Acute myeloid leukemia (AML) is a common hematologic malignancy with a mortality rate of approximately 50% (1, 2). Treatment has improved in recent years with the introduction of new drugs that target specific proteins altered by distinct genetic aberrations. This progress and the increasing knowledge of risk stratification urge for more accurate genetic testing at diagnosis and relapse. Treatment decisions are currently made in line with the risk classification of the European LeukemiaNet (ELN), which is based on disease-specific genetic alterations (3).

In a recent study, we compared standard cytogenetic methods with optical genome mapping (OGM) as a novel diagnostic method for structural genome analysis in AML and myelodysplastic syndrome (MDS) (4). Briefly, ultrahigh–molecular weight (UHMW) DNA is prepared and labeled genome-wide and sequence-specific with a fluorochrome dye. After being loaded on a chip and linearized through nanochannels by electrophoresis, the labeled DNA is captured by a fluorescence microscope and processed by different software algorithms against the human GRCh37/hg19 labeling pattern. Furthermore, different pipelines are used to detect structural variants (SVs) and copy number variants (CNVs). The coverage of this method is about 300×, and the mosaicism detection level currently is about 2% for SVs and 8% for CNVs, with a substantial number of CNVs also covered in the SV pipeline (4). Besides a substantial gain of relevant information by OGM compared to classical karyotyping, here, we detected a DDX3X: MLLT10 gene fusion in a 21-year-old female AML patient. To date, description of DDX3X: MLLT10 in AML is restricted to a few cases in male patients (5). Also, in acute lymphoblastic leukemia (ALL), where the fusion is more commonly involved, it is mainly found in male individuals (6, 7). To the best of our knowledge, this is the first time this aberration is described in a female AML patient. Strikingly, DDX3X is a gene that escapes X-inactivation in women (8). Accordingly, Brandimarte et al. (6) hypothesized that DDX3X translocations might be a leukemic driver due to the lack of a second gene copy in male individuals (6, 9). Apart from that, both genes, DDX3X and MLLT10, separately have been observed to play a role in a whole range of different hematologic and solid malignancies (10–12).

Based on cytomorphology, immunophenotypic analysis, and histopathology, the disease in our patient was classified as CD33+, CD34+, CD38+, CD117+ AML with aberrant CD7 expression. By OGM, different genetic alterations related to T-lineage commitment were unraveled. Interestingly, the disease course appeared to be worse than expected. To improve our understanding of the aberrations and their contribution to leukemogenesis, we further analyzed the bone marrow sample by fluorescence in situ hybridization (FISH), whole-exome sequencing (WES), and consecutive OGM analysis. The purpose of this report is to provide insights into the benefits of a “next-generation diagnostic workup“ for hematologic diseases using two different genome-wide high-resolution technologies and to find out whether its application might result in additional information useful for clinical decision-making.

Genetic standard diagnostics, such as karyotyping and PCR panels, are presented in the Results section and were performed at the University Hospital Knappschaftskrankenhaus Bochum and in collaborating laboratories. The patient gave written informed consent for genetic diagnostics and publication of results.

Acute leukemias have undergone tremendous changes regarding diagnostics and to a lesser extent treatments over the last years. We have recently described OGM as a potential tool to detect SVs on a genome-wide level with sufficient depth and high concordance to currently used methods as classical karyotyping (4). By implementing OGM in our diagnostic algorithm in combination with WES as part of an exemplary next-generation diagnostic workup, we could detect a DDX3X: MLLT10 gene fusion, to the best of our knowledge, for the first time in a female AML patient. Both genes, DDX3X and MLLT10, have been previously described to be involved in hematological and non-hematological neoplasia (10, 11).

In detail, DDX3X has previously been described as a regulator of RNA metabolism (21). Its role as a tumor suppressor and simultaneously as an oncogenic driver is complex and yet to be fully understood (10). The helicase interacts with important key pathways including p53 and kirsten rat sarcoma virus homologue (KRAS) (22–25). Somatic mutations were found in a variety of cancers including medulloblastoma and hematological neoplasia such as acute and chronic lymphocytic leukemia (26, 27). DDX3X appears to be epigenetically silenced in renal cell carcinoma and has been discussed as a potential therapeutic target (28, 29).

Rearrangements involving the MLLT10 gene are recurrent in ALL (more common in T-lineage ALL) and less often in AML (typically pediatric) (12, 30, 31). Regardless of the partner gene, MLLT10 rearrangements appear to be associated with an adverse outcome in AML (5). MLLT10 is involved in histone modification by regulating the function of the histone methyltransferase DOT1L, the only methyltransferase known to methylate H3K79. In MLLT10-rearranged leukemia, DOT1L induces the transcription of genes involved in cell cycle progression (especially genes in the Homeobox A Cluster) (32–34). This might in the future have clinical implications, as a recent phase I trial with pinometostat, as a specific inhibitor of DOT1L, showed some promising results (35).

As in most ALL cases, reports of DDX3X: MLLT10 fusions in AML are restricted to a few cases in male patients (5, 6). Apart from the DDX3X: MLLT10 gene fusion, OGM could also unravel deletions in ARPP21, NF1, and SUZ12. SUZ12 is part of the polycomb repressive complex 2 (PRC2) that in turn is responsible for the methylation of H3K27 (36, 37). This methylation enables DOT1L with its complex to bind and exert its function as a transcription initiator for H3K79 (34). Thus, the SUZ12 deletion and the DDX3X: MLLT10 gene fusion might act synergistically in histone modification and could explain the aggressive course of disease ( Figure 3 ).

ARPP21 is known to positively regulate PHF6-mRNA. It has a physiological counterpart, miRNA128, which is among others located at the 3′ end of ARPP21 (20). OGM here clearly shows that the deletion does not affect miRNA128 and its intronic polymerase III-dependent open reading frame (19). Altogether, this might lead to a downregulation of PHF6-mRNA, which is a known tumor suppressor (38, 39).

Intriguingly, most of the findings uncovered by our next-generation diagnostic workup have rarely been associated with AML but are more common in early T-lineage ALL. In pediatric T-lineage ALL, DDX3X is the second most common fusion partner for MLLT10 after PICALM (40). Furthermore, SUZ12 and PHF6 have been described in the context of T-ALL, with PHF6 being one of the most frequently mutated or deleted genes in T-lymphoblastic leukemia and less often in AML and other myeloid neoplasia (38, 41, 42). Furthermore, OGM showed deletion SVs in the TCR regions representing clonal V(D)J recombinations. Clonal recombination of TRD and TRB could be indicative of an early stage in T-cell development. Combined with the immunophenotypic findings of CD34 and CD38 as well as lack of CD1a expression, these findings support the hypothesis that the leukemic blasts are in a pro-thymocyte (DN2) state (43, 44).

Lineage attribution today is mostly based on immunophenotyping, and AML and ALL are further classified according to genetic alterations relevant for further prognosis. This in turn leads to treatment protocols that 1) are lineage-specific, 2) are more or less intensive according to the genetic risk stratification, and 3) might include targeted therapies if actionable variants are detected. In the case described here, despite an aberrant CD7 expression in flow cytometry, routine diagnostics including cytology (Auer rods), immunohistology (myeloperoxidase and lysozyme expression), immunophenotyping by flow cytometry ( Figure 1 , Supplementary Figure S1 ), and the mutational profile (FLT3-ITD, CEBPA) led to the diagnosis of an AML. This is in line with the WHO 2016 classification, and even the older criteria of the European Group for the Immunological Classification of Leukemias (EGIL) would not have diagnosed a mixed-phenotype acute leukemia (MPAL) but an AML with aberrant coexpression of lymphatic markers due to the CD7 expression (45, 46). However, although being by far more prevalent in AML, neither Auer rods nor FLT3-ITD or CEBPA variants are exclusive to myeloid differentiation but have been previously described in ALL, especially in early T-cell precursor (ETP)-ALL (47–50).

In clinical practice, commitment to a diagnosis is necessary for defining the optimal treatment. Despite the arguably best available treatment protocol by adding FLT3-targeted therapy to standard “7 + 3” induction, treatment response was unsatisfying, without sufficient blast clearance. However, FLAG-containing regimens with consolidating allogeneic transplantation and sorafenib maintenance treatment in case of activating FLT3 mutations not only are regular salvage approaches in refractory AML but also have been described as a successful therapy in ETP-ALL (51).

The immunophenotypic profile (including immunohistochemistry and flow cytometry) in this case was indicative of a myeloid differentiation ( Figure 1C , Figure 4 ). Treatment with FLAG-Eto as salvage therefore was a straightforward approach. Compared to that, the initial diagnosis of a T-lineage disease not only would have led to a different treatment protocol but also might have changed the approach in the treatment-refractory situation with substances such as nelarabine. Thus, our here proposed broad genomic workup utilizing whole-genome cytogenetics and WES might in the future lead to a more distinct classification of disease and might help to overcome the flaws of immunophenotyping in certain situations. The detection of genetic changes typical for myeloid and early lymphoid differentiation as presented here could hint to a stem cell-like myeloid-lymphoid precursor, as has been very recently proposed by Genescà and Starza (52). This in turn could lead to more personalized approaches in treatment, omitting unwanted toxicities and improving the outcome for the patient (53).

Without OGM as a whole genome-based method with high resolution and deep coverage, the DDX3X: MLLT10 gene fusion and other genetic aberrations not covered by routine diagnostics would have been missed. In this work, FISH, WES, and Sanger sequencing were applied as conformational diagnostics. Notably, the FISH probes used are far from any routine use. Thus, not only does OGM have the potential to improve the detection of novel aberrations but also it renders multiple other methods somewhat uncalled for.

In summary, by implementing OGM as a cytogenomic method, we detected SVs in genes involved in histone modification (DDX3X: MLLT10, SUZ12), offering a functional explanation for aggressiveness of disease in this case. Furthermore, these and other detected variants (ARPP21, TCR variants) in combination with mutated myeloid genes also typical for ETP-ALL clearly hint lineage ambiguity, which was missed by routine diagnostic procedures relying on immunophenotyping for lineage discrimination. In conclusion, a next-generation diagnostic approach as proposed here has the potential to not only detect novel genetic variants but also improve risk stratification and thus individualized treatment.

The data presented in the study are deposited under http://zenodo.org under accession number 6421158 387.

This study was reviewed and approved by Ethik-Kommission der Medizinischen Fakultät der Ruhr-Universität Bochum Gesundheitscampus 33. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

VN-E, MT, WG and DBV prepared the manuscript. FD, TM, VN-E, DBV, RS and KD collected patient data. KL, MT and JL analyzed WES data. TL and SK performed and analyzed FISH experiments. HN, RS and DBV contributed to conception and design of the study. All authors contributed to the article and approved the submitted version.

We thank FORUM of the Faculty of Medicine at the Ruhr-University Bochum for funding MT (medical doctoral thesis program) and VN-E as part of the Female Clinician Scientist program.

Thanks to Iris Over for expert technical assistance and the patient for participating in the study.

DV received speaker’s honoraria from Roche, consultant’s honoraria from Pfizer, Bristol Myers Squibb and Gilead as well as travel support and congress registration fees from Gilead and Celgene.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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