KMT2A Rearrangements Research Articles

Outcomes of acute lymphoblastic leukemia with KMT2A (MLL) rearrangement: the MD Anderson experience

AbstractAcute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23)-KMT2A-AFF1 is associated with a poor prognosis. The impact of KMT2A rearrangements other than t(4;11) is uncertain, and the benefit of allogeneic stem cell transplantation (HSCT) is unclear. We reviewed adult patients with ALL treated at our institution from 1984 to 2019 and identified 50 out of 1102 (5%) with KMT2A rearrangement, including 42 (84%) with t(4;11)/KMT2A-AFF1 and 8 (16%) with other gene partners. The median age was 45 years (range, 18-78 years); median white blood cell count was 109.0 3 109/L (range, 0.5-1573.0). The complete remission (CR) rate was 88%, and the rate of measurable residual disease negativity by flow cytometry at CR was 41% (76% overall during follow-up). At the last follow-up, 14 patients were alive. The 5-year overall survival (OS) rate was 18% (95% confidence interval [CI], 9% to 35%), with no difference between t(4;11) and other KMT2A rearrangements (P 5 .87). In a 4-month landmark analysis, the 5-year OS rate was 32% (95% CI, 14% to 70%) in patients who underwent HSCT vs 11% (95% CI, 3-39) in others (P 5 .10). Our study confirms the poor prognosis of ALL with any KMT2A rearrangement and the role of HSCT in these patients.

Predictors of outcomes in adults with acute myeloid leukemia and KMT2A rearrangements

Acute myeloid leukemia (AML) with rearrangement of the lysine methyltransferase 2a gene (KMT2Ar) has adverse outcomes. However, reports on the prognostic impact of various translocations causing KMT2Ar are conflicting. Less is known about associated mutations and their prognostic impact. In a retrospective analysis, we identified 172 adult patients with KMT2Ar AML and compared them to 522 age-matched patients with diploid AML. KMT2Ar AML had fewer mutations, most commonly affecting RAS and FLT3 without significant impact on prognosis, except for patients with ≥2 mutations with lower overall survival (OS). KMT2Ar AML had worse outcomes compared with diploid AML when newly diagnosed and at relapse, especially following second salvage (median OS of 2.4 vs 4.8 months, P < 0.0001). Therapy-related KMT2Ar AML (t-AML) had worse outcomes compared with de novo KMT2Ar AML (median OS of 0.7 years vs 1.4 years, P < 0.0001). Allogeneic hematopoietic stem cell transplant (allo-HSCT) in first remission was associated with improved OS (5-year, 52 vs 14% for no allo-HSCT, P < 0.0001). In a multivariate analysis, translocation subtypes causing KMT2Ar did not predict survival, unlike age and allo-HSCT. In conclusion, KMT2Ar was associated with adverse outcomes regardless of translocation subtype. Therefore, AML risk stratification guidelines should include all KMT2Ar as adverse.

AML-117: The Genetic Landscape of Relapsed Pediatric Acute Myeloid Leukemia

<h3>Context:</h3> The genomic characterization of acute myeloid leukemia (AML) in adults, both at diagnosis and relapse, has been extensively reported. Previous work has shown a clear difference between AML in adults and children, yet the genetic changes associated with relapsed disease in children have yet to be fully described. <h3>Objective:</h3> To elucidate the genetic background of pediatric AML at relapse and identify gene alterations related to poor prognosis. <h3>Design:</h3> Transcriptomic and genetic analyses were performed on relapsed AML from multiple clinical studies held in St. Jude Children's Research Hospital from 2002 to 2020. No blinding was performed in this study. <h3>Patients or Other Participants:</h3> RNA sequencing was performed from 136 patients (median age of 9.2 years) with relapsed pediatric AML. Ninety-one of these samples were also studied by tumor–normal-paired whole-genome sequencing. Additional transcriptomic data from pediatric AML at diagnosis (n=417) were also obtained from previous reports to define the molecular alterations common in relapsed pediatric AML. The clinical outcome of each molecular category is confirmed using clinical data of the diagnosis cohort of the TARGET pediatric AML study. <h3>Results:</h3> Genomic profiling of relapsed pediatric AML demonstrated an increase in KMT2A (n=36, 26.5%) and NUP98 rearrangements (n=18, 13.2%) when compared to <i>de novo</i> pediatric AML, as well as an increase in WT1 mutations (n=33, 24.3%). Comprehensive characterization of pediatric AML with extension RNA transcriptome data revealed 18 molecular categories with unique expression patterns and mutually exclusive gene alteration patterns. These molecular categories included rare categories with structural variants involving oncogenes of MECOM (n=3, 2.2%) or BCL11B (n=2, 1.5%), which led to the upregulation and allele-specific expression (ASE) of each gene. The entire molecular categories showed unique distribution in the relapse cohort compared to the TARGET cohort (p<0.001) and were associated with clinical outcomes. <h3>Conclusions:</h3> Integrated genomic profiling of pediatric AML identified unique molecular categories associated with relapsed disease and poor outcome.

Assessing acute myeloid leukemia susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CCCTC-binding factor/cohesin binding sites.

An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of nonfusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to nonfusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin-binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in nonfusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons.

BTK, NUTM2A, and PRPF19 Are Novel KMT2A Partner Genes in Childhood Acute Leukemia.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with acute leukemias, especially in infants. KMT2A is rearranged with a big variety of partner genes and in multiple breakpoint locations. Detection of all types of KMT2A rearrangements is an essential part of acute leukemia initial diagnostics and follow-up, as it has a strong impact on the patients’ outcome. Due to their high heterogeneity, KMT2A rearrangements are most effectively uncovered by next-generation sequencing (NGS), which, however, requires a thorough prescreening by cytogenetics. Here, we aimed to characterize uncommon KMT2A rearrangements in childhood acute leukemia by conventional karyotyping, FISH, and targeted NGS on both DNA and RNA level with subsequent validation. As a result of this comprehensive approach, three novel KMT2A rearrangements were discovered: ins(X;11)(q26;q13q25)/KMT2A-BTK, t(10;11)(q22;q23.3)/KMT2A-NUTM2A, and inv(11)(q12.2q23.3)/KMT2A-PRPF19. These novel KMT2A-chimeric genes expand our knowledge of the mechanisms of KMT2A-associated leukemogenesis and allow tracing the dynamics of minimal residual disease in the given patients.

Abstract 2289: Empowering point-and-click genomic analysis with large pediatric genomic reference data on St. Jude Cloud

Abstract Next-generation sequencing-based genomic profiling is now a mainstay of pediatric oncology research and clinical testing. Correlating genomic features of patient cancer genomes with curated data extracted from large reference cohorts is critical for identifying molecular subtypes and underlying mutagenesis processes. To facilitate such investigation, we developed two user-friendly workflows on St. Jude Cloud, a data sharing ecosystem hosting genomic data for &amp;gt;10,000 pediatric cancer patients and survivors. These workflows leverage St. Jude Cloud comprehensive pediatric cancer genomic data, including 1,616 RNA-seq of 135 cancer subtypes and 958 whole genome sequencing (WGS) of 35 subtypes, to enable user analysis of their data in the context of St. Jude Cloud cohorts without a need to download large datasets. The RNA-Seq Expression Classification workflow enables a user to compare their patient RNA-Seq gene expression data with blood (832), brain (456), and solid tumor (319) pediatric cancer reference cohorts and PDX models (45), enabling subtype classification using t-Distributed Stochastic Neighbor Embedding (t-SNE). Reference cohorts include curated subtype-defining somatic alterations integrating genomic variant data with expression profile. Resulting interactive t-SNE plots can be explored and annotated - with options to highlight cancer subtypes or samples and display sample information (age of onset, clinical diagnosis, molecular driver). To demonstrate, we analyze PAWNXH, a Children's Oncology Group AML sample with a novel ZBTB7A-NUTM1 fusion and find it clusters with AML samples harboring KMT2A re-arrangements suggesting a potential mechanism of pathogenesis. Integrating PDX samples enables model selection for functional experiments by connecting patient subtypes with mouse models. The Mutational Signatures workflow identifies and quantifies COSMIC mutational signatures in user-uploaded somatic VCF files for comparison to reference pediatric cancer cohorts. The interactive interface enables rapid identification of signatures within the query cohort and facilitates comparison to the reference using a cohort-level summary view. Identified signatures may also be explored at the sample-level for both query and reference cohorts, enabling the user to identify samples with signatures of interest for further analysis. We show an example comparison of mutational signatures identified in pediatric and adult AML samples. These workflows enable users to leverage curated pediatric cancer data to make discoveries in their own samples. Enabling point-and-click analysis in St. Jude Cloud removes the barrier for non-computational researchers and eliminates the need to download large reference datasets for local analysis. Both workflows utilize post-processed rather than raw genomic data, reducing transfer costs for uploading user data to the cloud. Citation Format: Andrew Thrasher, Michael Macias, Alexander M. Gout, Delaram Rahbarinia, Xin Zhou, Samuel W. Brady, Clay McLeod, Michael C. Rusch, Xiaolong Chen, Soheil Meshinchi, Michael A. Dyer, Suzanne J. Baker, Martine F. Roussel, Jinghui Zhang. Empowering point-and-click genomic analysis with large pediatric genomic reference data on St. Jude Cloud [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2289.

Comprehensive Methylation Analysis in Pediatric Patients with Acute Myeloid Leukemia —the JCCG Study, JPLSG AML-05-

IntroductionThe genetic heterogeneity of acute myeloid leukemia (AML) makes it difficult to predict an appropriate prognosis of patients with AML. Currently, several gene mutations associated with the pathogenesis of AML have been identified owing to recent advances in massively parallel sequencing technologies. However, despite incorporating these molecular markers, the prognosis of many patients remains uncertain. Aberrant DNA methylation has frequently been observed in myeloid cells of patients with AML. Reportedly, DNA methylation patterns are associated with molecular features. Furthermore, a previous report has demonstrated that the methylation levels of polycomb target genes are associated with the outcome, implying that the definition of DNA methylation patterns of AML is expected to reveal clinical and molecular features and survival in patients with AML. However, there are few reports of DNA methylation in pediatric patients with AML. Thus, we analyzed genome-wide DNA methylation in pediatric patients with AML to reveal its association with clinical features, genetic alterations, and prognostic impact. Besides, our previous study revealed that the high expression of MECOM (also known as EVI1 ) or PRDM16 (also known as MEL1 ) was associated with inferior outcomes in patients with AML. We also investigated the correlation between DNA methylation and gene expression in pediatric patients with AML.MethodsBetween 2006 and 2010, 443 pediatric patients with de novo AML (0-17 years) participated in the Japanese AML-05 trial conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group. Of these, 369 samples were available, and 64 patients were enrolled in this study. The cytogenetic features of 64 patients were as follows: normal karyotype, 33; RUNX1-RUNX1T1 , 8; KMT2A rearrangement, 10; complex karyotype, 6; and other cytogenetics, 7. This cohort included 15 patients with FLT3 -internal tandem duplication (ITD), 8 with CEBPA biallelic mutations, 10 with high MECOM expression, and 30 with high PRDM16 expression. We performed comprehensive DNA methylation analysis using Infinium MethylationEPIC BeadChip (Illumina, Inc., San Diego, CA, USA) in 64 pediatric patients with AML to reveal the correlations among DNA methylation patterns, clinical features, genetic alterations, and prognostic impact in pediatric AML.Results and DiscussionUnsupervised hierarchical clustering based on methylation values of the 567 CpG sites, which showed most variable methylation values between 64 individuals, generated four clusters (clusters 1-4). The number of patients in clusters 1, 2, 3, and 4 was 23, 12, 13, and 16, respectively. Interestingly, these four clusters harbored their molecular features. Cluster 1 included patients with RUNX1/RUNX1T1 , normal karyotype with low PRDM16 expression, or KMT2A rearrangement with low MECOM expression. Cluster 2 included patients with FLT3 -ITD and/or normal karyotype with high PRDM16 expression. Cluster 3 included patients with normal karyotype with CEBPA biallelic mutations and cluster 4 patients with FLT3 -ITD, normal karyotype with high PRDM 16 expression, or KMT2A rearrangement with high MECOM expression. Differences in frequencies of common molecular aberrations among clusters were assessed with the Fisher-Freeman-Halton test. There was an unequal distribution of FLT3 -ITD and high PRDM16 expression (P = 0.003 for both), with concordantly high frequencies in clusters 2 and 4 and low in clusters 1 and 3. Clusters 2 and 4 tended to be correlated with older age, higher white blood cell counts at diagnosis, high relapse rate, and high mortality. These results suggest that DNA methylation can stratify the risk in pediatric AML. Conversely, no independent methylation marker was present to predict the adverse outcome, thereby indicating that an assortment of several methylation sites may be a useful marker for risk stratification.ConclusionWe investigated DNA methylation patterns in pediatric patients with AML and found four distinct clusters. Furthermore, our findings indicated that quantitative determination of DNA methylation percentage at specific CpG sites may be useful for risk stratification in pediatric AML. Considering the need of further analyses to reveal the significance of DNA methylation, we are analyzing the results currently and would like to discuss these data at the annual meeting. DisclosuresNo relevant conflicts of interest to declare.

Rapid Screening of Targetable Kinase Gene Fusions in Acute Lymphoblastic Leukemia

Background: Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by a similar expression profiling to ALL with BCR-ABL rearrangement, but due to different genomic alterations that activate cytokine receptor and kinase signaling. Diagnose of this type of leukemia is challenging and current diagnostic algorithms include a combination of different techniques, most of them time-consuming and expensive.Here we present a multi-probe Fluorescence In Situ Hybridation (FISH)- Panel based screening test that permit a rapid diagnosis of ALL (including Ph-like ALL).Methods: We analysed 42 adolescent and adult patients (13 - 72 years old) diagnosed with ALL without BCR/ABL and KMT2A rearrangements. We performed a one-slide custom designed FISH panel in parallel with chromosome banding analysis. The panel included probes for ETV6/RUNX1, KMT2A, BCR/ABL1, E2A, CRLF2/P 2RY8, CSF1R, EPOR, PDGFRB, ABL2, JAK2, and IGH probes (Custom chromoprobe multiprobe ALL, Cytocell).Results: There were no hybridation failures. We identified 30 patients with alterations (71%), 20 of them (67%) with normal karyotype or without metaphases. The most common alterations were E2A deletion (8/30, 27%) [3 patients in a context of E2A/PBX rearrangement], hiperdiplid karyotype (6/30, 20%), ABL2 gain (5/30, 17%), CRLF2/IGH rearrangement (4/30, 13%), ETV6 deletion (4/30, 13%), IGH rearrangement (3/30, 10%) and JAK2 rearrangement (2/30, 7%). All together, Ph-like defining abnormalities comprised 20% of cases.Conclusions: We have validated a rapid and easy test to improve ALL subtype diagnosis, including Ph-like ALL. The panel allowed for 24h-diagnosis in cases without or not assessable metaphases. In conclusion, this panel can be a complementary technique for the immediate identification of most common rearrangements in ALL, particullary those with treatment implications. DisclosuresSanz:Gamida Cell: Research Funding. Solano:Neovii: Honoraria.

Evaluation of Cancer Testis Antigens (CTAs) As Immuno-Therapeutic Targets in Pediatric AML

The Cancer Testis Antigen (CTA) superfamily of genes have long emerged as potential targets for immunotherapy and response monitoring due to their relative restrictive expression in testis and variable expression in some cancers. Much of the data on CTA expression in cancer have been in solid tumors with little data on CTA expression in leukemias. Identification of leukemia associated antigens (LAA) may provide attractive targets for immunotherapy or monitoring of residual disease.In order to discover AML associated CTAs, we performed transcriptome sequencing on 446 de novo pediatric AML samples and 20 normal bone marrows (NBM) from healthy donors and interrogated the expression of 245 candidate CTAs obtained from the CT database (http://www.cta.lncc.br). Differential expression using Limma (https://bioconductor.org/packages/release/bioc/html/limma.html) identified 32 CTA genes to be significantly differentially expressed with 7 being up-regulated compared to NBM and 15 genes with expression in NBM which are silenced in AML. The upregulated genes include: CCNA1, PRAME , CT45A5, IGF2BP3, SPAG6, TEX14, and ZNF165 and are collectively expressed in 330 of 446 patients (75%) with the highest contributors being PRAME and CCNA1 (Fig. 1A, Fig. 1B) . These 2 CTAs are expressed in every fusion group and constitute 65% of the total cohort, with a notable enrichment in core binding factor (CBF) patients (80%). High CCNA1 expression are present in 2/3 of patients with Inv(16), 71% of patients with a NUP98 fusion, 88% of DEK - NUP214 carriers, and 64% of cytogenetic normal cohort. PRAME is expressed in 79% of patients with a RUNX1 - RUNX1T1 fusion, 57% of CBFA2T3 - GLIS2 positives, and 20% of Inv(16) patients. While the oncoprint (Fig. 1A) shows that most patients express 1 or 2 CTAs, with some genes showing exclusivity, patients with a KMT2A fusion tend to express multiple CTAs. As a group, 77% of patients with KMT2A fusions express at least 1 CTA.To determine the underlying mechanism of dysregulated CTA expression in AML, we inquired whether promoter methylation status might be associated with CTA expression. We correlated the expression level of the 7 upregulated CTA genes with the methylation of their corresponding promoters. Overall there was a strong signal demonstrating anti-correlation of expression of the above mentioned genes with their promoter methylation. In specific, for PRAME, differences in methylation at the region chr22:22899991-22901267 explain over 87% of the variation in expression, and this locus is normally methylated in NBM (Fig. 1C). Likewise, for CT45A5, it's normally methylated in NBM, and demethylation of this locus explains about 61% of the variance in expression (Fig. 1C).Additionally, we evaluated the clinical implications of CTA expression, and demonstrated that CCNA1 expression was significantly associated with clinical outcome. The range of expression for CCNA1 in the AML samples was 0-561.4 TPM (mean = 31.8 TPM), whereas expression for NBMs ranged from 0.3-2.5 TPM (mean = 0.9) (p=8.45E-08). After binning expression values to determine the best cutpoint for high and low expressors of CCNA1, patients classified with high expression had statistically significant worse event-free (EFS, Fig.1D) and overall survival (OS) (p=<0.0001, p=0.0393 respectively, Log-Rank test). As a sub-group, patients with KMT2A rearrangements and high CCNA1 expression also show prognostic significance with correlation of poor outcome (EFS, p=0.0009, Fig. 1E). In addition, cytogenetically normal (CN-AML) patients with FLT3 ITD had a much higher overall expression of CCNA1 than did CN-AML without FLT3 ITD (p=0.009, Mann-Whitney test).In this analysis we identify candidate CTA genes that are cumulatively expressed in the majority of AML cases and can provide novel therapeutic options by targeting with modified T-cell receptor therapies. They can also be used as biomarkers for response monitoring. Further, as aberrant expression of these genes are regulated by altered methylation, use of hypomethylating agents might enhance therapeutic efficacy by increasing target expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Analysis of Mixed Phenotype Acute Leukemia (MPAL) By Whole Genome Sequencing

Background: Mixed phenotype acute leukemias (MPAL) comprise a rare subgroup of acute leukemias with significant expression of lineage-specific antigens of more than one cell lineage not allowing assignment to a single cell lineage. While two MPAL subgroups are genetically defined according to the WHO 2016 classification, i.e. MPAL with BCR-ABL1 and MPAL with KMT2A rearrangement, the majority of cases are grouped as MPAL, B/myeloid, NOS or MPAL, T/myeloid, NOS and lack any specific or characteristic genetics so far.Aims: Molecular characterization of MPAL by whole genome sequencing (WGS).Patient cohort and methods: Diagnostic peripheral blood or bone marrow samples of 12 MPAL patients (median age 64 years, range 38-96; 5 male, 7 female) were selected for WGS with 90x coverage. One had MPAL with BCR-ABL1, 5 had MPAL, B/myeloid, NOS and 6 had MPAL, T/myeloid, NOS. Library preparation was performed using Truseq DNA PCR-Free HT sample preparation kit (Illumina, San Diego, CA). Sequencing was performed on NovaSeq instruments (Illumina). Tumor-only data was aligned by BaseSpace (BS) WGS app with default parameters and (structural) variant calling was performed with the Tumor/Normal app using an unmatched reference DNA (Promega, Fitchburg, WI). Data was subsequently loaded into BS Variant Interpreter to filter and prioritize variants of interest. For the structural variants we looked at all high confidence variant calls passing filters with at least 5 paired reads and 3 split reads with a somatic QScore of >30. In addition, all calls were eliminated with a population frequency of >1% according to the DGV (Database of Genomic variants). We performed mutation signature analysis by looking at the distribution of base substitutions across the sample with a 3mer context (including the base before and after the substitution) to decompose somatic mutation patterns and compared the results to the COSMIC signature database. For mutation analysis variants with a read frequency of >0.2% and ExAC population frequency of <1% were considered. For the first set of analysis we filtered down the variant list to the coding region of 65 genes described mutated in hematologic malignancies. For the second set of analysis we considered also genes beyond these 65 genes but required that variants had an entry in the COSMIC database and were classified with a “damaging” score by PolyPhen-2 and SIFT data bases.Results: Regarding the first set of analysis we found a median of 6 (range 2-7) mutations per patient, 63 overall, involving 35 of the 65 investigated genes. The majority were missense mutations (n=31), frameshift mutations (n=14) and splice-site mutations (n=13). The genes listed in table 1 were found most frequently mutated. Except for RUNX1 none of the frequently encountered mutations affected the case with MPAL with BCR-ABL1 while there was no restriction of mutations to either of the two other MPAL subgroups. 11 of these 63 mutations had been previously analyzed by routine diagnostics applying next generation sequencing (NGS) yielding identical results.Regarding the second set of analysis without limitation on the above mentioned 65 genes we found a median of 83 (range 63-129) mutations per patient, 1079 overall, involving 736 genes. Specifically, four candidate genes were found mutated in at least five patients (table 2). NBPF1 has been described as tumor suppressor, PRAMEF2 belongs to the PRAME family of genes associated with various malignancies and as a prognostic factor in B-ALL, MUC12 is a highly polymorphous gene involved in a variety of cancer types and described to be epigenetically regulated and thus a marker for early cancer detection, ARSD has been described overexpressed and prognostic in CLL. Again, mutations were not restricted to one MPAL subgroup.We subsequently conducted a context-sensitive mutation signature analysis with the SNV data, using 96 substitution patterns rather than only 6 to increase the complexity. In all instances we found a highly similar pattern across all 12 samples with a strong bias towards C>T substitutions (figure 1).Conclusion: WGS is capable of reproducing routine NGS diagnostics and of identifying mutations not yet described for MPAL which might be used to better understand and categorize this disease. [Display omitted] DisclosuresKern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Biology and Prognostic Markers in Young Children with Acute Myeloid Leukemia -the Jccg Study, JPLSG AML-05-

[Introduction] Acute myeloid leukemia (AML) in children aged < 12 months of age was conventionally defined as infant AML. On cytogenetic and molecular analysis, 11q23/ KMT2A rearrangement (KMT2A -R) was found to be the most frequent (approximately 50%) cause of infant AML. On prognostic analysis, infant AML was reported to have favorable outcome compared with AML in older children, and only KMT2A -R and RBM15-MKL1 were known to be intermediate-risk prognostic factors in infants. Although infants have been treated in the same regimens as older children, in some clinical trials, drug dosage for infants has been reduced to avoid severe toxicities. However, new findings in young children with AML have been recently accumulated. A recent report by an international expert panel of pediatric AML has proposed AML in children aged < 24 months of age as a distinct age-subgroup because their morphologic and cytogenetic/molecular characteristics were considered to be similar to those of infants (Creutzig et al, 2012). Moreover, recent studies have identified novel inv(16)(p13.3q24.3)/ CBFA2T3-GLIS2 and t(11;12)(p15;p13)/ NUP98-KDM5A fusion genes in AMKL. In this study, we investigated biology and prognosis of children aged < 5 years, to identify unique characteristics of young children with AML and propose a novel risk-stratification for them.[Patients and Methods] A total of 369 children with AML were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. In this study, we analyzed 133 children aged < 5 years (36%). We screened fusion genes of CBFA2T3-GLIS2, NUP98-KDM5A, RBM15-MKL1, KMT2A -Rs, NUP98-NSD1, FUS-ERG, DEK-NUP214, RUNX1-RUNX1T1, and CBFB-MYH11, and gene mutations of KIT, NRAS, KRAS, WT1, NPM1, KMT2A -PTD, FLT3 -ITD, CEBPA, ASXL1, ASXL2, and CSF3R .[Results] In clinical characteristics, no change was observed in the sex ratio with age. The WBC count at diagnosis was the highest in children aged 2 years (24-35months), and a trend of lower WBC in children aged > 2 years (> 35 months). On FAB classification, the proportion of M5 and M7 was observed to be decline with age. Among fusion genes, KMT2A -R was the most frequently identified fusion gene (23%), followed by RUNX1-RUNX1T1, CBFB-MYH11, and CBFA2T3-GLIS2 (13.5%, 9.8%, and 8.3%, respectively). Conversely, neither NUP98-NSD1 nor DEK-NUP214 was found. Although the frequencies of KMT2A -R and CBFA2T3-GLIS2 tended to decrease with age, the frequency of RUNX1-RUNX1T1 significantly increased with age. Among gene mutations, KIT and NRAS were the most frequent, and gene mutations other than ASXL1 and KMT2A -PTD were recurrently identified. When correlation of fusion genes with gene mutation was analyzed, 21 (68%) of 31 children with CBF-AML had gene mutations, whereas only 8 (16%) of 51 children with non-CBF-AML had gene mutations (p < 0.001). For fusion genes, 28 (90%) of 31 KMT2A -R-positive children were aged < 3 years (< 36 months) (90%), and 17 (94%) of 18 RUNX1-RUNX1T1 -positive children were aged > 2 years. Thus, according to these molecular characteristics of young children with AML, we defined children aged < 3 years as the youngest subgroup with distinct biology (n = 99). We further performed prognostic analyses of the youngest subgroup. According to recent previous studies that reported poor prognosis of CBFA2T3-GLIS2 and NUP98-KDM5A in AMKL, these fusions were combined to the same group. The Kaplan-Meier analyses revealed that CBFA2T3-GLIS2 / NUP98-KDM5A -positive children had the poorest outcome in OS, EFS, and CIR (38%, 13%, and 88%, respectively). Surprisingly, children with CBF-AML had the second poorest outcome in EFS and CIR (45% and 55%, respectively), although this subgroup had an excellent OS (100%), indicating that children with CBF-AML in the youngest subgroup have a high-risk of relapse and might need more intensified therapy as an initial treatment.[Conclusions] The youngest subgroup of pediatric AML had a unique biology. Children with CBFA2T3-GLIS2 and NUP98-KDM5A had the poorest outcome, and children with CBF-AML also had a high relapse rate. Because this subgroup is known to have a high incidence of severe side effects and late effects, a novel risk-stratified therapy restricted to young children is required. DisclosuresNo relevant conflicts of interest to declare.

Transplacental transfer of congenital B‐cell acute lymphoblastic leukemia to the maternal vasculature

To the Editor: Monozygotic twin infants with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) demonstrate near-uniform disease concordance due to prenatal transfer of pre-leukemic cells via placental vascular anastomoses.1-4 However, despite several historical reports recognizing the presence of fetal nucleated cells in the maternal circulation during pregnancy,5 fetal–maternal dissemination of leukemic clones has not been reported. We present a neonate with congenital KMT2Ar-ALL with documentation of metastasis to the maternal decidual placental vessels without clinical consequence. A newborn infant born at 37 4/7 weeks gestation was found to have leukocytosis (white blood cells 72.1 × 103/μl) with 62% blasts characterized by convoluted nuclear contour, prominent nucleoli, and scant deep-blue cytoplasm. Flow cytometry revealed expression of HLA-DR, CD15, CD19, CD22, CD34, cytoplasmic TdT, and cCD79a, consistent with B-cell ALL. G-banding karyotyping analysis revealed t(4;11)(q21;q23) with a dominant clone (five metaphase cells) containing a (4;11)(q21;q23) translocation and a subclone (one cell) containing an additional copy of der(4). Placental microscopy demonstrated clusters of blasts in both chorionic surface (fetal) vessels and decidual (maternal) vessels (Figure 1). Fluorescence in situ hybridization (FISH) analysis showed these clusters to be positive for the KMT2A rearrangement in both fetal and maternal vessels. These findings indicate that malignant fetal cells had infiltrated the maternal placental vasculature. Unlike in older children and adolescents, the prognosis of ALL in the neonatal age group is dismal.6 Despite intensive therapy, our patient experienced a highly complicated clinical course with eventual decompensation from Enterococcus faecalis bacteremia and infectious rotavirus leading to death on day 47 of life. Notably, throughout the patient's nearly 7-week clinical course, his mother did not demonstrate any clinically apparent findings to suggest sequelae of transplacental leukemia cell transfer. The discovery of leukemia cells of shared clonal lineage between monozygotic twins with ALL supports the so-called “intraplacental metastasis hypothesis”1 and confirms the capacity for pre-leukemic cells to spread from one twin to the other.7 These pre-leukemic cells are thought to transfuse via monochorionic placental vascular anastomoses rather than across the placental membrane barrier itself. Although placental involvement of congenital neuroblastoma has been reported,8 numerous reports of pregnant patients with hematologic malignancies demonstrate a noteworthy absence of clinically apparent transplacental “metastases” to exposed neonates.9-12 For example, in a report of prenatally diagnosed maternal ALL with leukemia cells detected in the cord blood, these cells were efficiently cleared and did not result in leukemic engraftment in the neonate.13 In this report, numerous lymphoblasts characterized by the KMT2A rearrangement were observed in the decidual (maternal) placental blood vessels, suggesting substantial cross-placental infiltration or local proliferation following infiltration. Our findings suggest the capacity for pre-leukemic cells to circumvent the placental membrane barrier as an additional means of transplacental penetration. The absence of a clinical impact on maternal health suggests that infiltrated malignant fetal cells undergo extraplacental maternal immune clearance and do not result in clinical consequence to the exposed mother. The authors declare that there is no conflict of interest.

Interrogation of novel CDK2/9 inhibitor fadraciclib (CYC065) as a potential therapeutic approach for AML

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6–8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

Pre-B cell receptor expression in B-lineage acute lymphoblastic leukemia: A report from the Children’s Oncology Group.

e19006 Background: The surface expression of mature B-cell markers have led to the development of immunotherapies against B-lineage lymphoblastic leukemia/lymphoma (B-ALL/B-LLy). Relapsing clones that have altered surface antigen expression are common means of treatment failure with immunotherapies. The elimination of the pan-B cell repertoire by current B-cell immunotherapies contributes to immune-compromise. A promising target is the pre-BCR surrogate light chain, comprised of the VpreB1 (CD179a) and Lamda5 (CD179b) subunits. Surrogate light chain is expressed on pro- and pre-B cells where it governs preBCR-mediated autonomous survival during B-cell maturation. Gene expression analyses have shown that CD179a is expressed in a sub-set of 10 to 15% of B-ALL cases. Because immunotherapies targeted to restricted stages of B-cell development may overcome the limitations of pan B-cell ablation, we tested the hypothesis that CD179a is more commonly expressed on B-lymphoblasts than previously thought. Methods: Utilizing an annotated set of 36 standard (AALL0331) and high-risk (AALL0232) B-ALL cases accrued to Children’s Oncology Group AALL03B1, we adapted the COG minimal residual disease (MRD) flow panel to include two additional PE- and FITC-conjugated mAbs against CD179a (Biolegend and i2Pharma). We assessed CD179a expression in 16 cases for which we had Day 28 end-induction samples, pre-selected to have ≥1% MRD, as determined by the COG Reference laboratories. Cases with ≥20% CD179a surface expression were determined to be positive for statistical comparisons. All analyses were performed on a 6-color Becton-Dickinson flow cytometer in a CLIA/CAP certified laboratory. Results: Thirty-four cases were arrested at the CD10-positive pre-B stage, and two cases at the CD10-negative pro-B stage. One or both mAbs showed that CD179a was present in ≥20% of the B-lymphoblast population, ranging from 20.2% to 90.6% for all 36 diagnostic samples. All cases expressed CD179a in the end-induction B-lymphoblast population. Compared to gene-expression based predictions, we found a significant difference between expected versus observed flow-based CD179a positivity (two-sided Fisher’s exact test, P&lt; 0.001). We found that CD179a expression was observed in cases having E2A-PBX3, KMT2A, BCR-ABL1 and other re-arrangements that typify mixed phenotype acute leukemias (MPALs). Conclusions: Our results show that CD179a is commonly expressed in B-ALL, regardless of stage, NCI risk features, or molecular aberrations. Because the productively assembled preBCR mediates autonomous survival signaling in pro- and pre-B cells, it may also contribute to the mechanistic basis of MRD in B-ALL. Immunotherapies directed against the CD179a component of the preBCR may spare the immune-compromise that occurs with pan B-cell ablation, and prevent the emergence of therapy-resistant disease in B-ALL/B-LLy.

Minimal residual disease at end of induction and consolidation remain important prognostic indicators for newly diagnosed children and young adults with very high-risk (VHR) B-lymphoblastic leukemia (B-ALL): Children’s Oncology Group AALL1131.

10004 Background: Children and young adults with very high risk (VHR) B-acute lymphoblastic leukemia (B-ALL) [13-30 years of age with any features or 1-30 years of age with adverse prognostic features including KMT2A rearrangements, iAMP21, hypodiploidy (&lt;44 chromosomes/DNA index &lt; 0.81), central nervous system disease, end of induction (EOI) minimal residual disease (MRD) &gt;0.01%, or induction failure] collectively have a predicted 4-year disease free survival (DFS) of approximately 70%. Whether patients with VHR B-ALL who are MRD positive at EOI and become MRD negative at the end of consolidation (EOC) will have improved survival versus patients remaining MRD positive at EOC is unknown. Methods: Patients with newly diagnosed NCI high risk B-ALL enrolled on AALL1131 or NCI standard risk B-ALL enrolled on AALL0932 and classified as VHR at EOI were treated on the VHR stratum of AALL1131 which sought to improve DFS with intensive post-Induction therapy using fractionated cyclophosphamide (CPM), etoposide (ETOP) and clofarabine (CLOF).Patients were randomly assigned post-Induction to Control Arm (CA) with modified augmented BFM CPM + fractionated cytarabine + mercaptopurine, Experimental Arm 1 (Exp1) with CPM + ETOP, or Experimental Arm 2 (Exp2) with CLOF + CPM + ETOP during Part 2 of Consolidation and Delayed Intensification. Doses of vincristine and pegaspargase were identical on all arms. Exp2 was permanently closed September 2014 due to excessive toxicities, and these patients are excluded from this report. MRD was measured by 6-color flow cytometry at EOI and for those who consented at the EOC. Results: 4-yr DFS for all patients (n=823) with VHR B-ALL was 76.8 ± 2.0%. As we reported previously, 4-year DFS was not significantly different between CA and Exp 1 (85.5 ± 6.8% versus 72.3 ± 6.3%; p=0.76; Burke, Haematologica 2019). 4-yr DFS for patients who were EOI MRD &lt;0.01%, (n=325) versus &gt;0.01 (n=498) was 83.3% ± 2.6% vs 72.0% ± 2.8%, p=0.0013. 4-Year DFS of Patients EOI MRD &gt; 0.01%. Conclusions: MRD is a powerful prognostic indicator in VHR B-ALL with inferior outcomes in patients who are EOI MRD positive. Among patients who were EOI MRD positive treated on Exp1, outcomes were similar for EOC MRD negative and EOC MRD positive, though numbers were small. In contrast, patients who were EOI MRD positive treated on CA that were EOC MRD negative had significantly improved DFS compared to those that were EOC MRD positive. The CA remains the standard of care for COG ALL trials. With this therapy, patients with VHR B-ALL that are EOI MRD positive and EOC MRD negative have significantly improved DFS compared to those that remain MRD positive at EOC. Clinical trial information: NCT02883049. [Table: see text]

Therapy-related acute myeloid leukemia with KMT2A-SNX9 gene fusion associated with a hyperdiploid karyotype after hemophagocytic lymphohistiocytosis

Therapy-related acute myeloid leukemia (t-AML) following treatment with topoisomerase-II inhibitors has been increasingly reported. These compounds (e.g. etoposide) promote DNA damage and are associated with KMT2A rearrangements. They are widely used as first-line treatment in hemophagocytic lymphohistiocytosis (HLH). Here we describe a newborn who developed t-AML after HLH treatment. We provide detailed clinical, cytogenetic, and molecular characteristics of this patient, including the identification of a novel gene fusion – KMT2A-SNX9 – in t-AML. Considering the dismal outcome of this case, we discuss the side-effects of etoposide administration during HLH treatment in infants.

Mesothelin is a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia

AbstractIn an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN–DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

Infantile leukemia-What factors determine its distinct biological nature? Clinicopathological study of 78 cases.

Infantile leukemia encompasses a heterogeneous group which needs stratifying for treatment selection. We collected 78 cases of infantile leukemia and retrospectively analyzed their clinicopathological data. Infantile leukemia featured a ratio of acute myeloid leukemia (AML) to B-lymphoblastic leukemia (B-ALL) of 1:2, with a better survival for AML than B-ALL (median survival 36 vs 24 months). When stratified by age, "early" infantile B-ALL (2-6 months) showed a high rate of KMT2A rearrangement (100%), similar to the rate seen in congenital B-ALL (1 month) (100%) and higher than seen in "late" infantile B-ALL (≥7 months) (68%). The three categories of infantile B-ALL exhibited an age-dependent increase in survival (median survival 8.5, 24, and >24 months, respectively). The age-dependent survival benefit remained after excluding the cases negative for KMT2A rearrangement. Conversely, infantile AML lacked an age-dependent pattern of survival. The clinical outcome of infantile leukemia depends on the type of leukemia. Given the age-dependent survival, infantile B-ALL can be divided into three subcategories.

Is acute lymphoblastic leukemia with mature B-cell phenotype and KMT2A rearrangements a new entity? A systematic review and meta-analysis

The association between mature B-cell phenotype and KMT2A rearrangements in acute lymphoblastic leukemia is a very rare finding. It identifies a group of patients with similar clinical and biological characteristics that clearly differs from the entity B-cell lymphoblastic leukemia/lymphoma with t(v;11q23)/KMT2A-rearranged, which typically presents an immature pro B-cell phenotype. We describe the clinical-biological characteristics and disease outcome of three pediatric ALL patients with these features treated at our institution, and review 28 cases described in the literature. Most cases occur in children under 2 years-old, presenting a mature B-cell phenotype that uniformly expresses cytoplasmic and surface IgM with lambda light chain restriction, with heterogeneous co-expression of immaturity antigens. Patients do not have MYC rearrangements and all show KMT2A abnormalities, with 76% presenting t(9;11)(p21;q23)/MLLT3-KMT2A. These patients have an unfavorable clinical outcome and a 48% relapse rate. In-depth knowledge of this disease entity is needed to improve outcome.

Long-term follow-up of salvage therapy using a combination of inotuzumab ozogamicin and mini-hyper-CVD with or without blinatumomab in relapsed/refractory Philadelphia chromosome-negative acute lymphoblastic leukemia.

The outcome of patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) is poor. The combination of inotuzumab with low-intensity mini-hyper-CVD (mini-hyper-CVD; cyclophosphamide and dexamethasone at 50% dose reduction, no anthracycline, methotrexate at 75% dose reduction, cytarabine at 0.5 g/m2 × 4 doses) chemotherapy has shown encouraging results. The sequential addition of blinatumomab might improve outcome in patients with R/R ALL. We used lower intensity chemotherapy, mini-hyper-CVD (cyclophosphamide and dexamethasone at 50% dose reduction, no anthracycline, methotrexate at 75% dose reduction, cytarabine at 0.5 g/m2 x 4 doses) compared to conventional hyper-CVAD. Ninety-six patients with a median age of 37 years (range, 18-87 years) were treated. Overall, 77 patients (80%) responded, 55 (57%) of whom achieved complete response. The overall measurable residual disease negativity rate among responders was 83%. Forty-four (46%) patients underwent later allogeneic stem cell transplantation. Veno-occlusive disease of any grade occurred in 10 (10%) patients. The rates were 13% with the original schedule and 3% with the use of lower-dose inotuzumab and sequential blinatumomab. With a median follow-up of 36 months, the median overall survival (OS) was 13.4 months, with 3-year OS rates of 33%. The 3-year OS rate for patients with CD22 expression ≥70% and without adverse cytogenetics (KMT2A rearrangements, low hypodiploidy/near triploidy) was 55%. The combination of inotuzumab and low-intensity mini-hyper-CVD chemotherapy with or without blinatumomab shows sustained efficacy in patients with R/R ALL.