Background: The International Consensus Classification (ICC) introduced MDS/AML as a novel myeloid disease entity defined by 10-19% blasts in the absence of AML-defining recurrent genetic abnormalities. MDS/AML patients should be eligible for AML-like treatment approaches in clinical trials. It has recently been shown that the AML-based risk classification according to European Leukemia Net (ELN) 2022 criteria fails in MDS/AML while an MDS-based risk classification according to Molecular International Prognostic Scoring System (IPSS-M) was applicable (Huber S et al. EHA 2023). Our aim was to refine the ELN 2022 criteria based on genome sequencing data to better reflect the outcome in this overlap category of MDS and AML. Methods: Non-therapy-related cases of 403 MDS/AML and 686 AML patients classified according to ICC were included. Bone marrow samples were analyzed by cytomorphology, cytogenetics and targeted NGS panel sequencing (median coverage 1500x). Overall survival (OS) was assessed in all AML patients and in a subcohort of 137 MDS/AML patients (median follow-ups: 5.6 and 10.2 years, respectively) in whom also whole genome sequencing (median coverage 100x) was performed. Results: MDS/AML patients had a median age of 74 years (female/male: 153/250). They were subclassified according to ICC as MDS/AML with mutated TP53 (14%), with myelodysplasia-related (MR) gene mutations (67%) or MR cytogenetic abnormalities (5%), or not otherwise specified (14%). Cytogenetic aberrations were detected in 173/403 (43%) cases including del(5q) (18%), -7/del(7q) (13%), and complex karyotypes (16%). Molecular aberrations were detected in 376/403 (93%) cases with a median number of 3 mutations. Most frequent gene mutations were ASXL1 (40%), TET2 (32%), SRSF2 (32%), and RUNX1 (29%); TP53 was mutated in (14%) (details in Figure 1A). Grouping of the MDS/AML cohort according to the ELN 2022 guidelines showed: no MDS/AML patient fulfilled criteria for the favorable ELN risk group, only 14% were classified as intermediate risk, and the vast majority (86%) as adverse risk. The survival of MDS/AML patients substantially differed from a bona fide AML cohort, in particular for adverse risk (Figure 1B left). To more adequately risk stratify MDS/AML patients, we aimed to modify current ELN criteria. Notably, in multivariate Cox regression analysis in addition to -7/del(7q) (hazard ratio/HR: 5.5) and mutated RUNX1 (HR: 2.2), also mutations in EZH2 (HR: 2.2) and in SF3B1 (HR: 0.3) remained significant prognostic markers for OS (all p<0.05). Following this, we defined three hierarchically defined risk groups for MDS/AML patients i) adverse (criteria: complex karyotype, -7/del(7q), mutation in TP53, RUNX1, EZH2), ii) intermediate (criteria: del(5q), mutation in ASXL1, SRSF2, U2AF1, ZRSR2, BCOR or STAG2) and iii) favorable (criteria: sole SF3B1 mutation or none of the abnormalities defined above). Overall, 149 MDS/AML patients (37%) were re-grouped compared to ELN 2022 categories resulting in 49% adverse, 35% intermediate and 16% favorable cases. Survival analysis revealed strong prognostic separations of the three new MDS/AML risk categories with median OS of 1.3 years for adverse, 3.1 years for intermediate and 8.1 years for favorable (overall p<0.001; Figure 1B right). The general survival prediction within MDS/AML was improved in the modified version compared to the original ELN 2022 (corrected Akaike information criterion using Cox models: 876 for modified vs. 902 for ELN 2022). Of note, the outcome of adverse risk MDS/AML according to the modified ELN was comparable to the OS of adverse risk AML according to the original ELN (median OS: 1.3 vs 0.7 years; p=0.304). Conclusion: ForMDS/AML patients, AML-based risk classification according to ELN 2022 guidelines is not applicable as the vast majority of cases are categorized into the adverse risk group based on mutations in splicing factor genes and other secondary-type mutations. Modification of the ELN risk categories reflects the partly MDS-based biology of the disease and substantially improves the prognostic stratification. Most importantly, patients with adverse outcome potentially benefitting from inclusion into clinical trials for novel substances are identified. The validity of the modified risk classification for MDS/AML patients undergoing specific treatment regimens remains to be confirmed in forthcoming studies.

Background: As reported in our preliminary work (Blood (2022) 140 (Supplement 1): 9142-9143) myeloid neoplasms (MN) with MYC-positive double minutes (dmin) are mostly AML and often show a cytomorphological proximity to APL. We here now present detailed genotypical and phenotypical characteristics to address the question if MN with MYC dmin might represent a distinct hematologic entity. Aim: In-depth characterization of cytomorphological, mutational, transcriptional and clinical features of 76 MN with MYC dmin and analysis of the amplified chromosomal region and its effect on gene expression (GE). Methods: We analyzed 60 bone marrow (BM) and 16 peripheral blood (PB) samples of 76 pts with MN and MYC dmin (36 female, 40 male; median age 75 yrs, range 44-89 yrs). The diagnosis was established following WHO guidelines. Dmin and MYC amplification were assessed by chromosome banding analysis and FISH. Cytomorphologic examination included assessment of APL-like features, i.e. high number of atypical hypergranulated promyelocytes, high number of Auer rods, faggot(-like) cells and pseudo Chediak-Higashi (PCH) granules. Molecular genetic analyses comprised a targeted NGS panel (all pts, median coverage 1500x), WGS (16 pts, median coverage 100x) and WTS (40 pts, 50 Mio reads). Mut frequencies and GE levels were compared to AML cases without MYC dmin (“non dmin” AML). Survival data were available for 41 pts (median follow-up: 12 months). Results: According to WHO 2022 most of the 76 cases were AML-MR (myelodysplasia-related) (55/76, 72 %, 9 of these post MDS or post MDS/MPN). Three (4 %) cases each were classified as AML with maturation or without maturation, while one case with AML was not further classifiable due to insufficient material quality and lack of defining markers. Other diagnoses comprised MDS with biallelic TP53 inactivation (4/76, 5 %), MDS-IB1 (1/76, 1 %), CMML-2 (4/76, 5 %), CMML-1 (1/76, 1 %), MDS/MPN (3/76, 4 %) and MPN in blast phase (1/76, 1 %). BM blast count was ≥10 % in 52/60 (87 %) pts, likely underestimated in the remaining 8 samples due to lack of particles, and all 16 pts where only PB was available showed >2 % blasts. Out of 50 pts with fully assessable cytomorphology 48 (96 %) presented with a highly dysplastic granulopoiesis, often including severe dysplasia in other myeloid lineages, independent of myelodysplasia-related genetic markers. Twenty-nine of 50 (58 %) pts were APL-like (≥2 APL-like features), but with a higher degree of maturation than APL. A complex karyotype (ck, ≥3 aberrations in addition to MYC dmin) was present in 25/76 pts (33 %), whereas 21/76 (28 %) pts presented with MYC dmin as the only cytogenetic aberration. TET2 mut, which represented the most frequent mut in MYC dmin pts (55/76, 72 %), often were biallelic events (36/55 pts, 65 %). TET2 mut were strikingly overrepresentated compared to “non dmin” AML (19 %) (p<0.001) (Figure A) and often accompanied by trisomy 4, representing the most frequent chromosomal gain (12/76, 16 %). TP53 mut (24/75, 32 %) and U2AF1 mut (20/75, 27 %) were almost always mutually exclusive (except for 2 pts). While TP53 mut were strongly associated with ck (84 % vs. 6 % in non ck, p<0.001), U2AF1 mut were associated with APL-like cytomorphology (48 % vs. 5 % (1 case) in non APL-like cases, p=0.001). Both mut were highly overrepresented in MYC dmin pts compared to “non dmin” AML ( TP53: 32 % vs. 11 %, p<0.001; U2AF1: 27 % vs. 4 %, p<0.001) (Figure A). WGS analysis revealed an amplified chromosomal region with a size varying from 4.3 - 5.6 Mb and a commonly amplified region of 4.3 Mb (Chr 8:126,422,001-130,697,000). This region encompasses 6 protein coding genes ASAP1, CYRIB, GSMDC, LRATD2, POU5F1B and MYC as well as several non-coding RNAs including the long non-coding RNA PVT1. An effect of the amplified region on GE was confirmed by overexpression of MYC (p<0.001) and PVT1 (p<0.001) compared to “non dmin” AML (Figure B). The median overall survival of 41 evaluable pts was 16 months. Only TP53 mut was independently associated with inferior survival (HR: 11.0, p=0.001). Conclusion: MN with MYC dmin are often AML-MR according to WHO 2022 definition, but even if not, show consistent features of severe dysplasia. They exhibit characteristic mutational patterns and distinct GE profiles, which are affected by a commonly amplified chromosomal region. Thus, we suggest MN with MYC dmin as a distinct genetically defined entity.

Background: MDS/AML has recently been introduced as novel myeloid disease entity by the International Consensus Classification (ICC) with 10–19% blasts in the absence of AML-defining recurrent genetic abnormalities. MDS/AML is not recognized as separate entity according to the in parallel published 5th edition of the WHO classification (WHO 2022) where it largely overlaps with MDS with increased blasts 2. A main argument of the ICC to introduce the MDS/AML category was a potential eligibility of these patients for either MDS or AML clinical trials. Since predefined risk groups are commonly used as inclusion criterion for clinical trials, we asked whether AML- and/or MDS-based risk stratification was appropriate for MDS/AML patients. Aims: Evaluate prognostication in MDS/AML patients using current MDS- (Molecular International Prognostic Scoring System (IPSS-M)) and AML-based (European Leukemia Net (ELN) 2022) risk prediction systems. Methods: Non-therapy-related cases of 137 MDS/AML, 626 MDS (including 116 MDS/AML), and 686 AML patients were included. Bone marrow samples were analyzed by cytomorphology, immunophenotyping, cytogenetics, and whole genome (median coverage 100x) and transcriptome (50 Mio reads) sequencing. Results: MDS/AML patients had a median age of 74 years (female/male: 58/79). They were subclassified as MDS/AML with mutated TP53 (14%), with myelodysplasia-related (MR) gene mutations (72%) or MR cytogenetic abnormalities (4%), or not otherwise specified (10%). We first focused on the MDS-based risk prediction using the recently published IPSS-M. As expected, resulting categories for MDS/AML patients showed a clear skewing towards high risk groups (45% Very High, 29% High, 10% Moderate High, 7% Moderate Low, 9% Low and 0% Very Low) compared to a bona fide MDS cohort (14%, 12%, 7%, 11%, 41% and 15% respectively). However notably, not all MDS/AML patients were classified as very high or high risk. Importantly, we also observed a clear prognostic separation for OS of MDS/AML patients according to the different IPSS-M risk groups (p<0.001), and the OS of the respective groups was well comparable to bona fide MDS cohorts (Figure 1A). The fit of the model reflected by the concordance (c) index was similar for MDS/AML (0.7125), the total MDS cohort (0.7155) and a down-sampled sex-matched MDS cohort (0.7166). Next, we grouped our MDS/AML cohort according to AML-based risk classification using ELN 2022 guidelines. Notably, no MDS/AML patient fulfilled criteria for the favorable ELN risk group per definitionem, only 9% were classified as intermediate risk, and the vast majority (91%) as adverse risk. While within MDS/AML the intermediate risk group showed longer OS than the adverse risk group, the survival of MDS/AML patients substantially differed from a bona fide AML cohort (Figure 1B). In particular, the OS of MDS/AML patients classified as adverse risk according to ELN 2022 was significantly and clinically meaningfully longer compared to corresponding adverse risk AML patients (median OS: 1.9 vs. 0.7 years; p<0.001). Summary/Conclusion: For MDS/AML patients, MDS-based risk assessment according to IPSS-M is fully applicable despite a skewing towards high risk categories. In contrast, AML-based risk classification according to ELN 2022 guidelines is not applicable for MDS/AML. The substantially better survival of MDS/AML patients compared to adverse risk AML despite more intensive therapy in the later and higher age in the former raises concerns about a potential justification of a general inclusion of MDS/AML patients in a clinical trial designed for adverse risk AML.Keywords: ELN

In parallel to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO 2022), an alternative International Consensus Classification (ICC) has been proposed. To evaluate the impact of the new classifications on AML diagnoses and ELN-based risk classification, we analyzed 717 MDS and 734 AML non-therapy-related patients diagnosed according to the revised 4th WHO edition (WHO 2017) by whole genome and transcriptome sequencing. In both new classifications, the purely morphologically defined AML entities decreased from 13% to 5%. Myelodysplasia-related (MR) AML increased from 22% to 28% (WHO 2022) and 26% (ICC). Other genetically-defined AML remained the largest group, and the abandoned AML-RUNX1 was mainly reclassified as AML-MR (WHO 2022: 77%; ICC: 96%). Different inclusion criteria of AML-CEBPA and AML-MR (i.a. exclusion of TP53 mutated cases according to ICC) were associated with differences in overall survival. In conclusion, both classifications focus on more genetics-based definitions with similar basic concepts and a large degree of agreement. The remaining non-comparability (e.g., TP53 mutated AML) needs additional studies to definitely answer open questions on disease categorization in an unbiased way.

Background: B-cell precursor acute lymphocytic leukemia (BCP-ALL) carrying KMT2A::AFF1 rearrangements is an aggressive disease with unsatisfactory response to treatment and poor prognosis. KMT2A is a histone methyl transferase involved in the establishment of epigenetic memory. In the fusion protein encoded by the KMT2A::AFF1 fusion gene, the catalytic SET domain of KMT2A mediating H3K4 trimethylation is lost. Instead, the fusion protein interacts with DOT1L and mediates H3K79 methylation. The subversion of the catalytic activity of KMT2A arrests affected cells in an immature stage of development that may precede V(D)J recombination. Aim: Investigate the immune repertoire of KMT2A::AFF1 rearranged BCP-ALL and evaluate the feasibility of measurable residual disease (MRD) detection using immunoglobulin (Ig) gene rearrangements. Patients and Methods: The cohort includes 290 adult and 89 pediatric BCP-ALL patients. The diagnosis was established following WHO classification. WTS (5x107 reads, 2x101bp) data were generated on HiSeq and NovaSeq instruments. Ig gene rearrangements were analyzed using MiXCR and NCBI-IgBlast. Results: We analyzed Ig heavy-chain variable region (IGHV) gene rearrangements in BCP-ALL samples. We observed that the immune repertoire of leukemic cells carrying KMT2A::AFF1 is less diverse than that of other BCP-ALL samples. We investigated the expression levels of IGHV genes using WTS data. We found that IGHV genes located proximally to the IGHD/IGHJ cluster of genes on chr14 were more likely to be expressed in samples carrying KMT2A::AFF1 (Fig 1A). This difference was particularly significant (p<10-18) for IGHV6-1, the IGHV gene closest to the IGHD/IGHJ cluster (Fig 1B). Proximally biased V(D)J recombination is indicative of incomplete Ig locus contraction. Locus contraction is regulated by PAX5, which suppresses WAPL, a negative regulator of chromatin loop extrusion. Chromatin looping of IGHV genes ensures a balanced immune repertoire by bringing distant IGHV genes in proximity to the V(D)J recombination center. We found that in samples with KMT2A::AFF1 fusion genes WAPL expression levels are significantly (p<10-10) higher than in other subtypes of BCP-ALL. Analysis of the Ig gene rearrangements in KMT2A::AFF1 samples revealed abundant expression of alleles encoding non-productive V(D)J rearrangements or IGHV alleles in germline configuration. Only 35% of complementarity determining region 3 covering reads encoded productive rearrangements in KMT2A::AFF1 samples while in other types of BCP-ALL 67% of reads are productive (p<10-6). In samples with chronic lymphocytic leukemia, a tumor of post-germinal center B cells, this percentage is generally 90% or more. Among the non-productive V(D)J rearranged alleles, in-frame stop codons were equally overrepresented as out-of-frame V-J fusions when compared to their abundance in other types of BCP-ALL (p<10-15 in both cases). As light chain rearrangements require signaling from a productive heavy chain, the significantly (p<9x10-7) lower expression of light chain alleles detectable in KMT2A::AFF1 samples supports the notion that the abundant expression of non-productive alleles is not an artefact. Clonal expansion as measured by the Gini index for the number of reads per clonotype is distributed uniformly between 0 (polyclonality) and 1 (monoclonality). Monoclonality was observed in only 10% of cases. Furthermore, some DJ-rearrangements with identical non-templated linker sequences were found to be recombined with different IGHV genes, indicating ongoing V(D)J recombination and clonal evolution. Conclusions: Our data suggest that the KMT2A::AFF1 rearrangement locks the cell of origin at a point of ongoing V(D)J recombination. V(D)J recombination appears to be induced prematurely in these cells. Poor locus contraction could explain the proximally biased V(D)J recombination pattern. The non-sense mediated decay pathway involved in down regulating the expression of non-productive alleles seems to be poorly functional. Consequently, the cells express unusually high amounts of non-productive alleles or alleles in germline configuration that are clonally evolving and derived frequently from IGHV6-1. These features distinguish KMT2A::AFF1 BCP-ALL from other BCP-ALL subtypes and make MRD detection in KMT2A::AFF1 BCP-ALL based on specific Ig gene rearrangements highly error prone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: The IGHV mutation status (IGHVms) is used for classifying CLL as mutated or unmutated depending on the identity of the expressed IGHV gene to the germline template with 98% identity used as cutoff. CLL patients with unmutated IGHV have a worse prognosis. WGS and WTS are comprehensive techniques identifying all genetic alterations in a single approach. Aim: Evaluate the accuracy of WGS and WTS in determining the IGHVms in a clinical setting. Patients and Methods: The cohort comprised 216 CLL patients. The diagnosis was established following WHO classification. WGS (100x, 2x151bp) and WTS (5x107 reads, 2x101bp) data were generated on NovaSeq instruments. The identification of IGHV rearrangements (IGHVr) using NGS data was performed using IgCaller for WGS data, and MiXCR for WTS data. The tools were used with default settings. For each sample, we used the IGHVr clonotype supported by the largest number of reads for further analyses. IGHVr calls were considered matching when the same IGHVr was called in routine diagnostics based on Sanger sequencing of PCR fragments. IMGT/V-QUEST was used to obtain the IGHVms for sequences obtained from PCR fragments and MiXCR. IgCaller reports its own IGHVms. Results: While MiXCR successfully identified an IGHVr for 98% of samples (211/216), IgCaller made an IGHVr call for only 76% of samples (164/216). In four samples, neither tool could identify an IGHVr. In cases where no IGHVr was identified by MiXCR or IgCaller, routine diagnostics found a mutated IGHV in 5/5 cases and 49/52 cases, respectively. When an IGHVr was detected using NGS data, it was in good agreement with routine data for both tools. MiXCR produced calls concordant with routine data in 92% of samples with mutated IGHV (92/100) and in 98% of samples with unmutated IGHV (109/111). For IgCaller, the percentage of concordant calls was 95% for mutated samples (52/55) and 98% of unmutated samples (106/108). For samples with matching IGHVr calls, we compared the estimates of sequence identity to the germline (Fig.1). The correlation coefficient R2 between the measures provided by IgCaller and routine was 0.74 while the corresponding coefficient for MiXCR was 0.91. Failure to identify an IGHVr was dependent on the IGHVms. The mean percentage of sequence identity to the germline in samples without an IGHVr call made by IgCaller was 92.9% compared to 98.2% in samples where IGHVr calling succeeded (p=3x10-24). The corresponding numbers for MiXCR were 97.0% in samples with IGHVr call and 93.3% in samples without IGHVr call (p=0.03). MiXCR was able to successfully identify IGHVr also in samples with high mutation burden, in which IgCaller showed limitations. IgCaller works with WGS data that, in the present study, have been sequenced at a coverage of 100x. MiXCR utilizes RNASeq data and the number of reads covering the rearranged IGHV genes is one to two orders of magnitude higher. Indeed, down sampling the number of reads utilized by MiXCR to 10% and 1% of all reads reduced the number of samples with IGHVr calls to 189/216 and 132/216, respectively. Taken together, the number of samples where both IGHVms and IGHVr calls were concordant with routine results was 129/216 for IgCaller and 180/216 for MiXCR. In 112/216 samples, all three methods yielded identical results. We found 7 samples with concordant IgCaller and MiXCR calls that nevertheless differed from routine results. These samples showed germline identities close to the 98% cut-off, which led to discordant IGHVms calls between NGS and routine. This observation holds for both tools individually: When the IGHVr call is concordant with routine, discordant IGHVms calls indicate a weakly mutated IGHV. Conclusions: Detection of IGHVms and IGHVr using NGS data is feasible but cannot guarantee results identical to routine diagnostics yet, particularly in CLL patients with mutated IGHV. WTS produced better results than WGS. Down sampling of WTS data to a level of sequence coverage at IGHV loci comparable to WGS data eliminated this difference. High quality NGS data are critical. For weakly mutated IGHV, discordant IGHVms calls are common. Thus, using either tool alone or in combination cannot be recommended for routine diagnostics use. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: Until now, myeloid malignancies are classified according to the revised 4th edition of World Health Organization Classification of Haematolymphoid Tumours, published in 2017 (WHO 2017). During the last years, substantive work has been done in the field of genetics, leading to dynamic changes with respect to defining specific sub-entities. Thus, the up-coming 5th edition of WHO Classification (WHO 2022) emphasizes a genetic basis for defining diseases. Amongst other changes, the blast cut-off between MDS and AML with defining genetic abnormalities (DGA) is largely abandoned. In parallel to the WHO 2022, the International Consensus Classification (ICC) sets the blast cut-off for AML-DGA to 10%, while cases with 10-19% blasts without DGA are assigned as a new category MDS/AML. Aim: Evaluate the impact of the up-coming WHO 2022 guideline on the classification of AML and MDS patients and quantify differences in disease categorization compared to ICC. Methods: 1451 non-therapy-related cases with MDS or AML diagnosed according to WHO 2017 were included. Bone marrow samples were analyzed by cytomorphology, immunophenotyping, cytogenetics, and whole genome (median coverage 100x) and transcriptome (50 Mio reads) sequencing. Results: 746 patients were diagnosed as AML according to WHO 2022. Overall, the group of AML-DGA (excluding MR) remained similar to the WHO 2017 with 65% but changed its composition. Major additional contributors were AML with KMT2A-r (n=44) now including 18 cases (41%) with a different partner gene but MLLT3, AML with MECOM-r (n=64) now including 28 cases (44%) not comprising GATA2, and the newly recognized categories AML with NUP98-r (n=5) and AML with other DGA (n=1, KAT6A::CREBBP). In addition, 8 further cases were classified as AML with mutated NPM1, and 5 cases as AML with mutated CEBPA. In contrast, the now abandoned AML with mutated RUNX1 was mainly re-classified as AML-MR (37/48). AML-MR increased from 22% as defined as AML-MRC by WHO 2017 to 28% in WHO 2022. The largest contributor to this increase were mutations in the defining genes solely leading to classification as AML-MR in 44% (92/210) of cases. Notably, cyto- and molecular genetics without medical history were sufficient for AML-MR classification in all but one patient. Complementary to these findings, the morphologically defined subgroups were substantially reduced from 13% AML-NOS to 5% AML with differentiation. 705 patients were diagnosed as MDS according to WHO 2022. The largest changes included the newly defined MDS-biTP53 which was mainly composed from MDS-EB (30/40; 75%) (Fig. 1A). When considering the main diagnoses in comparison to the WHO 2017, reclassification from MDS to AML according to WHO 2022 was a rare event affecting <1% of cases. In total, 12 MDS samples, 8 of them EB-2, were upstaged to AML based on DGA (MECOM-r: n=5; KMT2A-r: n=1; NPM1: n=6). According to ICC, the cohort would comprise 742 AML, 572 MDS, and 137 MDS/AML cases leading to a reclassification at this level in 10% of cases mainly due to the new ICC category MDS/AML which largely corresponded to MDS-EB2 according to WHO 2017 and to -IB2 and partly -biTP53 according to WHO 2022. In addition, 8 former MDS-EB-2 cases were categorized as AML according to ICC criteria (NPM1: n=4; CEBPA: n=4). The overlap of cases upstaged to AML according to ICC and WHO 2022 were 4/16 patients due to NPM1 mutations and ≥10% blasts (Fig. 1B). Conclusions: The new classifications consistently follow the idea of a more genetics-based definition substantially reducing purely morphologically defined AML, introducing new genetic subgroups, and making a comprehensive genetic analysis mandatory for diagnosis of AML and MDS. Basic concepts of classification are similar between WHO 2022 and ICC. However, differences in the exact diagnostic criteria lead to non-comparable diagnoses in a subset of patients. While this can be explained within the new ICC category MDS/AML, a small number of patients (~ 1%) will be differently classified as AML or MDS based on the phrasing of the definitions. It is beyond the scope of our study to objectively give preference to one classification over the other. Nevertheless, it is predictable that the parallel usage of two different classifications would confuse the diagnostic language for physicians and patients. A unified commonly accepted classification is essential for comparability of diagnostic data in- and outside of clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia associated with severe thrombophilia and characterized by complement-mediated lysis of erythrocytes lacking glycosylphosphatidylinositol (GPI)-anchored proteins. In the majority of cases, GPI deficiency is caused by somatic mutations in the PIGA gene. Presence of PNH clones is associated with acquired aplastic anemia (AA) and can be found in patients with myelodysplastic syndrome (MDS) or rarely other myeloid neoplasms (MN). Flow cytometric analysis for deficiency of GPI-anchored proteins on multiple cell lineages detects PNH clones, and PIGA mutational analysis is not mandatory to establish the diagnosis. In contrast, molecular genetic analysis of targeted gene panels is widely used in the diagnostic workup of MN. We hypothesized that the inclusion of PIGA into the myeloid gene panel could identify obscure cases with PNH clones irrespective of the initial clinical suspicion.

In AML patients, recurrent mutations were shown to persist in remission, however, only some have a prognostic value and persistent mutations might therefore reflect a re-established premalignant state or truly active disease causing relapse. We aimed to dissect the nature of co-mutations in NPM1 mutated AML where the detection of NPM1 transcripts allows highly specific and sensitive detection of complete molecular remission (CMR). We analysed 150 consecutive patients who achieved CMR following intensive treatment by next generation sequencing on paired samples at diagnosis, CMR and relapse (38/150 patients). Patients with persistence or the acquisition of non-DTA ( DNMT3A , TET2 , ASXL1 ) mutations at CMR (23/150 patients, 15%) have a significantly worse prognosis (EFS HR = 2.7, p = 0.003; OS HR = 3.6, p = 0.012). Based on clonal evolution analysis of diagnostic, CMR and relapse samples, we redefine pre-malignant mutations and include IDH1 , IDH2 and SRSF2 with the DTA genes in this newly defined group. Only the persistence or acquisition of CHOP-like (clonal hematopoiesis of oncogenic potential) mutations was significantly associated with an inferior outcome (EFS HR = 4.5, p = 0.0002; OS HR = 5.5, p = 0.002). Moreover, the detection of CHOP-like mutations at relapse was detrimental (HR = 4.5, p = 0.01). We confirmed these findings in a second independent whole genome sequencing cohort.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

According to WHO classification [1], diagnosis of hypereosinophilia still requires a persistent ( 6 months) eosinophil count of at least 1.5 10e9/L in peripheral blood, whereas the 2012 recommendations of an international consensus group [2] have refined criteria demanding eosinophil count of 1.5 10e9/L on two examinations with an interval of at least four weeks, or >20% of eosinophilic bone marrow infiltration. Various benign and malignant conditions can be related to chronic hypereosinophilia [2,3]. While the former particularly include a broad spectrum of reactive disorders, the latter are less frequent and include several kinds of clonal diseases related to hypereosinophilia, with eosinophils being part of the malignant clone particularly in myeloid neoplasms. Malignant conditions include ‘myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2’ (MLN-eo), ‘chronic eosinophilic leukemia, not otherwise specified’ (CEL, NOS), lymphoproliferative disorders (mostly T-cell lymphoma), myeloproliferative neoplasm/MPN-eo, myelodysplastic syndrome/MDS (or MDS/MPN)-eo, systemic mastocytosis or, occasionally, acute leukemia [4]. The remaining cases of hypereosinophilia, in which neither a reactive nor a malignant condition could be confirmed, can be provisionally summarized as ‘hypereosinophilia of undetermined significance (HEUS),’ following the recommendations of the consensus group. ‘HEUS’ is a diagnosis of exclusion, which means that the aforementioned potential causes of hypereosinophilia should have been eliminated upfront, and also demanding the absence of symptoms. Among others bone marrow cytomorphology/histology, flow cytometry, chromosome banding analysis, and fluorescence in situ hybridization or PCR for typical gene rearrangements (e.g. PDGFRA, see above) are essential to exclude definable underlying neoplasms, as required by WHO [1]. Concerning hypereosinophilia cases that are supposed to be HEUS, little is known about causal mutational patterns, possibly indicating clonal hematopoiesis or hematological neoplasm missed by conventional diagnostics. Today, panel sequencing of multiple genes is an established approach to confirm the suspicion of clonal disease in uncertain cases of cytopenia or myeloproliferative disorders and therefore, can also be used to identify clonal disease affecting the eosinophilic lineage. More recent publications reported mutations in JAK2 V617F or KIT D816V in a small percentage of HEUS patients [5], as well as, rare mutations in other genes commonly associated with myeloid neoplasms [6–8]. We defined a cohort of 154 well-characterized HEUS cases who were referred to our laboratory between 2009 and 2017. Patients’ written informed consent regarding genetic analysis and research studies was available, in accordance with the Declaration of Helsinki. The study was approved by our internal institutional review board. In all cases, bone marrow samples were available (and in addition peripheral blood or written information on differential blood count) and conventional cytomorphology, flow cytometry, chromosome banding analysis, and FISH/ PCR for typical gene rearrangements did not reveal any evidence of (B-/T-) lymphatic neoplasm, ‘MLN-eo’, chronic myeloid leukemia/CML or MDS-eo. Then we excluded cases with potential reasons of reactive hypereosinophilia mentioned in available medical reports (such as carcinoma disease, lymphadenopathy, or parasitosis). As a next step, we defined a study cohort of 52 cases of particularly distinct hypereosinophilia, demanding a leukocyte count of at least 10,000/ml and an eosinophil count of at least 30%. Choosing these thresholds, we assured to select cases with an eosinophil count at least twice as high as demanded in WHO classification ( 1.5 10e9/L) for diagnosis of hypereosinophilia: median absolute leukocyte count in our final study cohort was 23,200/ml (range: 10,500–91,600/ml), median eosinophil count 56% (range: 36–80%), median age 65 years (range: 24–84 years). Subsequently, we applied a NGS panel including analysis of 39 genes typically mutated in myeloid neoplasms (Table 1) [9]. DNA from bone marrow was isolated using MagNA Pure 96 (Roche Diagnostics, Mannheim, Germany).

Background: Progression of chronic myeloid leukemia (CML) is frequently accompanied by cytogenetic evolution, with an extra copy of the Philadelphia chromosome, trisomy 8 and 19, and isochromosome (17p) commonly detected. Translocations involving 11q23 chromosomal region have been rarely reported in CML. The few reported patients with blast crisis (BC) of CML carrying an 11q rearrangement have insufficient responses to tyrosine kinase inhibitors (TKIs) and possess a poor prognosis. Case report: We report the case of a 30-year-old man with CML who had a fulminant myeloid BC 4 months after initiation of first-line therapy with the TKI dasatinib, despite showing an optimal response at the 3-month timepoint. Despite cytoreductive therapy with hydroxyurea and 3rd-generation TKI ponatinib, the patient died within 10 days after the diagnosis of BC. Cytogenetic analyses revealed additional genetic aberrations including trisomy 8 and t(9;11)(p21;q23) involving the mixed lineage leukemia (MLL) gene. Conclusion: The presence of 11q23 rearrangements in the relapse clone in BC of CML most likely accounts for the adverse clinical outcome. Thus, in the case of rapid and unexpected BC, the presence of 11q rearrangements should be tested together with other additional chromosomal alterations, and immediate addition of chemotherapy to the TKIs should be evaluated.