WT1 Mutations Research Articles

Genotype-phenotype correlation of WT1 mutation-related nephropathy in Chinese children.

This study aimed to analyze the clinical characteristics of nephropathy associated with WT1 gene mutations in Chinese children and explore the relationship between genotype and clinical phenotype. Cases diagnosed at the Guangzhou Women and Children's Medical Center, were combined with those retrieved from PubMed and China National Knowledge Infrastructure (CNKI) databases from January 2015 to June 2022 and integrated into a study cohort; grouped according to gene mutation sites, clinical phenotype, and renal pathological types. The clinical characteristics between groups were compared, and the relationship between genotype and age of onset, clinical phenotype, and pathological type were retrospectively analyzed. The center enrolled 15 confirmed children: seven cases of non-simple nephropathy, including Denys-Drash syndrome (DDS) and Frasier syndrome (FS); eight cases of isolated steroid-resistant nephrotic syndrome (ISRNS); and 13 cases (86.7%) that progressed to end-stage renal disease (ESRD). The initial hemoglobin and bicarbonate levels of patients with clinical non-simple nephropathy were significantly lower than those with simple nephropathy, whereas the serum creatinine levels were higher than those of patients with simple nephropathy. A total of 75 cases of nephropathy associated with WT1 mutations in the study cohort met the inclusion and exclusion criteria. The most common clinical manifestations of WT1 mutations in this cohort were DDS (29/75, 38.7%) and ISRNS (37/75, 49.3%). A renal biopsy was performed in 43 patients, and the common types of renal pathology were focal segmental glomerulosclerosis (23/43, 53.5%) and DMS (13/43, 30.2%). Within the cohort, there were 12 cases (16.0%) in the exon 8 mutation group, 32 (42.6%) in the exon 9 group, 19 (25.3%) in the intron 9 group, and 12 (16.0%) in other gene site mutation groups. Common sites of WT1 mutations in Chinese children were exons 9 and intron 9. Exon 8 mutations were uniquely correlated with the age of onset within three months [5/7; 71.4%; Adjusted standardized residual (AR) = 4.2]. The renal survival time in the exon 8 mutation group was the shortest (P = 0.003). The molecular and biological characteristics of WT1 mutation-related nephropathy determine the clinical type, pathological features, and renal survival time of the disease; and there was a strong correlation between the genotype and clinical phenotype.

Understanding WT1 Alterations and Expression Profiles in Hematological Malignancies

WT1 is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression for WT1 mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/without WT1 mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences of WT1 into a lentiviral expression vector, we developed a unique tool that can stably overexpress the four WT1 isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.

Framework of clonal mutations concurrent with WT1 mutations in adults with acute myeloid leukemia: Alliance for Clinical Trials in Oncology study

Framework of clonal mutations concurrent with WT1 mutations in adults with acute myeloid leukemia: Alliance for Clinical Trials in Oncology study

Identification of a novel NPM1 mutation in acute myeloid leukemia.

e19019 Background: Nucleophosmin (NPM1) is predominant nucleolar localization. NPM1-mutant acute myeloid leukemia (AML) account for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 WHO classification. The majority of NPM1 mutations reported affect exon 12 (classical type A mutation), account for 75-80% of adult NPM1-mutated AML cases, which generate an extra nuclear export signal (NES) in the C-terminal leading to aberrant cytoplasmic localization. Similar aberrant cytoplasmic translocation has also been reported in NPM1 mutants that identified in exons 9 and 11. Identification of novel non-type A mutants is paramount for diagnosis, risk stratification, and prognosis for targeted population. Methods: Somatic mutation analysis is performed on 566 AML patients using next-generation sequencing (NGS). Immunofluorescence and immunohistochemistry were used to identify the cytological localization of mutant NPM1. HEK-293T cells transiently transfected with plasmids for ectopic expression of the GFP-mutated NPM1 fusion proteins were treated with the specific Crm1/XPO1 inhibitor leptomycin B and to evaluate the NES dependence of their subcellular localization. Results: We identified one AML patient with NPM1 mutant located on exon 5. This patient was a 59-year-old female with de novo AML, with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCCCTGGAACTGGGGAAC, named MutSong) in the middle of exon 5 [variant allele fraction (VAF), 11.9%]. It generated a new NPM1 mutant protein (p.135insALELGN), and containing a leucine-rich NES. Notably, different with all NPM1 mutant in other exons reported so far, our exon 5 mutant retained the functional c-terminal but insert one leucine-rich NES, similar finding was recently reported in NPM1 mutants that identified in exon 5 but on different position. This NPM1 mutant in exon 5 resulted in cytoplasmic localization both by confocal and immunohistochemistry in AML patient sample. HEK-293T overexpressing the new GFP-NPM1 exon 5 fusion protein indicated the aberrant localization in the cytoplasm and partially in nucleoli. Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B, indicated that the novel NES generated by exon 5 mutant is responsible for cytoplasmic localization. This patient carried WT1, IKZF1, JAK2, and NUP98 mutations, and was intravenously treated with daunorubicin plus cytarabine for induction followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, the patient died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT. Conclusions: Our finding of the novel NPM1 mutation in exon 5 supported that beside exon 12, exon 5 mutant is another NPM1 “born to be exported” mutant critical for leukemogenesis.

UBTF tandem duplications define a distinct subtype of adult de novo acute myeloid leukemia

Tandem duplications (TDs) of the UBTF gene have been recently described as a recurrent alteration in pediatric acute myeloid leukemia (AML). Here, by screening 1946 newly diagnosed adult AML, we found that UBTF-TDs occur in about 3% of patients aged 18–60 years, in a mutually exclusive pattern with other known AML subtype-defining alterations. The characteristics of 59 adults with UBTF-TD AML included young age (median 37 years), low bone marrow (BM) blast infiltration (median 25%), and high rates of WT1 mutations (61%), FLT3-ITDs (51%) and trisomy 8 (29%). BM morphology frequently demonstrates dysmyelopoiesis albeit modulated by the co-occurrence of FLT3-ITD. UBTF-TD patients have lower complete remission (CR) rates (57% after 1 course and 76% after 2 courses of intensive chemotherapy [ICT]) than UBTF-wild-type patients. In patients enrolled in the ALFA-0702 study (n = 614 patients including 21 with UBTF-TD AML), the 3-year disease-free survival (DFS) and overall survival of UBTF-TD patients were 42.9% (95%CI: 23.4–78.5%) and 57.1% (95%CI: 39.5–82.8%) and did not significantly differ from those of ELN 2022 intermediate/adverse risk patients. Finally, the study of paired diagnosis and relapsed/refractory AML samples suggests that WT1-mutated clones are frequently selected under ICT. This study supports the recognition of UBTF-TD AML as a new AML entity in adults.

The diagnostic significance of cerebrospinal fluid cytology and circulating tumor DNA in meningeal carcinomatosis.

The objective of this research is to investigate the clinical application value of cerebrospinal fluid (CSF) cytology and circulating tumor DNA (ctDNA) in lung adenocarcinoma (LUAD) meningeal metastasis-meningeal carcinomatosis (MC), and to further explore the possible molecular mechanisms and drug treatment targets of LUAD meningeal metastasis by next-generation sequencing (NGS). We retrospectively analyzed LUAD with MC in 52 patients. CSF cytology was carried out using the slide centrifugation precipitation method and May-Grüwald-Giemsa (MGG) staining. Tumor tissue, plasma and CSF ctDNA of some MC patients were detected by NGS. Of the 52 MC patients, 46 (88.46%) were positive for CSF cytology and 34 (65.38%) were positive for imaging, with statistically significant differences in diagnostic positivity (P < 0.05). In 32 of these patients, CSF cytology, cerebrospinal fluid ctDNA, plasma ctDNA and MRI examination were performed simultaneously, and the positive rates were 84.38, 100, 56.25, and 62.50% respectively, the difference was statistically significant (P < 0.001). Analysis of the NGS profiles of tumor tissues, plasma and CSF of 12 MC patients: the mutated gene with the highest detection rate was epidermal growth factor receptor (EGFR) and the detection rate were 100, 58.33, and 100% respectively in tumor tissues, plasma and CSF, and there were 6 cases of concordance between plasma and tissue EGFR mutation sites, with a concordance rate of 50.00%, and 12 cases of concordance between CSF and tissue EGFR mutation sites, with a concordance rate of 100%. In addition, mutations not found in tissue or plasma were detected in CSF: FH mutation, SETD2 mutation, WT1 mutation, CDKN2A mutation, CDKN2B mutation, and multiple copy number variants (CNV), with the most detected being CDKN2A mutation and MET amplification. CSF cytology is more sensitive than traditional imaging in the diagnosis of meningeal carcinomatosis and has significant advantages in the early screening and diagnosis of MC patients. CSF ctDNA can be used as a complementary diagnostic method to negative results of CSF cytology and MRI, and CSF ctDNA can be used as an important method for liquid biopsy of patients with MC, which has important clinical significance in revealing the possible molecular mechanisms and drug treatment targets of meningeal metastasis of LUAD.

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia.

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Somatic, Genetic and Epigenetic Changes in Nephrogenic Rests and Their Role in the Transformation to Wilms Tumors, a Systematic Review.

To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed.

Gene expression studies of WT1 mutant Wilms tumor cell lines in the frame work of published kidney development data reveals their early kidney stem cell origin.

In order to get a better insight into the timing of WT1 mutant Wilms tumor development, we compared the gene expression profiles of nine established WT1 mutant Wilms tumor cell lines with published data from different kidney cell types during development. Publications describing genes expressed in nephrogenic precursor cells, ureteric bud cells, more mature nephrogenic epithelial cells and interstitial cell types were used. These studies uncovered that the WT1 mutant Wilms tumor cells lines express genes from the earliest nephrogenic progenitor cells, as well as from more differentiated nephron cells with the highest expression from the stromal/interstitial compartment. The expression of genes from all cell compartments points to an early developmental origin of the tumor in a common stem cell. Although variability of the expression of specific genes was evident between the cell lines the overall expression pattern was very similar. This is likely dependent on their different genetic backgrounds with distinct WT1 mutations and the absence/presence of mutant CTNNB1.

Restratifying the prognosis of acute myeloid leukemia patients with CEBPA double mutations based on CSF3R mutations and measurable residual disease

Objective: This investigation aims to assess the impact of CSF3R mutations and the presence of measurable residual disease (MRD) on the prognosis of patients with CEBPA double mutations who have acute myeloid leukemia (AML) . Methods: The prognostic significance of these two factors was examined in the present study, which included 66 patients with complete genetic mutations and sequential MRD information. Results: Following the second course of chemotherapy, the MRD status and CSF3R mutations of these patients were linked to their long-term prognosis. CSF3R mutated patients showed inferior relapse-free survival (RFS) (5-year RFS: 15.2% vs 38.7% , P=0.006) and overall survival (OS) (5-year OS: 18.2% vs 60.6% , P=0.038) compared with those with wild-type CSF3R. After the second course of chemotherapy, patients with negative MRD had an RFS of 64 months and an OS of not reaching, which was significantly longer than that of patients with positive MRD (15 and 48 months, and the P value were 0.004 and 0.050, respectively) . CSF3R mutations (HR=0.317, 95% CI 0.129-0.779, P=0.012) , WT1 mutations (HR=0.304, 95% CI 0.115-0.804, P=0.016) , and NRAS mutations (HR=0.153, 95% CI 0.061-0.385, P<0.001) were all independently associated with a poor prognosis for RFS, and CSF3R mutations and positive MRD tended to be independently associated with a poor prognosis for OS, according to the results of a Cox proportional-hazards model analysis (P values were 0.071 and 0.088, respectively) . The patients were divided into three groups based on their CSF3R mutation status and MRD status following treatment: wide-type CSF3R and negative MRD, mutated CSF3R or positive MRD, and mutated CSF3R and positive MRD, which showed significantly different RFS (P<0.001) and OS (P=0.006) . Conclusion: Both CSF3R mutations and positive MRD were associated with poor outcome in AML patients with CEBPA double mutations. An integrity model based on these two factors may be beneficial for accurately evaluating the prognosis of these patients.

UBTF-internal tandem duplication as a novel poor prognostic factor in pediatric acute myeloid leukemia.

The prognosis of pediatric acute myeloid leukemia (AML) has improved via stratification therapy. However, relapse or death occurs in 30%-40% of cases. Novel genetic factors for pediatric AML need to be elucidated to improve prognosis. We detected recurrent internal tandem duplication in upstream binding transcription factor (UBTF-ITD) in 1.2% (6/503) of Japanese pediatric patients with de novo AML. No UBTF-ITD was detected in 175 adult patients with AML or in 65 cell lines that included 15 AML, 39 acute lymphoblastic leukemia, five chronic myeloid leukemia, and six neuroblastoma cell lines. All UBTF-ITDs were found in exon 13 and shared a duplicated region. UBTF-ITD was more frequently detected in patients with trisomy 8, FLT3-ITD, WT1 mutation, and/or high PRDM16 expression (trisomy 8, 3/6; FLT3-ITD, 5/6; WT1 mutation, 2/6; and high PRDM16 expression, 6/6). Gene expression patterns of patients with UBTF-ITD were similar to those of patients with NUP98::NSD1 or FUS::ERG. Survival analysis of the AML-05 cohort revealed that patients with UBTF-ITD had worse outcomes than those without UBTF-ITD (3-year event-free survival, 20% vs. 55%; 3-year overall survival, 40% vs. 74%). Moreover, among the 27 patients with trisomy 8, all three patients with UBTF -ITD had a poor prognosis resulting in early events (relapse or non-complete remission) within 1 year. Our findings suggest that UBTF-ITD may be a novel and significant prognostic factor for pediatric patients with AML.

Pre-transplant gene profiling characterization by next-generation DNA sequencing might predict relapse occurrence after hematopoietic stem cell transplantation in patients affected by AML.

In the last decade, many steps forward have been made in acute myeloid leukemia prognostic stratification, adding next-generation sequencing techniques to the conventional molecular assays. This resulted in the revision of the current risk classification and the introduction of new target therapies. We wanted to evaluate the prognostic impact of acute myeloid leukemia (AML) mutational pattern on relapse occurrence and survival after allogeneic stem cell transplantation. A specific next-generation sequencing (NGS) panel containing 26 genes was designed for the study. Ninety-six patients studied with NGS at diagnosis were included and retrospectively studied for post-transplant outcomes. Only eight patients did not show any mutations. Multivariate Cox regression revealed FLT3 (HR, 3.36; p=0.02), NRAS (HR, 4.78; p=0.01), TP53 (HR, 4.34; p=0.03), and WT1 (HR 5.97; p=0.005) mutations as predictive variables for relapse occurrence after transplantation. Other independent variables for relapse recurrence were donor age (HR, 0.97; p=0.04), the presence of an adverse cytogenetic risk at diagnosis (HR, 3.03; p=0.04), and the obtainment of complete remission of the disease before transplantation (HR, 0.23; p=0.001). Overall survival appeared to be affected only by grade 2-4 acute GvHD occurrence (HR, 2.29; p=0.05) and relapse occurrence (HR, 4.33; p=0.0001) in multivariate analysis. The small number of patients and the retrospective design of the study might affect the resonance of our data. Although results on TP53, FLT3, and WT1 were comparable to previous reports, the interesting data on NRAS deserve attention.

Abstract B017: Dysregulation of ultra long-range DNA methylation canyon interactions in human acute myeloid leukemia

Abstract DNA methylation Canyons (DMCs) are genomic regions devoid of DNA methylation over 3.5kb in hematopoietic stem cells (HSCs). DMCs form ultra long-range interactions spanning decades of megabases specifically in HSCs. These ultra-long DMC interactions between DMCs relies on the strong Polycomb binding inside DMC regions. DMCs are hypermethylated and partially activated in cancer. However, its 3D genomic alterations have not been examined extensively. Here we profiled the high-resolution 3D genomic interactions in 7 primary human acute myeloid leukemia (AMLs). Focusing on DMCs' epigenome state, we found that DMCs in all AMLs are hypermethylated and lose the strong Polycomb enrichment within DMCs in comparison with HSC. Based on the DMC interactions, we discovered two subtypes of AMLs with distinct 3D genomic dysregulation of DMCs’ long-range interactions. In the first AML subtype, DMCs’ interactions are still strong as HSPC but gained leukemia-specific long-range interactions. This subtype is enriched for WT1 mutations and is characterized by the expression of CD34 on blast cells. This particular subtype displayed a global basal level increase in the H3K27me3 mark in the genome. This particular subtype of AML is also sensitive to the EZH2 inhibitor treatment. The EZH2 inhibitor treatment leads to the differentiation of blasts and reduces the colony-forming ability of this subtype of AML. Further dissection shows the EZH2 inhibitors could eradicate the leukemia-specific ultra-long range interactions. In the second subtype of AML, 3D genomic interactions between DMCs significantly lost in AMLs with high HOX cluster gene expression (MLL-r and NPM1c+ AMLs). We further found repressive DMC genes, which are developmental transcriptional factors(TFs) in organ formation are activated in these AMLs. Interestingly, these developmental TFs are involved in forming leukemia-specific long-range interactions independent of CTCF-mediated TADs and chromosomal loops. Among those TFs, IRX3 and IRX5 are frequently activated in MLL-r, NPM1c+ AML and MPAL. These two genes have also been shown before to immortalize bone marrow progenitor cells and block differentiation of these progenitor cells. The loci of these two genes form strong long-range connections to an upstream FTO enhancer mimicking the 3D genomic structure observed in developing muscle and brains. Citation Format: Xue Qing D. Wang, Fan Feng, Yali Dou, Jie Liu, Xiaotian Zhang. Dysregulation of ultra long-range DNA methylation canyon interactions in human acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B017.

Distinct genetic landscapes and their clinical implications in younger and older patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clinically and genetically diverse diseases that impose patients with an increased risk of leukemic transformation. While MDS is a disease of the elderly, the interplay between aging and molecular profiles is not fully understood, especially in the Asian population. Thus, we compared the genetic landscape between younger and older patients in a cohort of 698 patients with primary MDS to advance our understanding of the distinct pathogenesis and different survival impacts of gene mutations in MDS according to age. We found that the average mutation number was higher in the older patients than younger ones. The younger patients had more WT1 and CBL mutations, but less mutated ASXL1, DNMT3A, TET2, SF3B1, SRSF2, STAG2, and TP53 than the older patients. In multivariable survival analysis, RUNX1 mutations with higher variant allele frequency (VAF) and U2AF1 and TP53 mutations were independent poor prognostic indicators in the younger patients, whereas DNMT3A and IDH2 mutations with higher VAF and TP53 mutations conferred inferior outcomes in the older patients. In conclusion, we demonstrated the distinct genetic landscape between younger and older patients with MDS and suggested that mutations impact survival in an age-depended manner.

Tandem Duplications of the UBTF gene in Adult AML: A Rare but Recurrent Alteration Associated with Myelodysplasia and Poor Outcome

The upstream binding transcription factor (UBTF) is a nucleolar protein that regulates the transcription of ribosomal genes as a component of the RNA Pol I pre-initiation complex. Recently, tandem duplication mutations of the UBTF gene (UBTF-TDs) were identified as recurrent alterations in pediatric AML (Umeda et al., 2022), potentially representing a new subtype-defining lesion. UBTF-TDs were found to be associated with normal karyotype (NK) or trisomy 8 (+8), FLT3-ITD and WT1 mutations as well as poor prognosis. Data on the role of UBTF-TD in adult AML are limited. In the present study, we therefore analyzed the prevalence and prognostic significance of UBTF-TDs in a large cohort of adult AML patients (pts). Methods: Pts included were registered and treated in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003, AML60+, SORAML) or the SAL-registry. Screening for UBTF-TDs was done by PCR covering exon 13 of the UBTF gene with 6-FAM labelled primers followed by high-resolution fragment analysis using genomic DNA isolated at diagnosis. Mutations were confirmed by Sanger sequencing. All UBTF mutant samples were further analyzed by targeted next generation sequencing (Archer Myeloid Panel) for the presence of co-occurring alterations. Results: Somatic mutations in UBTF were identified in 52 (1.2%) of 4247 pts. Mutations varied in size (3 to 309 bps), all yielding in-frame insertions, duplications or deletions in exon 13 of the UBTF gene. UBTF-TD AMLs frequently presented with a normal karyotype (46%) or +8 (36.5%). Similar to findings in pediatric pts, the co-mutation profile showed significant differences between UBTF-mut and UBTF-wt pts, i.e. UBTF-TDs were significantly associated with FLT3-ITD (48%, p<.001) or WT1 mutations (52%, p<.001), while DTA co-mutations were exceedingly rare (DNMT3A 1.9%; TET2 3.8%; ASXL1 3.8%) (Fig. 1A). Furthermore, UBTF-TDs and several class-defining lesions, i.e. reciprocal translocations such as RUNX1::RUNX1T1,DEK::NUP214 or mutations in CEBPA or NPM1 were mutually exclusive. The prevalence of UBTF-TDs showed a strong negative correlation with increasing age, ranging from 10% in patients <20 years (yrs) to 0.05% in patients >70 yrs, respectively. Accordingly, pts with UBTF-TDs were significantly younger (median age 41 yrs; range 17-77 yrs) than wt pts (med. 57 yrs; range 15-90 yrs, p<.001). Association with clinical parameters revealed significantly lower hemoglobin levels and platelet counts at diagnosis for UBTF-mut pts compared to UBTF-wt pts (median Hb 8.7 vs. 9.2 g/dL; p=.02; median PLT 31.5 vs 53 x 109/L; p=.003). Although UBTF-TDs mainly affected younger pts, cytomorphologic assessment revealed a clear association with myelodysplastic changes in about 60% of patients. Univariate analysis showed a significantly higher rate of complete remissions (CR) in UBTF-mut pts (92.3%) compared to UBTF-wt pts (70.8%) (p<.001). However, UBTF-mut pts had shorter overall (OS) and event-free survival (EFS). This effect was particularly pronounced in the subgroup of pts under 60 years of age (median OS 20.4 months in UBTF-mut pts vs. 45.3 months in UBTF-wt pts, p=.079; median EFS 6.7 months in UBTF-mut pts vs. 11 months in UBTF-wt pts, p<.001) (Fig.1B), who represented the majority (45/52, 87%) of UBTF-TD patients, and in patients with co-mutations of the WT1 gene (data not shown). AlloSCT performed in CR1 in 11 pts (21%) did not improve outcome. In multivariable analyses, the presence of UBTF-TD was associated with a trend for shorter OS (HR: 1.39; 95% CI 0.98-2, p=.06) and significantly worse EFS (HR: 1.68; 95% CI 1.24-2.3; p<.001). Conclusions: This analysis of a large cohort of adult patients with newly diagnosed AML revealed that UBTFmutations can be found in about 1% of all patients with AML, but are considerably more common in young adults. As in pediatric patients, they are associated with a specific cytogenetic and molecular profile, i.e. high rate of +8, FLT3-ITD and WT1 mutations. As a novel finding, adult UBTF-TD patients frequently showed signs of myelodysplasia. The distinct molecular genotype and the fact that UBTF-TDs were associated with a dismal outcome, even in patients treated with alloSCT in CR1, support the classification of this group as a new disease entity and indicate that novel treatment strategies are needed in this subgroup of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

UBTF tandem Duplications Account for a Third of Advanced Pediatric MDS without Genetic Predisposition to Myeloid Neoplasia

Introduction: Recurrent tandem duplications (TD) in exon 13 of the upstream binding transcription factor gene (UBTF) have recently been identified in 4% of pediatric AML (Umeda et al. 2022). UBTF-TD AMLs were associated with adolescent age, normal karyotype, trisomy 8, as well as FLT3-ITD and WT1 mutations. Response to chemotherapy was poor and probability of overall survival (OS) at 5 years only 44%. Here, we evaluate the role of UBTF-TD in pediatric MDS. Methods: We studied 104 consecutive patients (pts) diagnosed with primary MDS with excess blasts below the age of 18 years enrolled in the prospective studies 98 or 2006 of the European Working Group of MDS in Childhood (EWOG-MDS) in Germany between July 1, 1998 and June 30, 2021. Pts with MDS secondary to chemo- or radiation therapy, acquired aplastic anemia and pts with classical inherited bone marrow failure disorder had been excluded from the analysis. High molecular weight DNA was extracted from stored bone marrow (BM) samples or archived smears. Screening for UBTF-TD was performed by PCR using 6-FAM-labelled primers covering exon 13 of the UBTF gene followed by high resolution fragment analysis. For samples showing aberrant signals indicating UBTF-TDs, Sanger sequencing confirmed the presence of a mutation. All UBTF mutant samples were also analyzed using targeted next generation sequencing (Archer Myeloid panel) for the presence of co-occurring alterations. Results:UBTF-TDs were detected in 25 of the 104 pts (24%; 16 males/9 females), while 79 pts (76%; 45 males, 34 females) showed no UBTF mutation (UBTF-wt). Variant allelic frequencies of UBTF-TD ranged from 11%-79% (median 41%). Median age at diagnosis in UBTF-TD and UBTF-wt pts was not different (11.0 vs. 9.8 years). UBTF-TD pts had higher absolute neutrophil counts (2.7 vs. 0.9 x109/L, p=0.01) and more often normoblasts on peripheral blood smears (92% vs 43%, p<0.01). Blast percentage in BM was similar with <10% blasts in 40% and 38% of UBTF-TD and wt pts, respectively. BM cellularity was normal or increased in 92% of UBTF mutated pts compared to 72% of UBTF-wt pts (p<0.01). Cytogenetic analyses revealed striking differences according to UBTF mutational status with the following karyotypes (TD vs wt): normal (72% vs 33%), -7 (0% vs 30%), del(7q) (0% vs 2%), +8 (24% vs 9%), +8 plus other (0% vs 4%), random (4% vs 7%) and structural complex (0% vs 7%). Probability of OS at 5 years for pts with UBTF-TD was inferior (0.50, 0.28-0.72) to that of pts with UBTF-wt (0.73, 0.62-0.84; p=0.03). Of the 104 pts, 30 pts had a genetic predisposition to myeloid neoplasia, i.e. GATA2 deficiency (n=15), RUNX1 deficiency (n=7), SAMD9/SAMD9L disease (n=6) or neurofibromatosis type 1 (n=2). Of note, none of the pts with predisposition harbored a UBTF-TD. To study the impact of UBTF-TD on outcome of hematopoietic stem cell transplantation (HSCT), pts with germline disease were excluded; of the 72 pts available for HSCT analysis (25 UBTF-TD and 47 UBTF-wt). Therapy prior to HSCT in UBTF-TD and UBTF-wt pts was comparable and consisted of AML-type therapy in 32% vs 21%, less intensive chemotherapy 8% vs 15% and no therapy in 60% vs 64% pts, respectively. Type of donor, stem cell source and conditioning regimen did not differ between the 2 groups, 74% of pts received a busulfan, cyclophosphamide and melphalan conditioning. Event-free survival (EFS) and relapse incidence (CIR) at 5 years of the total cohort were 0.55 (0.39-0.71) and 0.23 (0.15-0.36), respectively. There was no difference in EFS between the 25 UBTF-TD (0.43, 0.22-0.64) and 47 UBTF-wt pts (0.60, 0.45-0.75). However, in pts with normal karyotype, presence of an UBTF-TD was associated with an inferior EFS (0.35 vs. 0.77, p=0.03) and higher CIR compared to wt pts (0.36 vs. 0.06, p=0.03). Co-occurrence of WT1 mutations among UBTF mutated pts was associated with higher CIR (p=0.03); other mutations were less frequent (Figure). Discussion: We identified UBTF-TD in a third of all pediatric pts with advanced primary MDS and absence of germline predisposition. In addition, we noted UBTF-TD at a high frequency in pediatric pts with MDS-related AML (3/15 pts UBTF-TD). In regards to the recent discovery in AML, we conclude that UBTF-TD mutations define a novel subgroup of myeloid neoplasia presenting frequently as pediatric MDS. The high CIR following HSCT particularly in patients with concurrent WT1 mutations outlines an urgent need for new therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinico-Genomic Landscape of Induction Failure in Childhood and Young Adult T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy comprising 10-15% of childhood ALL. Previously, we found that Induction Failure (IF) occurs in approximately 10% of T-ALL cases, 3-fold more than in B-ALL, with only 50% achieving long-term survival (O'Connor et al, JCO 2017). Despite the preponderance of IF in T-ALL, little is known about the factors that predict treatment resistance and influence outcome, or how best to treat these patients. To address this, we present the largest cohort of T-ALL IF reported to date, comprising all IF cases on two large multi-national consecutive randomized trials, UKALL2003 and UKALL2011. Methods The UKALL2003 and UKALL2011 trials recruited 711 patients with T-ALL between 2004-2019; 688 of these patients were assessable at the end of induction (EOI). We supplemented existing trial data with new clinical data, and combined whole genome sequencing (WGS) and RNA-sequencing (RNAseq), to comprehensively characterize the clinical and genetic landscape of T-ALL IF. Results Of 688 T-ALL patients, 10.3% experienced IF. IF was significantly associated with increasing age occurring in 5.6% of patients below 10 years, 12.9% of those aged 10-16 years and 20% of patients over 16 years (p<0.001). Survival was significantly worse in IF (5yr Event-free survival (EFS) 47.9% (95%CI: 35.8 - 59.1) vs. 84.0% (95%CI: 80.7 - 86.8); HR:5.14 (95% CI: 2.82-6.07), p<0.001; 5yr Overall survival (OS) 52.1% (95%CI: 39.7 - 63.1) vs. 90.2% (95%CI: 87.4 - 92.5); HR: 6.38 (95% CI: 4.17-9.76), p<0.001). There was a clear trend towards increasing use of nelarabine over the study period; IF patients treated on UKALL2003 commonly remained on standard trial chemotherapy (74.2%; 26/35), while IF patients treated on the later UKALL2011 trial were more frequently treated with nelarabine and HSCT (68.6%; 24/35, p=0.0007). However, this did not translate into an improved outcome for IF patients, with no clear benefit of either HSCT or Nelarabine therapy. WGS revealed a picture of remarkable genetic heterogeneity, with 25 different initiating lesions converging on 10 subtype-defining T-ALL genes, rather than a single unifying driver of IF (Figure 1). Allocation of cases to conventional T-ALL subtypes found almost half were of the HOXA subtype, significantly more than in responsive T-ALL (p<0.001). This was consistent with immunophenotypic characterisation showing a significant enrichment of the Early T-cell precursor (ETP) subtype, present in 48% of IF cases vs. 10% of non-IF cases (p<0.001), indicating a less differentiated, more stem cell-like leukemia. Although the overall proportion of TAL1 cases was lower in IF, there was an unexpected dominance of non-coding TAL1 enhancer mutations, accounting for 87.5% of TAL1 cases. Importantly, TAL1 cases had a particularly dismal outcome with an OS of 12.5% (0.6-42.3). Notably, none of the 7 patients with a TAL1 non-coding driver lesion survived, and 2 never achieved remission, identifying this as a very high-risk genetic lesion in the context of IF. Mutational profiling found a reduction in mutations in several highly recurrent T-ALL genes (NOTCH1, FBXW7, CDKN2A, LEF1) coupled with an increase in mutations in WT1 and MED12, both of which have been associated with disease resistance in other leukemias. In addition, 2 genes not previously reported in T-ALL were recurrently mutated in IF with mutations in CHD4 in 5 cases and KAT6A in 4 cases, suggesting they may be novel drivers of refractory disease. Survival analyses also showed a significantly poorer survival in patients with mutations in MYCN and NRAS. WGS of samples collected at the end of induction showed no new lesions demonstrating that disease at presentation is representative of true refractory leukemia and that IF does not occur as a result of chemotherapy-induced mutagenesis. Conclusions Our results confirm the poor prognosis of IF in T-ALL and highlight a lack of progress in treatment of this high-risk leukemia, with no clear improvement in outcome despite increased use of nelarabine and HSCT. Genomic analyses identified a marked enrichment for non-coding TAL1 enhancer mutations, characterizing an ultra-high-risk group of patients in the context of IF, in which there were no survivors. The lack of a readily targetable pathway of primary chemoresistance supports alternative strategies, such as those employing immunotherapeutic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a Pediatric AML Patient Derived Xenograft Program

Background: Survival rates for pediatric acute myeloid leukemia (AML) still lag far behind those for most other pediatric cancers. New agents are desperately needed, but new drug development has been hampered by a lack of clinically relevant pediatric AML patient-derived xenograft (PDX) models for research. Texas Children's Hospital (TCH) has the patient volume, a close collaboration between clinical groups and research laboratories, and the infrastructure to address this gap. Methods: TCH patients diagnosed with AML are offered participation in local research. Most samples from local patients are enriched for mononuclear cells (MNCs) and injected fresh on the day of collection. Additionally, cryopreserved bone marrow samples from patients who enrolled on Children's Oncology Group clinical trials and consented to bank tissue for research have been used for PDX generation. Leukemia cells are tail vein injected (2x10e5 cells per mouse) into immunodeficient mice without conditioning. Most models are generated in the NOD.scid.Il2Rγc-/-/SCFh/h/GM-CSFh/h/IL-3h/h (NSGS) strain. Mice are closely monitored for human CD45+/CD33+/CD3- (hAML) cells in peripheral blood by flow cytometry. Once moribund, the mice are humanely euthanized, and bone marrow, peripheral blood, and spleen are harvested and evaluated by flow cytometry for disease burden. Bone marrow cells are injected by tail vein into secondary recipients. Serially transplanting PDX models are validated and characterized by short tandem repeat (STR) fingerprinting and DNA/RNA sequencing. Results: Thus far, we have developed 24 serially transplanting pediatric AML PDX models representing a variety of clinical and cytomolecular categories. The patients from whom these models were derived ranged in age from 0.5-20 years of age at diagnosis (mean 9.4 years). Most are derived from samples collected at diagnosis. For AML005, we have a second serially transplanting model derived from relapse. Our collection includes 8 models with KMT2A fusions, 3 with NPM1 mutations, 4 with WT1 mutations, 3 with FLT3 internal tandem duplications or point mutations, 1 with a NUP98 rearrangement, and 1 CBFA2T3-GLIS2 model. Model identities are validated by comparing the PDX STRs, flow cytometry, and fluorescence in situ hybridization results with matched samples from the patient of origin. For 3 models we also have targeted DNA sequencing for 174 recurrent leukemia-associated mutations for both the patient and the corresponding PDX model. All mutations detected in the patient samples were also detected in the PDX models. The Pearson correlation coefficients comparing the variant allele frequencies for each patient-PDX pair range from 0.82 to 0.99, indicating that the models are faithful representations of the original disease even at the subclone level. A summary of the current collection of serially transplanting models is provided in Table 1. Over 15 additional samples have engrafted once and are currently in secondary recipients. Data on established and validated models are being made publicly available through the Baylor PDX Portal (https://pdxportal.research.bcm.edu), where investigators from Baylor and labs worldwide can access information about our collection and request viably frozen samples for their own research purposes. Our models have been distributed to labs around the world for preclinical studies of novel therapeutics. Conclusion: Through development and distribution of unique pediatric AML PDX models, we hope to create a renewable resource to speed preclinical research and expedite development of drugs for clinical testing for children with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

UBTF-TD Expression Is Necessary and Sufficient for Myeloid Cell Expansion

Children with acute myeloid leukemia (AML) have a high relapse rate and dismal overall survival. This can be attributed to a limited number of targeted therapies and a poor understanding of the molecular pathogenesis of AML in childhood. Our group recently identified a subtype of pediatric AML defined by recurrent somatic in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) (Umeda et al., 2022). UBTF-TDs are primarily found in children (~4% of cases at diagnosis), enriched in relapse (~9% of relapse cases), and associated with minimal residual disease (MRD) positivity. UBTF-TD mutations are mutually exclusive with known subtype-defining oncoproteins yet are associated with normal karyotype or trisomy 8 and tend to co-occur with WT1 mutations and FLT3-ITD. Wild-type (WT) UBTF is involved in nucleosome maintenance, chromatin remodeling, and rRNA biogenesis, but the consequences of the tandem duplications on UBTF function, and possibly leukemogenesis, are unknown. We found that expression of UBTF-TD in cord-blood CD34+ (cbCD34+) cells results in increased replating capacity compared to WT-UBTF overexpression as measured by colony forming unit (CFU) assay (UBTF-TD > 4 rounds, WT-UBTF < 2 rounds, q-value < 0.001) as well as increased cellular proliferation. UBTF-TD expression also maintains expression of the stem-cell marker CD117, decreases terminally differentiated myeloid cells (CD11b), and activates the HOXB gene cluster-recapitulating the transcriptional signature observed in patients with UBTF-TD alterations. We, therefore, hypothesized that UBTF-TD expression is a leukemia-initiating event and that leukemogenesis is dependent on UBTF-TDexpression. To assess the leukemic potential of UBTF-TD in vivo, we expressed UBTF-TD in cbCD34+ cells and transplanted them into humanized NSG-SGM3 mice (hSCF hGM-CSF, IL-3) to assess the leukemic potential of UBTF-TD. We observed a rapid in vivo expansion of UBTF-TD expressing cells with an average latency of 11.7 weeks. Peripheral blood immunophenotyping of these mice identified an expansion of myeloid cells (CD33+, CD11b+) and splenomegaly compared to wild-type controls (p-value = 0.0002). These data suggest that UBTF-TDis sufficient to promote myeloid expansion in vivo. To test whether human cbCD34+ myeloid cell expansion is dependent on UBTF-TD expression, we developed an in vitro inducible degradation model by integrating a targetable protein (FKBP12F36V) into our standard lentiviral overexpression vector. This system (dTAG) allows for rapid and direct control of UBTF-TD protein expression. Degradation is achieved by treating these cells with small molecules (i.e., dTAG-13) that recruit the FKBP12F36V-HA-UBTF-TD fusion protein to E3-ubiquitin ligase complex for ubiquitination and subsequent proteasome degradation. Our data shows that the FKBP12F36V tag has no effect on proliferation and replating capacity of UBTF-TD cbCD34+ cells. Treatment with 1 uM dTAG-13 quickly depleted FKBP12F36V-HA-UBTF-TD protein within 1 hour of treatment (fold change when compared to DMSO > 75%); this protein was completely undetectable by western blot after 4hrs. These cells displayed a reduction of CD117 surface marker expression (p-value < 0.0001) as measured by flow cytometry-highlighting a loss in stemness. Furthermore, long-term depletion of UBTF-TD resulted in decreased proliferation (p-value < 0.0001) and cell viability (p-value < 0.0001). This coincided with an increase in apoptosis as measured by Annexin V staining (p-value < 0.0001). Collectively, our data suggest that UBTF-TD expression is sufficient to promote myeloid cell expansion and a leukemic expression profile. This study will help guide therapeutic development in AML as our data shows that directly targeting UBTF-TD for degradation is sufficient to reverse UBTF-TD myeloid cell expansion. Umeda, M., Ma, J., Huang, B. J., Hagiwara, K., Westover, T., Abdelhamed, S., Klco, J. M. (2022). Integrated genomic analysis identifies UBTF tandem duplications as a recurrent lesion in pediatric acute myeloid leukemia. Blood Cancer Discov. doi:10.1158/2643-3230.BCD-21-0160

RUNX1 Mutations Have Distinct Prognostic and Clinical Implication in Pediatric AML: Results of the AML-BFM Study Group

Background: Runt-related transcription factor 1 (RUNX1) critically regulates hematopoiesis and contributes to leukemogenesis when mutated. In adult AML, RUNX1 mutations (RUNX1mut) have been identified as negative prognosticator associated with distinct clinicopathological features. These findings prompted the inclusion of RUNX1mut into the recent WHO classification as a new provisional entity in AML. However, the significance of RUNX1mut in pediatric AML remained to be validated. Methodology: To determine the impact of RUNX1mut (OMIM No. 151385) in pediatric AML, a German cohort of 512 AML patients (age <18 years) was retrospectively analyzed. Patients with diagnosis between 2015 and 2021 were enrolled to either the AMLR12 or AMLR17 register of the AML-BFM study group (Essen, Germany). Promyelocytic leukemia, Down-syndrome, and treatment related AML were excluded. Mutational screening of 52 different leukemia-specific genes was performed by next-generation sequencing (TruSight Myeloid panel, Illumina, San Diego, California, USA). RUNX1mut were confirmed by PCR and subsequent Sanger sequencing. Probability of overall survival (OS) and event-free survival (EFS) was determined using the Kaplan-Meyer method and log-rank test. Cox model was used for uni- and multivariable regression analyses. Results: In this cohort of childhood AML, 23 of 512 patients (4.5%) harbored 24 different RUNX1mut. RUNX1mut was associated with older average age at diagnosis (11.5 vs. 8.3 years, p = 0.01*) and male gender (63% vs. 50%, p = 0.06). Compared with non-mutated (wt) samples, RUNX1mut was more frequently allocated to myelomonocytic AML (FAB M4, 30% vs. 18%, p = 0.09), followed by AML with minimal differentiation and without maturation (FAB M0, 4.3% vs. 1.5%, p = 0.3 and FAB M1, 22% vs. 14%, p = 0.2), however without significance. Cytogenetically, RUNX1mut tends to correlate with -7/7q, (13% vs. 5%, p = 0.14) and most frequently accompanied by t(8;21) and trisomy 8, showing almost equal distribution (13% vs. 12%, p = 0.75 and 13% vs. 16%, p = 1.0). Together, except of -7/7q, no correlations between RUNX1mut and recurrent cytogenetic aberrations were observed. In the molecular analysis, FLT3-ITD was found to be the most frequently co-occurring mutation and significantly correlated with RUNX1mut (44% vs. 23%, p = 0.02*). Additionally, RUNX1mut was associated with WT1 mutations (26.1% vs. 12.4%, p = 0.1), but mutually exclusive of KIT, KRAS and NPM1 aberrations (10.8% vs. 0%, p = 0.15, 10.2% vs. 0%, p = 0.15 and 9.2% vs. 0%, p = 0.25). Contrary to reports on RUNX1mut in adult AML, we could not confirm exclusiveness of the recurrent aberration CEBPA (13% vs. 6.2%, p = 0.18). A broad range of co-occurring mutations, cooperating with RUNX1mut was found (Figure 1 a). Overall, the number of mutations was significantly increased in RUNX1mut group compared with the RUNX1wt group (average number 2.9 (range 1-4) vs. 2.0 (range 1-6), p < 0.001***). Paired analyses of initial and relapse samples in 8 cases suggest instability of RUNX1mut which was present only at relapse in 5 out of 8 cases and at initial diagnosis in a single case. Together, these results point to RUNX1 being a late rather than an early event in the pathogenesis of pediatric AML. Noteworthy, RUNX1mut did not have an adverse effect on 5-year OS (83.3±8.9% vs. 78.2±2.1%, p = 0.33) (Figure 1 b) or EFS (53.5±11% vs. 61.6±2.5%, p = 0.53) in pediatric AML and was not correlated with a worse response rate. This was supported in multivariable analyses, where RUNX1mut was found to have no effect on OS or EFS independent of other prognostic relevant covariates. In subset analysis, the co-occurring mutations FLT3-ITD and WT1 had no additional prognostic impact on RUNX1mut AML. Same pattern without prognostic difference of RUNX1mut for OS and EFS was determined in subset analysis for stem cell transplantation, age, number of mutation and risk groups. Together, these results failed to establish any prognostic value of RUNX1mut in childhood AML. Conclusion: This is the most comprehensive study assessing the impact of RUNX1mut in a large pediatric cohort. Unlike adult AML (Gaidzik et al., 2011 and 2016), our results provide evidence for a different clinicopathologic impact of RUNX1mut in pediatric AML, arguing against its classification as a distinct entity in childhood AML. These results may broaden the perspective on the pathological significance of RUNX1mut for leukemogenesis in pediatric AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal