Distinct Genomic Landscape of Chinese Pediatric Acute Myeloid Leukemia

Although comprehensive genomic studies have revealed key genomic aberrations in pediatric acute myeloid leukemia (AML), knowledge about Chinese patients remains lacking. Here we report the genomic landscape of Chinese pediatric AML by analyzing the sequence mutations and fusions from transcriptome sequencing (RNA-seq) of 292 cases diagnosed through 2009 to 2018 in Shanghai Children's Medical Center. Informed consents were obtained from parents for all patients. A total of 1831 non-synonymous mutations that were predicted somatic and/or associated to pediatric cancer were identified in 972 genes, including 1597 single nucleotide variants (SNV), 210 insertion/deletion (indels) and 24 internal tandem duplications (ITD), with a median of 6 mutations per case (ranging 0 to 15). Among these abnormalities, 7 aberrations occurred in more than 5% of cases in current cohort, including mutations in KIT (n=54, 18.5%), FLT3 (n=46, 15.8%), NRAS (n=28, 9.6%), CEBPA (n=23, 7.9%), ASXL2 (n=20, 6.8%), KRAS (n=16, 5.5%) and CSF3R (n=15, 5.1%). 444 potential driver variations were identified affecting 66 genes by a combined strategy of mutation pathogenicity and hotspot analysis. Each patient carried a median of one driver mutations per case (ranging 0 to 7). In addition, RNA-seq identified 227 fusions involving 99 genes in 203 out of 292 patients (69.5%), and CBL exon8/9 deletion in 12 patients (4.1%). The most prevalent fusions detected in current cohort included RUNX1-RUNX1T1 (n=82, 28.1%), KMT2A rearrangements (n=45, 15.4%) and NUP98 rearrangements (n=17, 5.8%). Furthermore, novel gene rearrangements were identified in current study, including PTPRA-FUS, ZEB2-ATIC, MSI2-UBE3C (n=1 each). Distinct genomic aberration profile was revealed while comparing our results to the mutation profile characterized in Children's Oncology Group (COG)-National Cancer Institute (NCI) TARGET AML initiative representing the Western pediatric AML cohort. A total of 16 recurrently mutated genes were identified with significantly (two-sided fisher exact test) different mutation frequency. Among these, 7 genes mutated more frequently in Chinese patients, including KIT (18.5% vs 12.8% in Chinese and Western cohort, respectively. p=0.027), ASXL2 (6.8% vs 3.6%, p=0.043), CSF3R (5.1% vs 2.4%, p=0.044), JAK2 (3.4% vs 0.0%, p<0.001), DNM2 (2.7% vs 0.0%, p<0.001), KDM6A (2.1% vs 0.0%, p<0.001) and KMT2C (1.7% vs 0.0%, p=0.003). On the other hand, mutations in FLT3 (15.8% vs 33.0%, p<0.001), NRAS (9.6 vs 30.9%, p<0.001), KRAS (5.5% vs 12.8%, p<0.001), WT1 (2.4% vs 13.6%, p<0.001), NPM1 (2.4% vs 10.3%, p<0.001), PTPN11 (3.8% vs 8.1%, p=0.016), TET2 (1.0% vs 5.2%, p=0.001), CBL sequence mutation (0.0% vs 3.0%, p<0.001) and IKZF1 (0.3% vs 2.7%, p=0.018) were occurred less frequently in Chinese patients. Notably, the RAS signaling pathway as a whole was significantly less frequently mutated in Chinese patients (35.6% vs 71.0%, p<0.001). Furthermore, distinct associations between mutations and FAB subtypes were also observed. For example, NF1 mutations were significantly enriched with subtype M5 in Chinese patients (p=0.003), which was previously reported as co-mutated with CBFB-MYH11 fusion with associated with subtype M4. Survival analysis revealed key genomic aberrations associated with patient prognosis. Variants significantly (log-rank test) associated with better event free survival rate included mutations in CEBPA (p=0.023), NPM1 (p=0.026) and GATA2 (p=0.016). On the other hand, CBFA2T3-GLIS2 (p=0.028), nucleoporin gene family related fusions (including NUP98, NUP214 and NUP153, p<0.001), FUS related fusions (p=0.030), mutations in RUNX1 (p<0.001) and FLT3 (p=0.003) were associated with worse prognosis. A revised risk stratification model was proposed based on these associations observed. Characterized a first comprehensive genomic landscape of Chinese pediatric AML, our results reveal a distinct mutation profile as compared to the Western cohort, in terms of both mutation frequency and patterns of mutation co-occurrence. These findings further reveal the complexity of pediatric AML and highlight the importance of tailored risk stratification for Chinese patients in clinical management. Disclosures No relevant conflicts of interest to declare.

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Transcriptome analysis offers a comprehensive illustration of the genetic background of pediatric acute myeloid leukemia

Blood Advances ◽

10.1182/bloodadvances.2019000404 ◽

2019 ◽

Vol 3 (20) ◽

pp. 3157-3169 ◽

Cited By ~ 5

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yusuke Hara ◽

Genki Yamato ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Genetic Background ◽

Myeloid Leukemia ◽

Gene Rearrangements ◽

Rna Seq ◽

Pediatric Acute Myeloid Leukemia ◽

Runx1 Gene ◽

Key Points ◽

Pediatric Aml ◽

Gene Alterations ◽

Acute Myeloid

Key Points Using RNA-seq in pediatric AML patients, 5 gene rearrangements were newly identified, including NPM1 and RUNX1 gene rearrangements. RNA-seq unmasked the complexity of gene alterations in pediatric AML by identifying disease-causing alterations in nearly all patients.

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An Immune Checkpoint-Related Gene Signature for Predicting Survival of Pediatric Acute Myeloid Leukemia

Journal of Oncology ◽

10.1155/2021/5550116 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Feng Jiang ◽

Xin-Yu Wang ◽

Ming-Yan Wang ◽

Yan Mao ◽

Xiao-Lin Miao ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Immune Checkpoint ◽

Myeloid Leukemia ◽

Immune Cell ◽

Cox Regression ◽

Secondary Data ◽

Gene Signature ◽

Pediatric Acute Myeloid Leukemia ◽

Pediatric Aml ◽

Acute Myeloid

Objective. The aim of this research was to create a new genetic signature of immune checkpoint-associated genes as a prognostic method for pediatric acute myeloid leukemia (AML). Methods. Transcriptome profiles and clinical follow-up details were obtained in Therapeutically Applicable Research to Generate Effective Treatments (TARGET), a database of pediatric tumors. Secondary data was collected from the Gene Expression Omnibus (GEO) to test the observations. In univariate Cox regression and multivariate Cox regression studies, the expression of immune checkpoint-related genes was studied. A three-mRNA signature was developed for predicting pediatric AML patient survival. Furthermore, the GEO cohort was used to confirm the reliability. A bioinformatics method was utilized to identify the diagnostic and prognostic value. Results. A three-gene (STAT1, BATF, EML4) signature was developed to identify patients into two danger categories depending on their OS. A multivariate regression study showed that the immune checkpoint-related signature (STAT1, BATF, EML4) was an independent indicator of pediatric AML. By immune cell subtypes analyses, the signature was correlated with multiple subtypes of immune cells. Conclusion. In summary, our three-gene signature can be a useful tool to predict the OS in AML patients.

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Acute Differentiated Dendritic Cell Leukemia: A New Form of Pediatric Acute Myeloid Leukemia (AML) with MLL Translocations.

Blood ◽

10.1182/blood.v106.11.3263.3263 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3263-3263

Author(s):

Luca Lo Nigro ◽

Laura Sainati ◽

Anna Leszl ◽

Elena Mirabile ◽

Monica Spinelli ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Dendritic Cells ◽

Dendritic Cell ◽

Myeloid Leukemia ◽

Fusion Transcript ◽

Fish Analysis ◽

Rt Pcr ◽

Pediatric Acute Myeloid Leukemia ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Background: Myelomonocytic precursors from acute or chronic leukemias can differentiate to dendritic cells in vitro, but leukemias with a dendritic cell immunophenotype are rare, have been reported mainly in adults, and their molecular pathogenesis is unknown. Dendritic cells are classified as Langherans, myeloid and lymphoid/plasmacytoid cells, but leukemias arising from dendritic cells are unclassified in the FAB system. We identified a new entity of pediatric acute myeloid leukemia (AML) presenting with morphologic and immunophenotypic features of mature dendritic cells, which is characterized by MLL gene translocation. Methods and Results: Standard methods were used to characterize the morphology, immunophenotype, karyotype and MLL translocations in 3 cases of pediatric AML. The patients included two boys and one girl diagnosed with AML between 1–6 years old. Their clinical histories and findings included fever, pallor, abdominal and joint pain, adenopathy, hepatosplenomegaly, normal WBC counts but anemia and thrombocytopenia. and no evidence of CNS disease. The bone marrow aspirates were hypocellular and replaced completely by large blasts with irregular nuclei, slightly basophilic cytoplasm, and prominent cytoplasmic projections. There were no cytoplasmatic granules or phagocytosis. Myeloperoxidase and alpha napthyl esterase reactions were negative, excluding FAB M5 AML, and the morphology was not consistent with any standard FAB morphologic diagnosis. The leukemic blasts in all three cases were CD83+, CD86+, CD116+, consistent with differentiated myeloid dendritic cells, and did not express CD34, CD56 or CD117. MLL translocations were identified in all 3 cases. In the first case FISH analysis showed t(10;11)(p12;q23) and RT-PCR identified and a ‘5-MLL-AF10-3’ fusion transcript. In the second case FISH analysis showed t(9;11)(p22;q23) and RT-PCR identified and a ‘5-MLL-AF9-3’ fusion transcript. In the remaining case, the MLL gene rearrangement was identified by Southern blot analysis and RT-PCR showed an MLL-AF9 fusion transcript. The fusion transcripts in all 3 cases were in-frame. Remission induction was achieved with intensive chemotherapy, and all three patients have remained in durable remission for 30–60 months after hematopoietic stem cell transplantation. Conclusions. We have characterized a new pediatric AML entity with features of mature dendritic cells, MLL translocation and an apparently favorable prognosis. The in-frame MLL fusion transcripts suggest that chimeric MLL oncoproteins underlie its pathogenesis. The partner genes in all 3 cases were known partner genes of MLL that encode transcription factors. This study increases the spectrum of leukemias with MLL translocations. Comprehensive morphological, immunophenotypic, cytogenetic and molecular analyses are critical for this diagnosis, and will reveal its frequency and spectrum as additional cases are uncovered.

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Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-151671 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1170-1170

Author(s):

Anilkumar Gopalakrishnapillai ◽

Anne Kisielewski ◽

Yang Zhang ◽

Bruce Ruggeri ◽

Peggy Scherle ◽

...

Keyword(s):

Median Survival ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Research Funding ◽

Publicly Traded ◽

Pediatric Acute Myeloid Leukemia ◽

Patient Derived Xenograft ◽

Pediatric Aml ◽

Pdx Models ◽

Acute Myeloid

Abstract Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML. We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints. Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

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Genomic Landscape and Prognosis in Pediatric Acute Myeloid Leukemia: A Study on the French ELAM02 Trial

Blood ◽

10.1182/blood.v128.22.1676.1676 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1676-1676

Author(s):

Alice Marceau-Renaut ◽

Nicolas Duployez ◽

Christine Ragu ◽

Arnaud Petit ◽

Odile Fenneteau ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Driver Mutations ◽

Mutational Profiling ◽

Genomic Landscape ◽

Diagnostic Samples ◽

Pediatric Aml ◽

Generation Sequencing ◽

Acute Myeloid

Abstract Background. Acute Myeloid Leukemia (AML) is a rare and genetically heterogeneous disease that constitutes 15 to 20% of childhood leukemia. Despite major treatment improvement over the past decades pediatric AML remains a challenging disease with poor outcome compared to acute lymphoid leukemia (ALL). About 50% of these patients relapse after standard intensive chemotherapy. Molecular analysis pointed out the prognostic impact of gene mutation such as FLT3-ITD, NPM1 or CEBPA; and new categories of regulators like epigenetic modifiers. More recently mutational profiling studies revealed distinct molecular subgroups with prognostic significant and stratification in adult AML. Nevertheless cytogenetic and mutational profiles are quite different between adult and pediatric AML. Extensive genomic studies have not been reported to date in pediatric AML. In this context it is of importance to identify additional genetic or molecular abnormalities to better understand leukemogenesis and also to predict outcome and serve as novel therapeutic targets. Methods. We performed a mutational analysis on diagnostic samples from patients enrolled in the French National Multicenter ELAM02 trial. 438 patients with de novo AML (except AML3) were enrolled between march 2005 and December 2011 (median age: 8,22yrs [0-18.61]; median WBC: 15.4G/l [0.4-575]; cytogenetic subgroups: CBF-AML[n=97], NK-AML [n=109], MLL-AML[n=95], MRC2 other[n=77], MRC3 [n=55], failure [n=5]). Diagnostic samples were prospectively collected and 386 of the 438 patients (88%) were studied by next-generation sequencing (Miseq, Illumina with haloplex librairy and ion Proton, thermofischer with ampliseq librairy) including 36 genes frequently reported in myeloid malignancy. Two different technologies of next generation sequencing (NGS) were used, allowing direct validation. FLT3-ITD was detected and quantified by Genescan analysis. Results. We identified 579 driver mutations involving 36 genes or regions in 386 patients (mean 1.5 per case), with at least 1 driver mutation in 291 patients (75%) and 2 or more driver mutations in 44% of samples. The number of mutation identified at diagnosis in cytogenetic subgroup is significantly lower in MLL-AML (0.44 mutation/patient; p<10-4). Mutations involving genes from the tyrosine kinase pathways (i.e RAS, FLT3, KIT, PTPN11, JAK2, MPL, CBL) were the most frequent and represent 56.3% of all aberrations. Among them N-RAS was detected in 26.4% of all cases, followed by FLT3-ITD, KIT and K-RAS in 14.8%, 12.4% and 12.2% respectively. We identified 64 driver mutations in the group of transcription factors (CEBPA, RUNX1, GATA, ETV6), 60 in the combined group of chromatin modifier (ASXL1, EZH2, BCOR) and DNA methylation (DNMT3A, IDH, TET2), 59 in the group of tumor suppressor genes (WT1, PHF6, TP53) 36 mutations in NPM1 gene, and few mutations in cohesion and spliceosome sub-groups. Identified mutations are indicated in the figure according cytogenetic subgroups. Among the 438 patients, 398 (91%) were in complete remission (CR) after two courses (induction and first consolidation), the 5-year overall survival (OS) is 71.5% [65-78] and the 5-year leukemia free survival (LFS) is 56.6% [49.7-63.5]. In univariate analysis, we found that FLT3-ITD, mutations in RUNX1, WT1 and PHF6 were associated with reduced LFS (p=0.0003 for FLT3-ITD, p=0.01 for RUNX1, p=0.02 for WT1 and p=0.025 for PHF6) and reduced OS (p=0.0003 for FLT3-ITD, p=0.0003 for RUNX1, p=0.015 for WT1 and p=0.04 for PHF6). Mutations in NPM1 is associated with an improved 5-yr LFS (p=0.014) and 5-yr OS (p=0.005). Multivariate analysis revealed that FLT3-ITD, RUNX1 and PHF6 were independently associated with an adverse outcome and NPM1 with an improved outcome. Conclusions. We performed an extensive mutational study in de novo pediatric AML enrolled in the ELAM02 trial. We described the genomic landscape of 386 patients and showed the frequency of different mutations according cytogenetics. Interestingly we found mutations in genes involved in constitutional or pre-leukemic disease such as PTPN11, RUNX1, MPL or ETV6. We found that FLT3-ITD, RUNX1 and PHF6 mutations predict poor outcome although NPM1 mutations predict a better outcome. Mutational profiling reveals useful information for risk stratification and therapeutic decisions. Figure Figure. Disclosures Baruchel: Amgen: Consultancy.

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Transcriptome Analysis Revealed the Entire Genetic Understanding of Pediatric Acute Myeloid Leukemia with a Normal Karyotype

Blood ◽

10.1182/blood.v128.22.2850.2850 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2850-2850

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yuichi Shiraishi ◽

Shiraishi Yuichi ◽

Yusuke Hara ◽

...

Keyword(s):

Rna Sequencing ◽

Myeloid Leukemia ◽

Research Funding ◽

Normal Karyotype ◽

Gene Rearrangements ◽

Pediatric Leukemia ◽

Pediatric Acute Myeloid Leukemia ◽

Recurrent Mutations ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Background: Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with a current overall survival rate of less than 70%. The pathogenesis of AML is heterogeneous and can be caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations [e.g., t(8;21), inv(16), and MLL-rearrangements] have been well established as diagnostic and prognostic markers of AML. Furthermore, recurrent mutations in FLT3, KIT, NPM1, and CEBPA have been reported in both adult and pediatric AML. Recently, massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for the prediction of the prognosis. However, these mutations are rare in pediatric AML, suggesting that other genetic alterations exist in pediatric AML. In contrast, recent reports have described NUP98-NSD1 fusion as an adverse AML prognostic marker and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harboring NUP98-NSD1 fusion. Intriguingly, PRDM16 overexpression occurs in nearly one-quarter of all children, with AML involving NUP98-NSD1-negative patients. Moreover, this overexpression is enriched in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1, and MLL-PTD). Patients and Methods: To reveal a complete registry of gene rearrangements and other genetic lesions in pediatric AML with a normal karyotype, we performed transcriptome analysis (RNA sequencing) of 61 of 70 de novo pediatric AML patients with a normal karyotype using Illumina HiSeq 2000. We could not perform RNA sequencing in nine patients because of a lack of RNA quantity or quality. Among the 70 AML patients with a normal karyotype, 33 patients overexpressed PRDM16, which was found to be strongly associated with a poor prognosis in our previous studies. All patients were enrolled and treated with AML-05 in the study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG). We also analyzed the known genetic mutations associated with these patients using the data derived from RNA sequencing. Results: A total of 144 candidate gene rearrangements, which were not observed in normal samples, were identified in 51 of 61 samples. Many of the recurrent gene rearrangements identified in this study involved previously reported targets in AML, including NUP98-NSD1, NUP98-JARID1A, CBFA2T3-GLIS2, MLL-MLLT10, and MLL-MLLT3. However, several gene rearrangements were newly identified in the current study, including MLL-SEPT6, HOXA10-HOXA-AS3, PRDM16-SKI, and CUL1-EZH2. We have also performed the validation of these novel gene rearrangements using Sanger sequencing. Most of these gene rearrangements were found in patients with a high expressionof PRDM16. In contrast, CEBPA mutations were frequently observed in patients with a low expression of PRDM16. Known gene alterations, such as FLT3-ITD and MLL-PTD, and mutations of the RAS, KIT, CEBPA, WT1, and NPM1genes were also detected using RNA sequencing. Conclusion: RNA sequencing unmasked a complexity of gene rearrangements and mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from adult counterparts, regarding the spectrum of gene rearrangements and mutations. In the present study, we identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including some novel fusion genes. These findings suggest that gene rearrangements in conjunction with mutations also play essential roles in pediatric AML. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

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In Vivo Evaluation of Mesothelin As a Therapeutic Target in Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-123794 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1370-1370 ◽

Cited By ~ 1

Author(s):

Anilkumar Gopalakrishnapillai ◽

Allison Kaeding ◽

Christoph Schatz ◽

Anette Sommer ◽

Soheil Meshinchi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Combination Therapy ◽

Therapeutic Target ◽

Myeloid Leukemia ◽

Antibody Drug Conjugate ◽

Pediatric Acute Myeloid Leukemia ◽

Pediatric Aml ◽

Acute Myeloid ◽

Antibody Drug

Pediatric acute myeloid leukemia (AML) continues to have a cure rate of only 50% despite the use of highly intensive cytotoxic chemotherapy. Transcriptome sequencing of several AML samples by the NCI/COG TARGET AML Initiative identified mesothelin (MSLN) to be highly overexpressed in about one-third of pediatric AML (Tarlock et al., Blood, 128:2873, 2016). Because MSLN is not expressed in normal bone marrow samples (Fan et al., Blood, 130:3792, 2017) and only to a low level in other human organs and tissues, MSLN is an attractive therapeutic target for pediatric AML (Kaeding et al., Blood, 130:2641, 2017). The anti-MSLN antibody-drug conjugate (ADC) anetumab ravtansine (BAY 94-9343) generated by conjugating MSLN-antibody with tubulin inhibitor DM4 (Meso-ADC), and isotype control antibody conjugated with the same drug (Iso-ADC) were used to evaluate the efficacy of MSLN targeting in vivo. MSLN-overexpressing K562 (K562-MSLN) CML cells and MV4;11 (MV4;11-MSLN) AML cells were generated by lentiviral transduction of MSLN cDNA. Cell line-derived xenografts (CDX) were created by injecting the MSLN-transduced or parental (MSLN-) cells into NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when the median percentage of human cells in mouse peripheral blood was greater than 0.5%. K562-MSLN CDX mice treated with Meso-ADC (5 mg/Kg Q3dx3, i.v.) survived a median of 46 days longer than those treated with Iso-ADC (P=0.0011) and significantly longer than comparison groups, including K562-MSLN CDX mice treated with daunorubicin and Ara-C (DA, P=0.0008) or untreated (P=0.0018) (Fig. 1A). Median survival of K562 CDX mice treated with Meso-ADC, Iso-ADC, or untreated was similar (Fig. 1B). MV4;11-MSLN CDX mice treated with Meso-ADC exhibited complete remission and remained disease-free at 1 year post cell injection, with AML cell burden remaining <0.1% throughout the study period (Fig. 1C). In contrast, MV;11-MSLN CDX mice treated with Iso-ADC or untreated succumbed to disease at 72 and 38 days, respectively. Taken together, these results indicate that Meso-ADC was efficacious in reducing leukemia burden, and this effect required MSLN expression in target cells. We have generated a panel of patient-derived xenograft (PDX) lines by transplanting and serially propagating primary pediatric AML samples into NSG-SGM3 mice. The efficacy of Meso-ADC was also evaluated in a systemic PDX model using a MSLN+ PDX line (NTPL-146). NTPL-146 PDX mice treated with Meso-ADC (5 mg/Kg, Q3dx3 -x2 cycles) survived a median of 50 days longer than those treated with Iso-ADC (P=0.0018, Fig. 1D, arrows indicate time when each treatment cycle was initiated). In an independent experiment with NTPL-146 PDX mice, a survival benefit of Meso-ADC treatment over no treatment was observed after 1 cycle of Meso-ADC treatment (5 mg/Kg, Q3dx3, P=0.0019, Fig. 1E). Additionally, a combination therapy strategy with daunorubicin and Ara-C followed by Meso-ADC (DA -> Meso-ADC) resulted in improved median survival over Meso-ADC (P=0.0027) or DA treatment alone (P=0.0018) (Fig. 1E). The disseminated MSLN+ leukemia mouse models described herein support MSLN-targeted antibody-drug conjugate as a potential treatment strategy in MSLN+ AML. Furthermore, we provide the first in vivo demonstration of synergy between MSLN-targeted therapy and conventional chemotherapy in MSLN+ AML, warranting additional investigation to validate and optimize novel strategies for combination therapy. Figure 1 Disclosures Kaeding: Celgene: Employment. Schatz:Bayer AG: Employment. Sommer:Bayer AG: Employment, Equity Ownership.

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Identification of Gene Expression Signatures Accurately Predicting Cytogenetic Subtypes in Pediatric Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v112.11.1509.1509 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1509-1509 ◽

Cited By ~ 1

Author(s):

Brian V Balgobind ◽

Marry M van den Heuvel-Eibrink ◽

Renee X Menezes ◽

Dirk Reinhardt ◽

Iris H.I.M. Hollink ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Type I ◽

Pediatric Acute Myeloid Leukemia ◽

Genetic Aberrations ◽

Gene Expression Signatures ◽

Pediatric Aml ◽

Normal Cytogenetics ◽

Acute Myeloid

Abstract Pediatric acute myeloid leukemia (AML) is a heterogeneous disease, which is classified according to the WHO classification, based on morphology, immunophenotyping and non-random genetic aberrations. AML is hypothesized to arise from two different types of genetic aberrations, i.e. type-I (proliferation enhancing) mutations and type-II (differentiation impairing) mutations. To detect genetic aberrations multiple techniques such as conventional karyotyping, FISH and RT-PCR are being used. In addition to conventional karyotyping, the latter two techniques revealed a higher frequency of aberrations. Still, failures or false negative results should be taken into account. Recent studies have focused on the potential of gene expression profiling (GEP) to classify acute leukemias. To study the clinical value of classification by GEP, we first used a double-loop cross validation (CV) to avoid over-fitting of GEP data and, subsequently, addressed whether the identified GEP was suitable to classify pediatric AML cases in a second independent group of cases. Affymetrix Human Genome U133 plus 2.0 microarrays were used to generate gene expression profiles of 257 children with AML, with high blast counts, if necessary, after enrichment (~80% or more) and good quality RNA. Probe set intensities were normalized using the variance-stabilizing normalization (VSN) implemented in R (version 2.2.0). The patient group was divided into a test cohort (n=170) and an independent validation cohort (n=87). The test cohort was used to construct the classifier using two levels of CV: the minimum number of predictive genes was estimated using a 10-fold CV on random subsets of about 113 (~2/3 of total) patients; the accuracy of the obtained classifier is estimated on the remaining 57 (~1/3) patients. Candidate genes to represent the GEP in the classifier were those genes that discriminated AML subtypes according to an empirical Bayes linear regression model (Bioconductor package: Limma). To construct a reliable classifier it was sufficient to use 75 probe sets, representing the top 15 discriminating probe sets for MLL-gene rearranged AML, t(8;21), inv(16), t(15;17) and t(7;12). These subtypes represented ~50% of the included patients. The remaining patients either had normal cytogenetics, random aberrations or no data available (cytogenetic failure). Due to the heterogeneity of these remaining groups discriminative probe sets were not found. This classifier could reliably predict the 5 subtypes with a median accuracy of 93%. Validation of the classifier on the independent cohort confirmed that the sensitivity and accuracy was more than 99%. No gene expression signatures could be found for the molecular aberrations NPM1, CEBPa, MLL-PTD, FLT3, C-KIT, RAS or PTPN1, possibly due to the small number of cases. However, specific gene expression signatures were found for FLT3-ITD within the subset of cases with t(15;17) or normal cytogenetics. Importantly, a high expression of HOXB-cluster related genes was found in cases with FLT3-ITD and normal cytogenetics. In conclusion, GEP can correctly predict several important cytogenetic subtypes of pediatric AML, including cases that are currently classified using different cytogenetic techniques and cases with failed cytogenetic analysis. Prospective studies are needed to validate the use of GEP in the classification of pediatric AML, especially to provide information on its utility in clinical practice. Increasing numbers in rare subtypes may result in the discovery of genes discriminative for them, and may foster GEP as a new diagnostic tool.

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BCOR and BCORL1 Mutations In Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3815.3815 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3815-3815

Author(s):

Malou C.H. Hermkens ◽

Marry M. van den Heuvel-Eibrink ◽

Susan T.J.C.M. Arentsen-Peters ◽

Maarten W.J. Fornerod ◽

Andre Baruchel ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Genetic Background ◽

Myeloid Leukemia ◽

Disease Outcome ◽

Next Generation ◽

Genetic Syndrome ◽

Pediatric Acute Myeloid Leukemia ◽

Exon 4 ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Introduction In pediatric acute myeloid leukemia (AML) current survival rates are approximately 70%, but further improvements are required to improve disease outcome. Prognosis is correlated to early response to treatment and genetic aberrations (Creutzig et al, 2012). In approximately 20% no cytogenetic aberrations can be identified. In some of these cases repetitive aberrations, such as NPM1 mutations or cryptic translocations including NUP98-translocations (Hollink, 2011 and De Rooij, 2013) have been found. Recently, mutations in BCOR and BCORL1, both located on the X-chromosome, were found in adult AML using next generation sequencing. They both are transcriptional co-repressors, although with distinct binding targets (Tiacci, Heamatologica, 2012), and are thought to represent a novel mechanism of leukemogenesis. Somatic inactivating BCOR mutations were identified in 4% of adult cytogenetically normal (CN-) AML patients, predominantly located in exon 4, but also in other exons (Grossmann et al, 2011). Of interest, germline BCOR mutations cause the X-linked oculo-facio-cardio-dental genetic syndrome, which may occur due to its function as a co-repressor of the BCL6 gene. Somatic inactivating BCORL1mutations were found in 6% of adult AML patients (Li et al, 2011); all mutations were located in exon 4. Their exact role in AML and the targets of their co-repressive transcriptional activity has not been elucidated as yet (Tiacci, Haematologica, 2012). Methods We screened newly diagnosed pediatric AML patients for the presence of BCOR and BCORL1 mutations using direct sequencing of the complete coding sequence of both genes starting with a cohort of 86 patients including all cytogenetic subgroups patients, and later expanding this with an additional 146 patients for BCORL1screening of exon 4. This cohort was enriched for samples from CN-AML patients (56% and 21% respectively). Samples were obtained from the Dutch Childhood Oncology Group (DCOG; The Hague, The Netherlands), the AML-BFM-SG; Hannover, Germany and Prague, Czech Republic, and the Hôpital Robert Debré (Paris, France). Results A single BCOR mutation was found in 1 patient only with CN-AML. The mutation, p.A854T, was located in exon 4. The patient was a 4 year old boy with a FAB M1, WBC 354 x 109/L, who is alive 45 months after diagnosis. In addition, only 1 patient carried a BCORL1 mutation. The mutation, located in exon 4, p.G158X, caused a premature stop-codon. The male patient was diagnosed with secondary AML, aged 17 years, with normal cytogenetics and a WBC of 9.4 x 109/L, FAB M1, and died 3 months after diagnosis. Multiple recurrent SNPs were observed for both BCOR (rs5917933: 7/86 pts (91.9%); rs6520618: 15/86 (17.4%); rs144606152: 6/86 (7.0%)) and BCORL1 (rs4830173: 232/232 (100%); rs5932715: 36/232 (15.5%)), all in exon 4. No relation could be found between the presence of SNPs and disease outcome. Conclusions BCOR and BCORL1 mutations occur in less than 1% of pediatric AML patients. These data provide further evidence for the differences in genetic background between pediatric and adult AML. Separate next-generation studies should be performed to elucidate the genetic background of pediatric CN-AML. This project was funded by KIKA, project number 64, entitled: Aberrant signal transduction profiling in pediatric AML. Disclosures: No relevant conflicts of interest to declare.

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ETV6 Aberrations Are a Recurrent Event in Pediatric Acute Myeloid Leukemia with Poor Clinical Outcome

Blood ◽

10.1182/blood.v124.21.1012.1012 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1012-1012 ◽

Cited By ~ 2

Author(s):

Jasmijn de Rooij ◽

Eva Beuling ◽

Maarten Fornerod ◽

Askar Obulkasim ◽

André Baruchel ◽

...

Keyword(s):

Myeloid Leukemia ◽

De Novo ◽

Poor Clinical Outcome ◽

Pediatric Acute Myeloid Leukemia ◽

Genetic Abnormalities ◽

Pediatric Aml ◽

Split Signal ◽

Treatment Related Mortality ◽

Related Mortality ◽

Acute Myeloid

Abstract Pediatric acute myeloid leukemia (AML) is a heterogeneous disease and 30-40% of the patients still die. Prognosis is dependent on relevant genetic aberrations. Although many driving genetic alterations causing AML have been defined, in ~20% of the pediatric AML patients the oncogenic events remain unidentified. The ETS-Variant gene 6 (ETV6) encodes a transcription factor that functions as a tumor suppressor gene and is required for proper hematopoiesis in the bone marrow niche. Point mutations, deletions and translocations can lead to silencing of the gene, resulting in loss of transcriptional repression activity. ETV6 aberrations strongly associate with leukemia. In pediatric B-cell precursor acute lymphoblastic leukemia, translocation ETV6/RUNX1 occurs in ~25% of cases. Mutations in ETV6 are identified in ~25% of early immature T-cell ALL and also reported as event in adult AML (Van Vlierberghe et al, J Exp Med 2011; Barjesteh van Waalwijk van Doorn-Khosrovani et al, Oncogene 2005). We previously reported that pediatric AML patients can be divided in three clusters based on HOX-expression; (1) low HOXA/B expression, (2) high HOXA and low HOXB expression, and (3) high HOXA/B expression, and identified new repetitive genetic abnormalities in the third cluster, especially in NUP98. Cluster 1 is mainly represented by core-binding factor (CBF) AML, but in ~20% of these cases we did not find specific genetic abnormalities. Helton et al presented ETV6 aberrations in pediatric CBF-AML at ASH 2011, identified with whole genome sequencing, and with poor clinical outcome. We hypothesized that ETV6 aberrations might reduce the number of patients without known driving abnormality, especially in the low HOXA/B cluster. We screened a large representative de novo pediatric AML cohort for ETV6 mutations in exons 2-8 with direct sequencing, for ETV6 deletions by multiplex ligation-dependent probe amplification and for ETV6 translocations using split signal FISH, and analyzed outcome. In a well-characterized de novo pediatric AML cases with available gene-expression data, 6/275 (2.2%) patients had mutations affecting the predicted amino acid sequence of ETV6 and one had a silent mutation, 4/259 (1.5%) had an ETV6 deletion and 6/65 (9.2%) patients an MNX1/ETV6 translocation. Additionally, we identified 3 cases with a positive split signal FISH suggestive of a break in which ETV6 is involved, and a similar gene expression profile was found in these three cases. The aberrations of ETV6 were seen in patients of all three HOX-groups; n=9, n=6 and n=4 for cluster 1, 2 and 3 respectively. In patients with an ETV6 mutation (n=6) or deletion (n=4) 13 and 38 genes, respectively, were significantly up-regulated, including CLDN5,DPEP1 and BIRC7. This is consistent with the up-regulated genes in functional studies silencing ETV6 in LOUCY cells (Van Vlierberghe et al, J Exp Med 2011). High expression of BIRC7 has been associated with poor prognosis in adult acute leukemia (El-Mesallamy et al, Leuk Res 2011). The median age of patients with an ETV6-mutation or deletion (n=10) was 11.3 years (range 4.0-15.3) and 40% were female. Median WBC was significantly lower (15.1x109/L vs 47.0x109/L, p<0.01) in comparison to other pediatric AML cases. Other cytogenetic aberrations found in the ETV6-mutated or deleted cases were RUNX1/RUNX1T1 (n=3), PML/RARA (n=1), MLL/AF6 (n=1) and one case with an NPM1-mutation. Six out of ten patients encountered a relapse and one patient died of treatment-related mortality. The median age for patients with an ETV6-split signal FISH (n=9) was significantly lower compared to other pediatric AML cases (median 1.1 years vs 9.8, p<0.01), median WBC and sex did not differ. Five out of 9 encountered relapsed/refractory disease whereas 2 cases died of treatment-related mortality. The 3-yr pOS for all ETV6-aberrated patients taken together (n=19) was 37±11% vs 65±3% for the other pediatric AML patients (n=242, p<0.01); the 3-yr pEFS was 26±10% vs 46±3% (p=0.07), and 3-yr pCIR 47±13% vs 37±3% (p=0.24). We conclude that ETV6 aberrations are rare but recurrent in pediatric AML. ETV6 aberrations predict a poor survival, although there was no evidence for an increased relapse incidence in this small cohort. Disclosures No relevant conflicts of interest to declare.

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