Deletion of chromosome 5q occurs in 15%–20% of MDS patients and is associated with favorable prognosis if present as a single aberration or with only one additional cytogenetic aberration. The TP53 mutations, reported in 5%–10% of MDS, are enriched in del(5q) MDS (~20%), therapy-related MDS and MDS with complex karyotype and are associated with high-risk disease, AML transformation, treatment resistance and poor outcome. (1, 2) Recently, Bernard et al. showed that the number of TP53 aberrations is prognostic for death and leukemic transformation. (3) The PPM1D mutations are found in clonal hematopoiesis of indeterminate potential (CHIP) and appear more frequent in therapy-related MDS compared to de novo MDS (15% vs 3%). (4, 5) Activating PPM1D mutations are considered to act similarly to TP53 loss-of-function mutations. Loss of the C-terminal localization domain of PPM1D activates PPM1D and inhibits p53 activation. (6) However, the prevalence of PPM1D mutations, their impact and role in lenalidomide (LEN) resistance and disease progression in MDS with del(5q) still remains unknown. We performed a retrospective analysis of 234 patients ≥18 years old, with WHO 2016 defined del(5q) MDS (n = 175, 74.8%) or other MDS with del(5q) (n = 38, 16.2%) or sAML with del(5q) (n = 21, 9%) (supplementary methods). Patients with del(5q) alone or with one additional chromosomal abnormality except monosomy 7 or del(7q) and a blast count of <5% in bone marrow (BM) and < 1% in peripheral blood (PB) comprised the group of WHO 2016 defined del(5q) MDS. All other cases of MDS with del(5q) and complex karyotype, chromosome 7 abnormalities or blasts >5% in the BM or > 1% in PB were included in the group of other MDS with del(5q). Overall survival information was available for 216 of the 234 patients, specifically for 164 patients with WHO 2016 defined del(5q) MDS, 31 patients with other MDS with del(5q) and 21 patients with sAML with del5(q). There were 65 patients with WHO 2016 defined del(5q) MDS were treated with LEN and had information about treatment response available (Figure S1(A)). Del(5q) was the sole cytogenetic abnormality in 202 patients (86.3%). Ten patients (4.3%) harbored del(5q) and at least one additional chromosomal abnormality and 22 (9.4%) had a complex karyotype. The median age was 72.2 years (range 35–93). As expected, there was a female predominance (72.2%). Forty-four percent of the patients were transfusion-dependent at the time of diagnosis; 68 patients (29%) progressed to AML, and 19 (8.1%) underwent allogeneic hematopoietic cell transplantation (HCT) (Table S1). At time of diagnosis PPM1D mutations were detected in 13 of 234 (5.6%) MDS del(5q) patients, 11 of which had mutations in the hotspot region between amino acids 427 and 542 (Figure S1(B); supplementary methods and Table S2 for sequencing details). The mutation frequency was 6.3% (11 of 175) in patients with WHO 2016 defined del(5q) MDS, 5.3% (2 of 38) in patients with other MDS with del(5q) and 0% (0 of 21) in sAML from MDS with del(5q). One of the 13 PPM1D-mutated patients harbored a trisomy 8 in addition to del(5q), and two had a complex karyotype. Three PPM1D-mutated patients had a TP53 co-mutation (23%), including the two patients with complex karyotype and one with WHO 2016 defined del(5q) MDS. The PPM1D mutations co-occurred with CSNK1A1, SF3B1, ETV6, KIT, ASXL1, TET2 and DNMT3A mutations. Three of the 13 (23%) PPM1D-mutated patients had no additional mutations. Also, TP53 mutations were found in 35 of 234 (15%) patients. Twelve of the 35 (34%) TP53-mutated patients had a complex karyotype. We next investigated the prognostic impact of PPM1D mutations in 164 WHO 2016 defined del(5q) MDS patients (Tables S1 and S3). This cohort included 11 PPM1D-mutated patients, 16 PPM1D-wildtype/TP53-mutated patients and 137 PPM1D-/TP53-wildtype patients. All TP53 mutations were monoallelic in this group (supplementary methods). The PPM1D mutated patients were numerically older compared to PPM1D/TP53 wildtype patients (78.3 vs 71 years, p = .31). After a median follow up of 2.6 years, two of 11 (18.2%) PPM1D-mutated patients transformed to AML. The AML transformation rate was 6.3% for TP53-mutated/PPM1D-wildtype patients and 20.4% for PPM1D-/TP53-wildtype patients (Table S3). None of the 11 PPM1D-mutated patients and one of the 16 PPM1D-wildtype/TP53-mutated patients underwent HCT. The 2-year OS was 100% for PPM1D-mutated patients (n = 11) and PPM1Dwt/TP53mut patients (n = 16), and 85% for PPM1D-/TP53-wildtype patients (n = 137) with WHO 2016 defined del(5q) (Figure 1(A)). For multivariate analysis four variables were considered based on univariate analysis (age, sex, IPSS risk group, PPM1D mutation status). Only age and IPSS risk group were independent predictors of OS (Table S4). We then investigated the prognostic effect of PPM1D mutations in 52 patients with other MDS with del (5q) (n = 31) and sAML (n = 21) with del(5q) (Table S1). Note, PPM1D was mutated in two of 52 patients, both showing a concurrent TP53 mutation and complex karyotype. Thus, we could not evaluate the prognostic effect of PPM1D independently of a complex karyotype and a TP53 mutation. Nine patients had a monoallelic and six patients a biallelic TP53 aberration. Overall survival was shorter for the TP53mut monoallelic ± PPM1Dmut patients (n = 9) and significantly shorter for the TP53mut biallelic ± PPM1Dmut patients (n = 6) compared to TP53-wildtype and PPM1D-wildtype patients (n = 37; 2-y-OS 11% vs 0% vs 53%, respectively, Figure 1(B)). To evaluate the hematologic response to LEN in WHO 2016 defined del(5q) MDS we analyzed 65 LEN treated patients (Tables S1 and S5). Nine of 65 (13.9%) patients were TP53 (n = 5, 7.7%) or PPM1D-mutated (n = 4, 6.2%). Of 65 patients with WHO 2016 defined del(5q) MDS who were treated with LEN, 54 achieved hematologic response (83.1%) and 11 (16.9%) did not. Treatment response was independent of PPM1D (p = .35) (Figure 1(C)) or TP53 (p = .15) mutation status (Figure 1(D)). After a median follow up of 3.1 years, 40 of the 65 (61.5%) LEN treated patients became refractory or progressed to AML (Figure S2(A)). The median time to AML progression was 2.6 years. The rate of LEN resistance or disease progression was independent of the PPM1D (p = .62, Figure S2(B)) or TP53 (p = .38) mutation status (Figure S2(C)). Lastly, we investigated clonal evolution under LEN treatment. Follow-up samples were available after LEN treatment for 22 patients with MDS with del(5q) (19 of 22 with WHO 2016 defined del(5q) MDS) (Table S1), who either achieved a complete remission (n = 5) or developed resistance to LEN, which was followed by MDS progression (n = 7) or AML transformation (n = 10). All samples were screened at diagnosis, time of LEN resistance and/or time of AML transformation by NGS (supplementary methods and Table S6). Of the five patients achieving complete hematological remission four patients displayed no mutations, while one patient was PPM1D-mutated and ASXL1-mutated prior LEN. After 76 months on LEN, the VAF decreased from 27.6% to 4.8% for PPM1D and from 12.1% to 1.1% for ASXL1 in this patient. Of the 17 patients with LEN resistance or MDS/AML progression, two patients (11.8%) carried mutations in PPM1D and three patients (17.6%) in TP53 prior to LEN treatment (p = .64). At the time of LEN resistance or MDS/AML progression, we observed three (17.6%) PPM1D-mutated and eight (47.1%) TP53-mutated patients (p = .03) (Figure 1(E),(F)). The one novel PPM1D and the five novel TP53 mutations were not detected in the diagnostic sample at a median sequencing depth of 2528 reads (range 1393–12583 reads) and a median limit of detection of 0.72% (range 0.56%–1.77%). Two of eight TP53-mutated patients co-expressed PPM1D mutations. The prevalence of PPM1D-mutated and/or TP53-mutated patients increased from 29.4% prior LEN treatment to 52.9% (p = .09) at the time of LEN resistance/progression (Figure 1(G)). At the time of LEN resistance or AML progression, the VAF of PPM1D mutations increased from 10.2% to 23.3% and of TP53 mutations from 5.9% to 23.2% (Figure S2(D),(E)). This corresponds to a 2.5% and 3% increase of the VAF per year in PPM1D-mutated and TP53-mutated patients, respectively. Novel ETV6, RUNX1, WT1, U2AF1, SF3B1 and SRSF2 mutations were observed in patients with LEN resistance or MDS/AML progression (Figure S3(A)–(I)). In summary, we found a 5.6% and 15% prevalence of PPM1D and TP53 mutations prior to LEN treatment, respectively in 234 MDS/sAML patients with del(5q). All patients with WHO 2016 defined del(5q) MDS harbored a TP53 monallelic state. PPM1D and monoallelic TP53 mutations had no prognostic impact in MDS patients with WHO 2016 defined del(5q), while TP53 mutations, especially when biallelic, predicted poor OS in patients with sAML and other MDS with del(5q). Furthermore, neither the hematologic response to LEN nor MDS and AML progression risk was affected by PPM1D and TP53 mutation status in patients with WHO 2016 defined del(5q) MDS, although this analysis is preliminary due to the limited number of patients bearing these mutations. Lastly, we found that LEN resistance and disease progression were associated with the acquisition of novel TP53 and PPM1D mutations and a VAF increase suggesting that hematopoietic clones with these mutations are less inhibited by the selective pressure of LEN than PPM1D and TP53 wildtype clones and therefore expand over time. Future studies need to investigate whether sequential genetic analysis for the detection of clonal evolution is useful to identify patients at risk of adverse outcomes and to choose an appropriate treatment to prevent transformation to AML. We would like to thank all participating patients, contributing doctors and our technicians Blerina Neziri and Martin Wichmann for their excellent support. This work was supported by an ERC grant under the European Union's Horizon 2020 research and innovation program (No. 638035), by grant 70 112 697 from Deutsche Krebshilfe, DFG grants HE 5240/6-1 and HE 5240/6-2 and DJCLS 06 R/2017 from Deutsche José Carreras Stiftung. P.V. was supported by the Austrian Science Fund (FWF) SFB project F4704-B20. Open Access funding enabled and organized by Projekt DEAL. The authors declare no conflict of interest. Written informed consent from patients was obtained according to the Declaration of Helsinki. V.P and M.H designed the research; V.P, M.M., A.K.,R.G., R.S, C.K, P.K, J.S, S.K, M.H. performed the research; M.M, J.K.,A.M, G.G, C.F, C.G, K.S., A.G. C.T., U.G., T.S., G.K, C.K., B.S., N.K, D.H., K.D., W.S., P.V., A.G, F.T., T.H., U.P. contributed patient samples and clinical data; V.P, M.M., R.G, M.H. analyzed the data; V.P and M.H wrote the manuscript. All authors read and agreed to the final version of the manuscript. The study was approved by the review board of Hannover Medical School (ethical vote 5558/2010). All data are available from the corresponding author and in the supplementary data file. Appendix S1: Supporting Information Figure S1 Flow Diagram of analyzed patients and location of PPM1D mutations. (A) Molecular analysis of 234 patients with MDS or sAML and del(5q). Overall survival (OS) information was available for 216 of the 234 patients, i.e. for 164 MDS patients with WHO 2016 defined del(5q), 31 patients with other MDS with del(5q) and 21 patients with sAML with del5(q). Sixty-five patients with WHO 2016 defined del(5q) MDS were treated with lenalidomide (LEN) and had available information about treatment response (LEN cohort). For 22 patients with WHO 2016 defined del(5q) MDS or other MDS with del(5q), follow up (FU) samples after LEN treatment were available and they were included in the follow up cohort. (B) Schematic PPM1D gene structure with localization and frequency of mutations in 234 MDS/sAML patients with del(5q) based on reference PPM1D sequence ENST00000305921.7. Figure S2 Impact of PPM1D and TP53 mutations on the development of lenalidomide resistance or disease progression of patients with WHO 2016 defined del(5q) MDS. (A) Development of LEN resistance or disease progression of 65 patients with WHO 2016 defined del(5q) MDS treated with LEN. (B) Frequency of PPM1D mutated patients who developed LEN resistance and/or progressed under LEN treatment in comparison to PPM1D wildtype patients. (C) Frequency of TP53 mutated patients who developed LEN resistance and/or progressed under LEN treatment in comparison to TP53 wildtype patients. (D,E) Variant allele frequency of PPM1D (D) and TP53 (E) mutations prior LEN treatment and at the time of LEN resistance or AML progression. Figure S3 Clonal evolution under lenalidomide treatment. (A-J) Graphical illustration of the clonal evolution in PPM1D and/or TP53 mutated patients (n = 9) at the time of LEN resistance or disease progression under LEN treatment. Shown are time of diagnosis, time of LEN resistance or AML transformation and the variant allele frequency at each timepoint. Table S1 Comparison of clinical characteristics of MDS/sAML patients with del(5q). Table S2 Cycling conditions and primers for PPM1D sanger sequencing. Table S3 Comparison of clinical and molecular characteristics of 164 MDS patients with WHO 2016 defined del(5q)* and available survival information. Table S4 Univariate and multivariate analysis for OS in 164 MDS patients with WHO 2016 defined del(5q)* and available survival information. Table S5 Comparison of clinical and molecular characteristics of 65 MDS patients with WHO 2016 defined del(5q)* treated with lenalidomide and available response and survival information. Table S6 Genes covered by our custom myeloid panel for NGS analysis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
The utilization of human induced pluripotent stem cells (hiPSCs) for disease modeling and drug discovery is already reality, and several first-in-man-applications as cellular therapeutics have been initiated. Implementation of good manufacturing practice (GMP)-compliant protocols for the generation of hiPSC lines is crucial to increase the application safety as well as to fulfil the legal requirements for clinical trials approval. Here we describe the development of a GMP-compatible protocol for the reprogramming of CD34+ hematopoietic stem cells from peripheral blood (CD34+ PBHSC) into hiPSCs using Sendai virus-based reprogramming vectors. Three GMP-compatible hiPSC (GMP-hiPSC) lines were manufactured and characterized under these conditions.
Abstract Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners ( MNX1 and ETV6 ) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6 . The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking is a more common and overlooked mechanism for structural rearrangement-mediated gene activation in AML. Key points Expression analysis of over 1500 pediatric AML samples demonstrates MNX1 expression as a universal feature of t(7;12)(q36;p13) AML as well as in rare cases without t(7;12)(q36;p13) MNX1 is activated by an enhancer-hijacking event in t(7;12)(q36;p13) AML and not, as previously postulated, by the creation of a MNX1 :: ETV6 oncofusion gene.
The aim of this study was to determine the impact of the revised 5-group International Prognostic Scoring System cytogenetic classification on outcome after allogeneic stem cell transplantation in patients with myelodysplastic syndromes or secondary acute myeloid leukemia who were reported to the European Society for Blood and Marrow Transplantation database. A total of 903 patients had sufficient cytogenetic information available at stem cell transplantation to be classified according to the 5-group classification. Poor and very poor risk according to this classification was an independent predictor of shorter relapse-free survival (hazard ratio 1.40 and 2.14), overall survival (hazard ratio 1.38 and 2.14), and significantly higher cumulative incidence of relapse (hazard ratio 1.64 and 2.76), compared to patients with very good, good or intermediate risk. When comparing the predictive performance of a series of Cox models both for relapse-free survival and for overall survival, a model with simplified 5-group cytogenetics (merging very good, good and intermediate cytogenetics) performed best. Furthermore, monosomal karyotype is an additional negative predictor for outcome within patients of the poor, but not the very poor risk group of the 5-group classification. The revised International Prognostic Scoring System cytogenetic classification allows patients with myelodysplastic syndromes to be separated into three groups with clearly different outcomes after stem cell transplantation. Poor and very poor risk cytogenetics were strong predictors of poor patient outcome. The new cytogenetic classification added value to prediction of patient outcome compared to prediction models using only traditional risk factors or the 3-group International Prognostic Scoring System cytogenetic classification.
Acute myeloid leukemia with complex karyotype (CK-AML) is a distinct biological entity associated with a very poor outcome. Since complex karyotypes frequently contain deletions of the chromosomal region 12p13 encompassing the tumor suppressor genes ETV6 and CDKN1B, we aimed to unravel their modes of inactivation in CK-AML. To decipher deletions, mutations and methylation of ETV6 and CDKN1B, arrayCGH, SNP arrays, direct sequencing of all coding exons and pyrosequencing of the 5′UTR CpG islands of ETV6 and CDKN1B were performed. In total, 39 of 79 patients (49%) showed monoallelic deletions of 12p13 according to karyotypic data and 20 of 43 patients (47%) according to genomic profiling. Genomic profiling led to the minimal deleted region covering the 3′-UTR of ETV6 and CDKN1B. Direct sequencing revealed one novel monoallelic frameshift mutation in ETV6 while no mutations in CDKN1B were identified. Furthermore, methylation levels of ETV6 and CDKN1B did not indicate transcriptional silencing of any of these genes. ETV6 and CDKN1B had reduced expression levels in CK-AML patients with deletion in 12p13 as compared to CK-AML without deletion in 12p13, while the other genes (BCL2L14, LRP6, DUSP16 and GPRC5D) located within the minimal deleted region in 12p13 had very low or missing expression in CK-AML irrespective of their copy number status. ETV6 and CDKN1B are mainly affected by small monoallelic deletions, whereas mutations and hypermethylation play a minor role in CK-AML. Reduced gene dosage led to reduced gene expression levels, pointing to haploinsufficiency as the relevant mechanism of inactivation of ETV6 and CDKN1B in CK-AML.
