WT1 Gene Research Articles

A lasso and random forest model using flow cytometry data identifies primary myelofibrosis.

Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38- cells on CD34+cells, CD13dim+CD11b- cells in granulocytes, CD38str+CD19+/-plasma, and CD123+HLA-DR-basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38- on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

Outcome of illuminated microcatheter-assisted circumferential trabeculotomy following failed angle surgery in PAX6 aniridic glaucoma: a case report and literature review

BackgroundAniridia is a rare eye disorder with a high incidence of glaucoma, and surgical intervention is often needed to control the intraocular pressure (IOP). Here, we reported a case of illuminated microcatheter-assisted circumferential trabeculotomy (MAT) performed on an aniridic glaucoma patient following a previous failed angle surgery. The surgical procedures for aniridic glaucoma were also reviewed.Case presentationA 21-year-old man, diagnosed with aniridic glaucoma, came to our hospital consulting for the poor control of left eye’s IOP despite receiving goniotomy surgery 3 years ago. The IOP was 26 mmHg with maximum topical antiglaucoma eyedrops. The central cornea was opaque and the majority of iris was absent. The gonioscopy and ultrasound biomicroscopy (UBM) demonstrated that 360° anterior chamber angle was closed. The whole exome sequencing of peripheral blood confirmed a 13.39 Mb copy number loss at chromosome 11p15.1p13, containing PAX6 and WT1 gene. The 360° MAT surgery was performed on his left eye. At 1-year follow-up, the IOP was 19mmHg with 2 kinds of topical antiglaucoma medications, and the postoperative UBM demonstrated the successful incision of the anterior chamber angle.ConclusionsThe case presented here exhibited a case of aniridic glaucoma treated by MAT surgery. The MAT surgery may be an effective option for IOP control in aniridic glaucoma patients following a previous failed angle surgery.

Abstract 130: Multi-omic analysis of desmoplastic small sound cell tumors reveal distinct epithelial and neuroendocrine lineages

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive sarcoma subtype that is usually uncurable. All malignant cells harbor a pathognomonic EWSR1::WT1 fusion protein (FP), but FP-targeted agents are nonexistent, and less than 20% of patients survive beyond five years. Lacking the availability of FP-targeted drugs, we seek to understand how the FP regulates tumor cells to promote the characteristic multilineage phenotypes of epithelial and neuroendocrine (NE) cell fates to discover a therapeutic opportunity. Herein, we share the results of gene expression and chromatin accessibility analyses to explore fundamental mechanisms explaining epithelial and NE biology in DSRCT. We profiled 85,640 nuclei from fresh-frozen tissue of nine DSRCT patients post-chemotherapy using single-nucleus RNA-sequencing (snRNA-seq) and ATAC-sequencing (snATAC-seq). A subset of DSRCT patients with low or negative expression of AR expressed partial NE reprogramming, akin to NE prostate cancer (NEPC), exhibiting NE markers (SYP, ENO2, CHG, FOXA2, ASCL1, and SOX2). Another patient subset expressed epithelial markers (MUC1, MUC6, KRT18, KRT23, CDH1). One specimen appeared to exhibit both markers - implying a hybrid or a poorly differentiated phenotype. This suggests that DSRCT could be classified into three subtypes: 1) Epithelial/AR-positive, 2) NE-positive, and 3) hybrid AR/NE. We used CytoTRACE to predict differentiation states. As expected, normal cells (i.e., fibroblasts and immune cells) were the most differentiated. Neoplastic cells, identified by EWSR1::WT1 gene targets and copy number variations, had lower differentiation scores, indicative of a less differentiated phenotype. The NE subtype had the lowest differentiation score indicative of open chromatin and greater plastic capacity, previously implicated in NEPC. SnATAC-seq revealed that the WT1 motif was enriched in malignant cells. Epithelial/AR-positive subtypes were enriched in DNA-response elements for nuclear receptors (ARE, GRE, and PGR) and the forkhead box family of transcription factors (FOXA1, a known pioneer factor for AR). The NE subtype lacked the ARE, GRE, and PGR motifs but was enriched with FOXA1 and neural-specific motifs, including NEUROD1, ATOH1, OLIG2, and NANOG.We show significant heterogeneity in DSRCT phenotypes, where we observe both epithelial and NE lineages. Initially, we detected the Epithelial/AR-positive and NE-positive lineages by gene expression, but the snATAC-seq data also supports this. Although the emergence of the NE subtype in prostate cancer is generally in response to therapy, how it emerges in DSRCT is yet unknown. The CytoTRACE analysis suggests that NE dedifferentiated from the more differentiated Epithelial/AR-positive subtype, but this remains an active area of investigation. Finally, we are working to understand the Epithelial/AR and NE in DSRCT dimorphic states. Citation Format: Danh Truong, Emre Arslan, Veena Kochat, Sandhya Krishnan, Clement Agyemang, Margarita Divenko, Davis Ingram, Rossana Lascano, Akshay Basi, Javier Gomez, Hannah Beird, Chia-Chin Wu, Jared Burks, P Andrew Futreal, Alexander Lazar, Ravin Ratan, Najat Daw, Andrea Hayes, Kunal Rai, Joseph Ludwig. Multi-omic analysis of desmoplastic small sound cell tumors reveal distinct epithelial and neuroendocrine lineages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 130.

WT1-related disorders: more than Denys-Drash syndrome.

Historically, specific mutations in WT1 gene have been associated with distinct syndromes based on phenotypic characteristics, including Denys-Drash syndrome (DDS), Frasier syndrome (FS), Meacham syndrome, and WAGR syndrome. DDS is classically defined by the triad of steroid-resistant nephrotic syndrome (SRNS) onset in the first year of life, disorders of sex development (DSD), and a predisposition to Wilms tumor (WT). Currently, a paradigm shift acknowledges a diverse spectrum of presentations beyond traditional syndromic definitions. Consequently, the concept of WT1-related disorders becomes more precise. A genotype-phenotype correlation has been established, emphasizing that the location and type of WT1 mutations significantly influence the clinical presentation, the condition severity, and the chronology of patient manifestations. Individuals presenting with persistent proteinuria, with or without nephrotic syndrome, and varying degrees of kidney dysfunction accompanied by genital malformations should prompt suspicion of WT1 mutations. Recent genetic advances enable a more accurate estimation of malignancy risk in these patients, facilitating a conservative nephron-sparing surgery (NSS) approach in select cases, with a focus on preserving residual kidney function and delaying nephrectomies. Other key management strategies include kidney transplantation and addressing DSD and gonadoblastoma. In summary, recent genetic insights underscore the imperative to implement individualized, integrated, and multidisciplinary management strategies for WT1-related disorders. This approach is pivotal in optimizing patient outcomes and addressing the complexities associated with these diverse clinical manifestations.

WT1 exon 10 missense variant in a pediatric patient with focal segmental glomerulosclerosis with embryonal hyperplasia.

A 6-year-old boy was diagnosed with chromosomal abnormalities (48,XYY, + 21[11]/46,XY[19]) at 4months of age after a physical examination revealed an undescended testis and a dwarf penis. He also had mild renal dysfunction and severe proteinuria, and kidney biopsy at 2years of age revealed focal segmental glomerulosclerosis. Genetic analysis to investigate suspected WT1 gene abnormalities revealed a novel variant in NM_024426.6:exon10:c.1506T > A (p.(Asp502Glu)). His kidney function deteriorated rapidly, leading to the induction of peritoneal dialysis at 5years of age. Although this variant had not been previously reported, bilateral nephrectomy was performed to prevent any progression of the tumor. Histopathology showed all the glomeruli observed within the observation area to be completely sclerotic, while also showing evidence of embryonal hyperplasia. This case was not a hot spot for Denys-Drash syndrome, but it had a similar phenotype and pathology that could have been derived from a WT1 gene abnormality.

Polyarthritis due to metastatic calcinosis in a patient with new WT1 gene mutation: resolution after renal transplantation

Polyarthritis due to metastatic calcinosis in a patient with new WT1 gene mutation: resolution after renal transplantation

Desmoplastic small round cell tumor a case report of a rare intra-abdominal tumor of the young adult

Desmoplastic small round cell tumor is a rare and highly aggressive mesenchymal tumor described as a distinct clinico-pathological entity in 1989 by Gerald . These tumors occur mainly in the peritoneal cavity,although other primary sites, such as testicular, ovarian, thoracic, pulmonary, intracranial and head and neck regions have been reported. Clinical symptoms are non-specific. Diagnosis is based on histological analysis combined with immunohistochemical study. Histological study typically shows small round blue cells in nests separated by abundant desmoplastic stroma associated with a single chromosomal translocation t(11:22) (p 13; q 12) involving the EWSR1 and WT1 genes. The prognosis is particularly poor, with median survival ranging from 17 to 25 months. Management of this tumor remains difficult, and current regimens do not achieve a significant cure rate despite the use of aggressive treatments such as polychemotherapy.

Stabilization of G-quadruplexes in Intronic Hematopoietic-specific Enhancer of WT1 gene with G-quadruplex targeting ligands: in silico and in vitro techniques

Stabilization of G-quadruplexes in Intronic Hematopoietic-specific Enhancer of WT1 gene with G-quadruplex targeting ligands: in silico and in vitro techniques

Genotypic and phenotypic drug resistance patterns of Mycobacterium tuberculosis isolated from presumptive pulmonary tuberculosis patients in Ethiopia: A multicenter study.

The emergence of drug-resistant tuberculosis (DR-TB) has been a major obstacle to global tuberculosis control programs, especially in developing countries, including Ethiopia. This study investigated drug resistance patterns and associated mutations of Mycobacterium tuberculosis Complex (MTBC) isolates from the Amhara, Gambella, and Benishangul-Gumuz regions of Ethiopia. A cross-sectional study was conducted using 128 MTBC isolates obtained from patients with presumptive tuberculosis (TB). Phenotypic (BACTEC MGIT 960) and genotypic (MTBDRplus and MTBDRsl assays) methods were used for drug susceptibility testing. Data were entered into Epi-info and analyzed using SPSS version 25. Frequencies and proportions were determined to describe drug resistance levels and associated mutations. Of the 127 isolates recovered, 100 (78.7%) were susceptible to four first-line anti-TB drugs. Any drug resistance, polydrug resistance, and multi-drug resistance (MDR) were detected in 21.3% (27), 15.7% (20), and 15% (19) of the isolates, respectively, by phenotypic and/or genotypic methods. Mono-resistance was observed for Isoniazid (INH) (2, 1.6%) and Streptomycin (STR) (2, 1.6%). There were two genotypically discordant RIF-resistant cases and one INH-resistant case. One case of pre-extensively drug-resistant TB (pre-XDR-TB) and one case of extensively drug-resistant TB (XDR-TB) were identified. The most frequent gene mutations associated with INH and rifampicin (RIF) resistance were observed in the katG MUT1 (S315T1) (20, 76.9%) and rpoB (S531L) (10, 52.6%) genes, respectively. Two MDR-TB isolates were resistant to second-line drugs; one had a mutation in the gyrA MUT1 gene, and the other had missing gyrA WT1, gyrA WT3, and rrs WT1 genes without any mutation. The detection of a significant proportion of DR-TB cases in this study suggests that DR-TB is a major public health problem in Ethiopia. Thus, we recommend the early detection and treatment of DR-TB and universal full first-line drug-susceptibility testing in routine system.

Mutation of NPHS1, NPHS2, WT1, LAMB2, COL4A5 and other genes in children with idiopathic steroid resistant nephrotic syndrome

Background: Many children with idiopathic steroid resistant nephrotic syndrome have been reported worldwide due to mutation of NPHS1, NPHS2, WT1 and LAMB2 genes. This study aimed to determine the frequency of mutation of NPHS1, NPHS2, WT1, LAMB2, COL4A5 and other genes and their association with renal histopathological patterns of idiopathic steroid resistant nephrotic syndrome patients. Methods: This cross-sectional study was conducted on 25 patients with idiopathic steroid resistant nephrotic aged 1-17 years in the Department of Paediatric Nephrology, Bangabandhu Sheikh Mujib Medical University, Bangladesh, from July 2017 to June 2018. Next Generation Sequencing and mutation analysis were performed after DNA extraction from patients' venous blood lymphocytes. Histopathological study of renal tissue was done among 17 patients. Results: A little more than half (56%) of the patients were male. The mean age at the initial attack of nephrotic syndrome was 94.2 months. They mostly had minimal change disease (41%) and IgA nephropathy (12%). One subject had the NPHS2 gene mutation, histopathologically diffuse mesangial proliferative glomerulonephritis, and clinically stage-4 chronic kidney disease. Another subject had the COL4A5 gene mutation and focal segmental glomerulosclerosis. Both were male and had no familial renal disease, consanguinity, or hematuria. Conclusion: Children with idiopathic steroid resistant nephrotic syndrome showed NPHS2 and COL4A5 gene mutations. Histopathologically, they showed diffuse mesangial proliferative glomerulonephritis and focal segmental glomerulosclerosis.

EWSR1::WT1 Fusions in Neoplasms Other Than Conventional Desmoplastic Small Round Cell Tumor: Three Tumors Occurring Outside the Female Genital Tract

Desmoplastic small round cell tumor (DSRCT) is a high-grade, primitive round cell sarcoma classically associated with prominent desmoplastic stroma, coexpression of keratin and desmin, and a characteristic EWSR1::WT1 gene fusion. DSRCT typically arises in the abdominopelvic cavity of young males with diffuse peritoneal spread and poor overall survival. Although originally considered to be pathognomonic for DSRCT, EWSR1::WT1 gene fusions have recently been detected in rare tumors lacking the characteristic morphologic and immunohistochemical features of DSRCT. Here, we report 3 additional cases of neoplasms other than conventional DSCRCT with EWSR1::WT1 gene fusions that occurred outside the female genital tract. Two occurred in the abdominopelvic cavities of a 27-year-old man and a 12-year-old girl, whereas the third arose in the axillary soft tissue of an 85-year-old man. All cases lacked prominent desmoplastic stroma and were instead solid and cystic with peripheral fibrous pseudocapsules and occasional intervening fibrous septa. Necrosis was either absent (1/3) or rare (2/3), and mitotic activity was low (<1 to 3 per 10 hpf). In immunohistochemical studies, there was expression of smooth muscle actin (3/3) and desmin (3/3), rare to focal reactivity for EMA (2/3), and variable expression of CK AE1/AE3 (1/3). Myogenin and MyoD1 were negative, and C-terminus-specific WT1 was positive in both cases tested (2/2). All 3 tumors followed a more indolent clinical course with 2 cases demonstrating no evidence of disease at 20 and 44 months after resection. The patient from case 3 died of other causes at 14 months with no evidence of recurrence. DNA methylation profiling showed that the 3 cases clustered with DSRCT; however, they demonstrated fewer copy number variations with 2 cases having a flat profile (0% copy number variation). Differential methylation analysis with hierarchical clustering further showed variation between the 3 cases and conventional DSRCT. Although further study is needed, our results, in addition to previous reports, suggest that EWSR1::WT1 gene fusions occur in rare and seemingly distinctive tumors other than conventional DSRCT with indolent behavior. Proper classification of these unusual soft tissue tumors with EWSR1::WT1 gene fusions requires direct correlation with tumor morphology and clinical behavior, which is essential to avoid overtreatment with aggressive chemotherapy.

Genetic and epigenetic features of bilateral Wilms tumor predisposition in patients from the Children’s Oncology Group AREN18B5-Q

Developing synchronous bilateral Wilms tumor suggests an underlying (epi)genetic predisposition. Here, we evaluate this predisposition in 68 patients using whole exome or genome sequencing (n = 85 tumors from 61 patients with matched germline blood DNA), RNA-seq (n = 99 tumors), and DNA methylation analysis (n = 61 peripheral blood, n = 29 non-diseased kidney, n = 99 tumors). We determine the predominant events for bilateral Wilms tumor predisposition: 1)pre-zygotic germline genetic variants readily detectable in blood DNA [WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%), and BRCA-related genes (5%)] or 2)post-zygotic epigenetic hypermethylation at 11p15.5 H19/ICR1 that may require analysis of multiple tissue types for diagnosis. Of 99 total tumor specimens, 16 (16.1%) have 11p15.5 normal retention of imprinting, 25 (25.2%) have 11p15.5 copy neutral loss of heterozygosity, and 58 (58.6%) have 11p15.5 H19/ICR1 epigenetic hypermethylation (loss of imprinting). Here, we ascertain the epigenetic and genetic modes of bilateral Wilms tumor predisposition.

Expression level of Wilms' tumor 1 gene and its correlation with clinical features in patients with myeloproliferative neoplasms

Objective: To investigate the expression level of WT1 gene in patients with classical Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPN) and its correlation with clinical features. Methods: A retrospective study included 252 patients with newly diagnosed MPN in Zhongnan Hospital of Wuhan University from January 2015 to March 2023, including 128 males and 124 females, aged［M（Q1，Q3）］62 (53, 69) years. The WT1-positive group (n=93) and the WT1-negative group (n=159) were split based on the level of WT1 gene expression, and the variations in clinical indicators between the two groups were compared. Its levels of expression in each subtype and its relationships to thrombotic events and clinically significant variables were analyzed. As of March 31, 2023, the follow-up period [M (Q1, Q3)] was 12.0(6.5,21.0)months. The risk factors of thrombosis in MPN patients were analyzed by using the logistic regression analysis. Results: The WT1 gene expression level in the overall bone marrow samples of 252 patients with newly diagnosed MPN was 0.30% (0.10%, 1.10%). The expression level in primary myelofibrosis (PMF) patients was 1.45% (0.41%, 3.24%), which was higher than 0.15% (0.02%, 0.32%), 0.37% (0.16%, 1.09%) in essential thrombocythemia (ET) and polycythemia vera (PV) patients (both P<0.05). Positive correlations were found between WT1 gene expression levels and JAK2V617F gene mutation load, RDW, MPV (r=0.478, 0.346, 0.236, all P<0.01). While negative correlations between WT1 gene expression levels and PLT, LYM, PTTA, LDH were found (r=-0.339, -0.170, -0.206, -0.388, all P<0.01). Patients in the WT1-positive group exhibited a higher percentage of somatic symptoms, splenomegaly, positive JAK2V617F gene mutation, and higher levels of RDW, LDH, NEUT, and MPV compared to the WT1-negative group. In contrast, the proportion of triple-negative (negative for all three hot mutations of JAK2V617F, CALR and MPL) was lower, and the levels of PLT, LYM and PTTA were lower (all P<0.05). The thrombotic event rates of WT1-positive group and WT1-negative group were 32.3% (30/93) and 32.1% (51/159), respectively, and the difference was not statistically significant (P=0.883). Logistic regression analysis showed that male (OR=2.41,95%CI:1.02-5.71,P=0.046) and positive JAK2V617F gene mutation (OR=3.96,95%CI:1.50-10.42,P=0.005) were risk factors for thrombotic events in ET patients. Conclusions: WT1 gene expression is elevated in PMF patients and correlated with indicators of disease progression and transformation in MPN patients. It can be utilized as an auxiliary diagnostic indicator for classical MPN staging but is not correlated with the incidence of thrombotic events. Male and positive JAK2V617F gene mutation are risk factors for thrombotic events in ET patients.

Comprehensive Characterization of Evolution of Genomic Complexity By Structural Variant and Mutational Profiling in Myelodysplastic Syndrome Patients with Hypomethylating Agent Failure

Introduction MDS patients (pts) who develop resistance to hypomethylating agent therapy (HMA-F) have dismal outcomes. Genomic aberrations underlying progression to HMA-F remains poorly understood. By sequencing, studies have demonstrated acquisition of new mutations affecting transcription factors such as FLT3 and RAS-MAPK pathway genes in a subset of cases. Until now, evolution of genomic complexity in HMA-F by acquisition of new structural variants (SVs) has not been systematically investigated. This is due to lack of genome-wide technologies to quantitatively identify all types of SVs at a gene/exon-level resolution (deletions/insertions/inversions/fusions etc..). In this study, we optical genome mapping (OGM), a novel, non-sequencing-based approach for accurate high-resolution SV detection, combined with ultra-deep coverage targeted NGS to comprehensively analyze genome-wide changes affecting both SVs and somatic mutations using paired baseline and progression samples from 21 MDS pts who developed HMA-F. Methods We identified all MDS pts with sufficient (1.5 million) cryopreserved, viable bone marrow (BM) mononuclear cells at both diagnosis (baseline) and HMA-F. All pts underwent BM morphologic exam, traditional banding analysis (TBA), and 81-gene targeted NGS panel (2500X coverage; LOD: 2% VAF; myeloid genes). We excluded pts with AML-defining abnormalities by standard-of-care work-up (per WHO 2022 criteria) and CMML pts. OGM was performed using standard procedures: extraction of ultra-long DNA molecules, sequence-specific fluorophore labeling and stain, followed by loading on to nanochannels and imaging (Saphyr; Bionano Genomics). Rare Variant Pipeline was used for detection of clinically significant SVs [minimum 300X coverage; 5000 bp size cut-off). We used bidirectional paired genome-wide analysis for identifying unique SVs seen exclusively at either baseline or HMA-F. Results There were 21 pts [11 men; 10 women; median age: 73 (43-89)] with available cryopreserved cells for comprehensive mutational and SV profiling. Median BM blast% was 5 (1-18). The median time to HMA failure was 11 (6-21) months. All pts had at least 1 genomic aberration: 14 (66%) by TBA and 20 (95%) by NGS (median: 3 mutations per pt.). Seven (35%) pts had normal karyotype (NK), 6 (30%) pts had complex karyotype (CK). Most frequent gene mutations included TET2, ASXL1 TP53 [each seen in 7 pts, 33%], SRSF2 [6 (30%)], DNMT3A, RUNX1 and STAG2 (5 pts each, 25%). Baseline OGM identified multiple clinically significant SVs, 6 of which, seen across 4 (19%) pts, were cryptic by TBA. These included CBFB::MYH1 and KMT2A partial tandem duplication. OGM at HMA-F showed increased genomic complexity compared to baseline in 9 (43%) pts. Paired analysis comparing genome-wide SV burden at HMA-F with matched baseline sample revealed 306 new unique SVs at HMA-F not seen at baseline ( Fig 1). These included 30 deletions (8 pts), duplications (6 pts), inversions, intrachromosomal and intrachromosomal translocations (5 pts each). Recurrent aberrations included segmental deletions of chr 17, 1, 7, 9, 12 and 21, affecting ETV6, CDKN1B, IKZF1, RUNX1, FHIT and HOXA11 genes, exon-spanning duplication in RUNX1, and FANCA::NF1 and YTHDF3-SETBP1 fusions. Three (14%) pts (2 with CK, 1 with NK), showed a cataclysmic increase in the numbers of SVs (translocations/ inversions) indicative of chromoanagenesis ( Fig 2). In contrast, there were 29 SVs seen exclusively at baseline and not detected at HMA-F [intrachromosomal translocations affecting chr 3 ( FOXP1) and chr 7 ( CUX1), low-level IGH::BCL2, deletions and insertions]. Sequential NGS and TBA was available in 20 pts. At HMA-F, 13 (62%) pts developed additional mutations, most frequent in NRAS, followed by TP53, TET2, CSF3R, KIT, second mutation in CEBPA, DDX41 and WT1 genes. The median number of mutations per pt. was 4. The highest increase in SV burden by OGM was noted in pts who did not acquire new mutations by NGS at HMA-F (5/7; 71%). Seven (33%) pts showed cytogenetic clonal evolution at HMA-F by TBA. OGM identified new SVs at HMA-F in 2 (50%) pts who did not show clonal evolution by NGS or TBA. Conclusions HMA-F MDS is characterized by increased genomic complexity by acquisition of new somatic mutations (NGS) and SVs (OGM), highlighting their complementary role in MDS progression. OGM further highlighted cataclysmic genomic changes by chromoanagenesis in a subset of MDS pts at HMA-F.

Characterization of the Molecular Landscape of Pediatric Acute Myeloid Leukemia: A Retrospective AIEOP AML2013/01 Study

Background. Advances in genomic techniques have recently shed light on leukemia biology, recognizing that pediatric acute myeloid leukemia (AML) is a highly genetically heterogeneous disease. In the last two decades, genetic and cytogenetic abnormalities contributed to the stratification of patients in different classes of risk, where children receive a risk-adapted therapy. In the recently concluded AIEOP-AML2013/01 clinical trial, 371 Italian pediatric AML patients were allocated into three risk groups that were largely based on measurable residual disease at the end of induction therapy and on recurrent molecular markers with a recognized impact on children's outcome. Methods and Results. We screened by RT-PCR, RQ-PCR, capillary electrophoresis and Sanger sequencing 41 genetic markers on 371 RNA and DNA samples, identifying a molecular marker at diagnosis for 247 cases out of the 371 enrolled (67%). As far as gene mutations detected are concerned, we identified 24/371 cases harboring a mutation in exon 12 of NPM1 gene (6.5%), 16/371 a FLT3-ITD mutation (4.3%), and 7 harboring both NPM1 and FLT3-ITD mutations (2%). Moreover, we also found 2 cases having a partial tandem duplication affecting KMT2A gene (2/371, 0.5%), one of them occurring together with a FLT3-ITD mutation. For chromosomal aberrations, we confirmed that t(8;21) RUNX1::RUNX1T1 and inv(16) CBFB::MYH11 are recurrent translocations in pediatric AML, being found in 13.5% (50/371) and 12% (44/371) of cases respectively. Sixty-four (17,3%) AML cases were characterized by a KMT2A fusion with different partner genes, with the t(9;11) KMT2A::MLLT3 being the most recurrent (24/371, 6.5%), followed by t(10;11) KMT2A::MLLT10 (21/371, 5.7%), t(6;11) KMT2A::AFDN (10/371, 2.7%), t(11;19) KMT2A::ELL (6/371, 1.6%). In 14 patients, we found a NUP98 translocations (3.8%), and, in particular, 9 cases harbored the t(5;11) NUP98::NSD1 (2.4%, 5 of them together with a FLT3-ITD mutation), 4 the t(11;12) NUP98::KDM5A (1%) and only one case the t(2;11) NUP98::HOXD13 (0.3%). Other chromosomal aberrations were rarer in our cohort, with t(16;16) CBFA2T3::GLIS2 accounting for 2.4% of cases (9/371), t(6;9) DEK::CAN 1.3% (5/371, co-occurring with FLT3-ITD in 3/5 cases), t(1;22) RBM15::MKL1 1.1% (4/371), t(3;5) NPM1::MLF1 0.5% (2/371, one concomitant with FLT3-ITD), t(16;21) FUS::ERG 0.5% (2/371), t(16;21) RUNX1::CBFA2T3 0.3% (1/371), del(9) SET::NUP214 0.3% (1/371), t(8;16) KAT6A::CREBBP 0.3% (1/371), t(9;22) BCR::ABL1 0.3% (1/371). In addition, we retrospectively evaluated the incidence and prognostic relevance of mutations at specific loci of KIT, FLT3, ASXL1, WT1 and CEBPA genes. As concerns the KIT gene, we identified mutations at exon 8 or 17 in 16/371 AML (4.3%). Of note, all the 16 cases had a concomitant core-binding factor (CBF) rearrangement, and in particular 10/16 the t(8;21) RUNX1::RUNX1T1 translocation and 6/16 the inv(16) CBFB::MYH11, with a relative occurrence of 17% in the CBF-r AML subgroup (16 KIT mutated AML/94 CBF-r) and a slight, but not significant, impact on outcome. As regards exon 13 of ASXL1 gene and the tyrosine-kinase domain of FLT3 gene ( FLT3-TKD), our study confirmed a low prevalence of these mutations, which occurred in 2.5% and 3.4% of AML respectively. On the contrary, WT1 mutations (exons 6-9) occurred in a consistent portion of AML (45 mutated cases, 13.2%)did not affect the outcome of FLT3-ITD mutated AML. Conversely, by screening both at bZIP and NTD domains of the CEBPA gene, we identified 18 double-mutated patients (4.8%), and notably all these patients had no other molecular marker detected at diagnosis, reducing the proportion of negative cases from 33% to 28%. We found that CEBPA mutated cases are characterized by a favorable outcome (EFS 93% vs 58% for CEBPA wt, p=0.008), this finding corroborating the indication to allocate these patients to the standard risk group. Conclusion. Overall, in the prospective AIEOP AML2013/01 trial, we described the frequency of different molecular lesions and improved the genetic landscape of AML. The future use of NGS-based screenings could further optimize the characterization of the genetic landscape of childhood AML, thus refining the risk class patients stratification and modulation of treatment approaches.

Molecular Characterisation and Outcomes of 363 Adolescent and Young Adults with AML in Saudi Arabia Risk Stratified By PCR and NGS-Based Testing

Introduction Acute Myeloid Leukaemia (AML) is characterised by cytogenetic (CGN) abnormalities and or mutations in one or more of a number of genes that allow risk stratification and guide treatment. The mutational landscape of patients from Saudi Arabia, which has a young population, has not been well characterised. One study indicates a higher rate of core binding factor and monosomies. Mutational analysis by Next generation sequencing (NGS) has not had widespread utilisation hampering the risk stratification of AML in the country. The more recent introduction of NGS panels has allowed more risk stratification. Here we analysed patients and characterised the CGN and molecular abnormalities. Methods Data were collected from 363 patients, and their cytogenetic and molecular results were reviewed. Patients were divided into 2 cohorts: Cohort A only had PCR-based testing diagnosed or treated at King Faisal Specialist Hospital and research centre - Riyadh. PCR testing included mutation analysis for FLT3, NPM1, CEBPA, c-KIT, IDH1, IDH2, WT1, DNMT3A, and JAK2; quantitative PCR for RUNX1-ETO in a subset of patients. Cohort B had PCR-based mutation testing in addition to the Next-Generation Sequencing (NGS) myeloid panel, which was Illumina-based or Ion Torrent. The panel comprises 23 hotspot genes, 17 entire genes, 29 fusion drivers, and five expression genes associated with haematological malignancies. We risk-stratified patients according to the most recent testing method available. Cohort A and Cohort B were risk-stratified, according to ELN risk classification 2017. Survival analysis for these patients was conducted. Results The median age for all patients was 39 years (13-89). PCR testing included mutation analysis for FLT3 (93.4%), NPM1 (90.4%), CEBPA (69.7%), c -KIT (54.3%), IDH1 (57%), IDH2 (57.9%), WT1 (61.2%), DNMT3A (54.5.5%), JAK2 (4.7%) and quantitative PCR for RUNX1- ETO fusion (13.2%). Cohort A comprised 262 (72.7%) patients who underwent PCR-based mutation testing only. Cohort B consisted of 101 (27.8%) patients who underwent PCR-based mutation testing in addition to NGS myeloid panel analysis. In the entire cohort of 363 patients, we found FLT3-ITD mutations in 85 patients (23.4%) and NPM1 mutations in 68 (18.7%). 36 patients (9.9%) harboured both FLT3-ITD and NPM1 mutations together. In Cohort A, FLT3-ITD detected 26.7% of patients, and 20.2% had NPM1 mutation, while 40% had no detectable mutations by conventional PCR. On the other hand, FLT3-ITD was detected in 14.9% of patients by both NGS and conventional PCR and in 14.9% of patients with NPM1. When comparing Cohort A and Cohort B, Cohort B had more mutations in genes that play an important role in myeloid malignancies, which might have better risk stratification and precision, including TP53 in 13.9% of patients and RUNX1 in 18.8% of patients and ASXL1 in 9.9% of patients. Moreover, NGS pick up more mutations in CEBPA, c-KIT, WT1, IDH1 and JAK2 genes compared to conventional PCR. Within cohort A, 9.2% of patients had normal cytogenetic testing results with no detectable mutations, whereas in cohort B, only 1% of patients had normal karyotype and molecular testing. Furthermore, six mutations were detected only by NGS but not PCR in FLT3-ITD, CEBPA, c-KIT, IDH2, WT1 and DNMT3A in 5.9% of patients. Cohorts A and B were also categorised into ELN risk groups using PCR, sequencing, and cytogenetics testing. It was found that patients at adverse risk had shorter overall survival compared to those at intermediate or favourable risk in both cohorts, with p-values of 0.030 and 0.0007, respectively (Figure 1). The use of NGS was found to be more effective than conventional testing in identifying patients with adverse risk, with a hazard ratio for death of 0.22 (95% CI, 0.08989-0.5732) compared to a hazard ratio of 0.66 (95% CI, 0.4352 -1.031) for conventional PCR testing. Conclusion In this study, we present the mutational profile of AML in young Saudi patients and identify certain frequent mutations in this population. The study presents the largest molecular profiling to our knowledge of this population. The study emphasises the need for integration of NGS to enhance risk stratification and precision, enable better characterisation of high-risk patients, and improve the potential for targeted treatments.

MAJIQlopedia: an encyclopedia of RNA splicing variations in human tissues and cancer.

Quantification of RNA splicing variations based on RNA-Sequencing can reveal tissue- and disease-specific splicing patterns. To study such splicing variations, we introduce MAJIQlopedia, an encyclopedia of splicing variations that encompasses 86 human tissues and 41 cancer datasets. MAJIQlopedia reports annotated and unannotated splicing events for a total of 486175 alternative splice junctions in normal tissues and 338317 alternative splice junctions in cancer. This database, available at https://majiq.biociphers.org/majiqlopedia/, includes a user-friendly interface that provides graphical representations of junction usage quantification for each junction across all tissue or cancer types. To demonstrate case usage of MAJIQlopedia, we review splicing variations in genes WT1, MAPT and BIN1, which all have known tissue or cancer-specific splicing variations. We also use MAJIQlopedia to highlight novel splicing variations in FDX1 and MEGF9 in normal tissues, and we uncover a novel exon inclusion event in RPS6KA6 that only occurs in two cancer types. Users can download the database, request the addition of data to the webtool, or install a MAJIQlopedia server to integrate proprietary data. MAJIQlopedia can serve as a reference database for researchers seeking to understand what splicing variations exist in genes of interest, and those looking to understand tissue- or cancer-specific splice isoform usage.

THU540 Cribriform Morular Thyroid Carcinoma In A Young Female With Prior Exposure To Whole-Body Radiation For Metastatic Wilms’ Tumor

Abstract Disclosure: D. Caesario: None. M.S. Richardson: None. D. Carneiro-Pla: None. J.K. Fernandes: None. Introduction: Cribriform morular thyroid carcinoma (CMTC) is a rare thyroid cancer that is classically associated with familial adenomatous polyposis (FAP), but can also occur sporadically. We report a case of CMTC in a young female with history of whole-body radiation therapy (WBRT). Case Report: A 19-year-old female with past medical history of Wilms’ tumor (WT) with lung metastasis status post left nephrectomy and WBRT at the age of 6 was seen for newly diagnosed thyroid nodules. Physical exam was remarkable for thyromegaly. Thyroid function tests were normal. Thyroid ultrasound revealed 2 dominant TI-RADS 4 nodules measuring 1.9 x 1.7 x 2.1 cm on the left lobe and 0.9 x 0.8 x 0.8 cm on the right lobe. No suspicious lymphadenopathy. Fine needle aspiration biopsy on both nodules showed findings consistent with papillary thyroid carcinoma (PTC). No family history of thyroid disorders, malignancies, or FAP. She underwent total thyroidectomy with central neck node sampling. Thyroid gland surgical pathology result revealed CMTC positive for thyroid transcription factor-1 (TTF-1), β-catenin, and cytokeratins 5 and 6. It was negative for calcitonin. There was no evidence of invasion, extension, or lymph node metastasis. Genetic tests are currently pending. Discussion: CMTC was previously considered a variant of PTC, accounting for 0.2 to 6% of PTC cases. However, recent observations have challenged this view, and latest WHO update reclassified it as a distinct form of thyroid cancer. It is associated with an indolent clinical course and good prognosis. It almost exclusively affects young women. Both familial and sporadic cases are associated with mutations in the adenomatous polyposis coli (APC) gene. The APC protein is a negative regulator that controls β-catenin concentrations, which is involved in cell adhesion. Mutations in the APC gene may result in FAP and colorectal cancers. In cases associated with FAP, the tumors are usually multifocal and/or bilateral, while sporadic cases usually appear as single nodule. Tumor cells are always positive for TTF-1 and negative for calcitonin. They can also be positive for multiple cytokeratins, but strong nuclear and cytoplasmic staining for β-catenin is the hallmark of this tumor type. The case above occurred in a young female with no significant family history, but who underwent WBRT for WT as a child. Radiation is a known predisposing factor for thyroid cancer, and limited studies on early exposure to radiation revealed higher incidence of sporadic CMTC. However, even among FAP associated cases, CMTC diagnosis preceded that of FAP in up to 40% of the cases. Additionally, up to 20% of WT cases have mutations of the WT1 gene, and among these, at least half also carry acquired somatic mutations in CTNNB1, the gene encoding β-catenin. So far, there has been no reported case of CMTC in the background of WT. It will be interesting to see if she has these underlying mutations. Presentation: Thursday, June 15, 2023

AML-503 An Unusual Presentation of Acute Promyelocytic Leukemia (APML): Complex Cytogenetic Isochromosome 17 and WT1 Gene Mutation in the Absence of PML-RARA Fusion Protein

AML-503 An Unusual Presentation of Acute Promyelocytic Leukemia (APML): Complex Cytogenetic Isochromosome 17 and WT1 Gene Mutation in the Absence of PML-RARA Fusion Protein