation had a translocation affecting chromosome 4. In summary, our data argue in favour of using 5-azacitidine in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of personalised medicine for cancer patients. Disclosures Stoelzel: JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Jackson:Celgene, Amgen, Roche, Janssen, Sanofi: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background The combination of intermediate-dose cytarabine plus mitoxantrone (IMA) can induce high complete remission rates with acceptable toxicity in elderly patients with acute myeloid leukemia (AML). We present the final results of a randomized-controlled trial comparing IMA with the standard 7 + 3 induction regimen consisting of continuous infusion cytarabine plus daunorubicin (DA). Patients and methods Patients with newly diagnosed AML >60 years were randomized to receive either intermediate-dose cytarabine (1000 mg/m2 twice daily on days 1, 3, 5, 7) plus mitoxantrone (10 mg/m2 days 1-3) (IMA) or standard induction therapy with cytarabine (100 mg/m2 continuously days 1-7) plus daunorubicin (45 mg/m2 days 3-5) (DA). Patients in complete remission after DA received intermediate-dose cytarabine plus amsacrine as consolidation treatment, whereas patients after IMA were consolidated with standard-dose cytarabine plus mitoxantrone. Results Between February 2005 and October 2009, 485 patients were randomized; 241 for treatment arm DA and 244 for IMA; 76% of patients were >65 years. The complete response rate after DA was 39% [95% confidence interval (95% CI): 33-45] versus 55% (95% CI: 49-61) after IMA (odds ratio 1.89, P = 0.001). The 6-week early-death rate was 14% in both arms. Relapse-free survival curves were superimposable in the first year, but separated afterwards, resulting in 3-year relapse-free survival rates of 29% versus 14% in the DA versus IMA arms, respectively (P = 0.042). The median overall survival was 10 months in both arms (P = 0.513). Conclusion The dose escalation of cytarabine in induction therapy lead to improved remission rates in the elderly AML patients. This did not translate into a survival advantage, most likely due to differences in consolidation treatment. Thus, effective consolidation strategies need to be further explored. In combination with an effective consolidation strategy, the use of intermediate-dose cytarabine in induction may improve curative treatment for elderly AML patients.

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition characterized by somatic mutations in peripheral blood mononuclear cells (PBMC) of otherwise healthy adults that has been associated with increased risk of developing hematological malignancies. Clonal hematopoiesis has been shown to be present in patients with therapy-related myeloid neoplasms (therapy-related acute myeloid leukemia, t-AML) / therapy-related myelodysplastic syndrome, t-MDS) at the time of their primary cancer diagnosis and before exposure to treatment. Such clones expand under selective pressure from cytotoxic treatment for the primary cancer and can subsequently give rise to overt myeloid neoplasms. Somatic mutations in the gene encoding the TP53-inducible protein phosphatase Mg2+/Mn2+ 1D (PPM1D) were initially reported in PBMC of patients with solid tumors (breast, ovary, lung) and lymphoma. They are associated with older age and seem to reflect prior exposure to cytotoxic treatment. Moreover, the mutations, all of which are nonsense or frameshift mutations in exon 6, have been described as one of the most recurrent mutations in CHIP and to be frequent in t-MDS.

Abstract Background: Flow cytometry (FCM) has recently been recognized as an important supplementary tool in the diagnostic work-up of patients with MDS. Within the international MDS-flow working group of the ELN, different and also novel Flow scores have been evaluated and tested in rather small patients series. Aim: Currently available FCM scores have been prospectively compared concerning their potential diagnostic and prognostic impact in patients with proven or suspected MDS. Patients and Methods: Bone marrow (BM) samples from 616 patients (951 measurements) with proven or suspected MDS, 42 MPN patients, 30/18 MDS patients in cytomorphologic/cytogenetic CR, 22 patients with inappropriate BM aspirations as well as 48 age matched controls have been analyzed by FCM. For measurement and analysis a FACS-CantoII cytometer including FACS-DiVa software was used, performing an 8-color panel and measuring 200,000 events per sample. The following FCM scores were compared: FCSS (granulopoiesis / monopoiesis), Ogata-score (blasts), ELN-red score and Mathis score (both erythropoiesis), and the new iFScore (granulo-, mono-, erythropoiesis, and blasts). Overall survival (OS) was estimated with a median follow up of 21 month (range: 3.4-77 month) applying the Log-Rank-Test within SPSS software. Results: The new iFScore, evaluating granulo-, mono-, erythropoiesis and blasts, turned out to be the most comprehensive score with the best balance between sensitivity and specificity (81%; 78%). FCSS indeed showed a comparably high sensitivity (82%) but has a clearly lower specificity (60%). The Ogata-score and the red-scores showed a lower sensitivity (60%, 31%, 40%) but were very specific (92%, 97%, 89%). Interestingly, despite inappropriate bone marrow aspirations (w/o crumbs) in 22 patients the new iFScore accounted for a "MDS conformable" result in 72% of these cases. Next, the prognostic impact of the different FCM scores was evaluated. Of note, patients with MDS conformable FCM-scores showed a significantly shorter OS compared to patients with scores being not MDS conformable, new iFS, Ogata- and Mathis-score: p = 0.001 (ELN-red: p = 0.003; FCSS: p = 0.005). This also held true evaluating only untreated patients, with Ogata-score exhibiting the highest significance (p < 0.001). Remarkably, even when analyzing the subgroup of MDS-SLD/MLD with normal karyotype and without ringsideroblasts plus non-clonal cytopenias only, a high Ogata score predicted for a significantly lower OS (p = 0.035; median OS = 40 month vs. not reached). Thus, the group of patients with MDS conformable FCM showed significantly higher IPSS-R (p = 0.023) and more mutations (p = 0.0357). When evaluating MDS patients in cytomorphologic or even cytogenetic CR, 23% (17%) showed a MDS conformable iFS with significantly shorter OS (p = 0.025; p = 0.026). Finally, 49% or 35% of patients with MPN showed MDS conformable iFS or Ogata scores, respectively. This translated in a shorter OS in those patients (p = 0.048; p = 0.018). Conclusions: Currently available FCM scores have different sensitivity and specificity in diagnosing MDS, with the novel and comprehensive iFScore, capturing granulo-, mono-, erythropoiesis and blasts together, being superior to other FCM-scores. Additionally, iFS and Ogata score comprise significant prognostic information, even in lower risk MDS. Remarkably, FCM might be a tool to refine response evaluation in MDS and reveal significant dyspoietic features in a subset of MPN patients. Disclosures Platzbecker: Celgene: Research Funding.

INTRODUCTION:Acute myeloid leukemia (AML) rarely involves the central nervous system (CNS). Little is known about the clinical course in adult AML patients since most studies examined pediatric patients. Therefore, this study analyzed the data of patients treated in three prospective trials of the "Study Alliance Leukemia" (SAL) study group for CNS involvement.METHODS:In all, 3,261 AML patients included in the prospective AML96, AML2003, and AML60+ trials of the SAL study group were analyzed. Symptomatic patients underwent cerebrospinal fluid (CSF) puncture and CNS involvement was diagnosed depending on morphology and/or flow cytometry of the CSF. Cytogenetic, molecular, clinical, and laboratory parameters were analyzed in order to identify risk factors.RESULTS:A total of 55 patients had proven symptomatic CNS involvement. Significantly more patients revealed CNS involvement at relapse (34 patients, 2.9%) compared with first diagnosis (21 patients, 0.6%), p<0.001. CNS involvement at initial diagnosis had a significantly higher frequency in patients with complex aberrant karyotypes, high serum lactate dehydrogenase activity, French-American-British M5 subtype, FLT3-internal tandem duplication (ITD) mutations alone, and co-occurrence of a FLT3-ITD and NPM1 mutation. Furthermore, AML patients with CNS involvement at diagnosis had an inferior outcome compared with patients without CNS involvement even if treated with intrathecal chemotherapy with an overall survival of 11% versus 30% at 5 years, p=0.004.CONCLUSION:This study analyzed the largest data set of adult AML patients with proven CNS involvement reported so far. The data demonstrated very low prevalence of CNS involvement at initial diagnosis in adult patients with AML, and described new risk factors. In patients with risk factors, intense diagnostic and treatment strategies should be employed in the future.

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.

Donor cell leukemia: evidence for multiple preleukemic clones and parallel long term clonal evolution in donor and recipient

Recently, we have established a 5-parameter flow cytometry (FCM)-based score allowing for a precise prediction a deletion (5q) in therapy naïve MDS patients. The aim of this prospective study was to test, whether this FCM-based profiling is at least equal to the cytogenetics/FISH-based del(5q) detection for monitoring response to treatment.