Chromosome 22q11 Research Articles

Outcomes of Patients with Multiple Myeloma and 1q Gain/Amplification Receiving Autologous Hematopoietic Stem Cell Transplant: The MD Anderson Cancer Center Experience

Background: There is contradictory data regarding the prognostic impact of additional copy numbers of chromosome 1q (1q+) on the outcomes of newly diagnosed multiple myeloma (NDMM) patients, and there is scarce data in the context of autologous stem cell transplantation (autoSCT). In this report, we studied outcomes of NDMM patients with 1q+ who received induction with contemporary anti-myeloma agents, followed by autoSCT and post-transplant maintenance therapy. Methods: We conducted a retrospective single-center chart review analysis of adult NDMM patients with 1q21 gain or amplification (3 or ≥4 copies of 1q, respectively; 1q+) detected by fluorescence in situ hybridization (FISH) that received autoSCT between 2008-2018. Progression-free survival (PFS) and overall survival (OS) were the primary endpoints. Results: 213 NDMM patients with 1q+ were included in the analysis, with a median age of 62.5 years and 53% were male. Overall, 169 (79%) patients had 1q gain, while 44 (21%) patients had 1q amplification. The most commonly used induction and conditioning regimens were bortezomib, lenalidomide, and dexamethasone (VRD) (41%) and melphalan (77%), respectively (Table 1). At day 100 after autoSCT and at best post-transplant response, 78% and 87% of patients achieved ≥VGPR, 34% and 56% achieved CR, 38% and 50% achieved MRD negative ≥VGPR, respectively. The median PFS and OS for the entire cohort were 35.5 months and 81.4 months, respectively. 1q amplification was associated with inferior PFS compared to 1q gain (HR=2.03, 95% CI 1.36-3.03, p<0.001; Figure 1). On multivariable assessment (MVA) for PFS, MRD negative >VGPR before autoSCT and at day 100 post-transplant [(HR=0.56, 95% CI 0.36-0.86, p=0.009) and (HR=0.64, 95% CI 0.44-0.94, p=0.022), respectively] were associated with better PFS, whereas 1q amplification was associated with inferior PFS (HR=1.94, 95% CI 1.29-2.92, p=0.001). On MVA for OS, R-ISS stage III (HR=4.08, 95% CI 1.07-15.50, p=0.039) was associated with inferior OS, whereas achieving MRD negative >VGPR at best post-transplant response was associated with superior OS (HR=0.45, 95% CI 0.24-0.85, p=0.014). Notably, the percentage of cells with 1q+ was not associated with PFS nor with OS, both when evaluated as a continuous variable and when evaluated as a categorical variable using thresholds of either 30% or 50%. The presence of additional high-risk cytogenetic abnormalities did not adversely affect survival outcomes. Conclusions: Patients with NDMM and 1q+, especially 1q amplification, have poor survival outcomes, despite the use of contemporary induction regimens, upfront autoSCT and post-transplant maintenance. These patients may benefit from novel treatment modalities, such as CAR-T and bispecific antibodies, earlier in their disease course.

Paroxysmal Nocturnal Haemoglobinuria (PNH) Arising from Non-Canonical Mutations

Introduction Acquired somatic mutations in the X-linked PIGA gene are the most commonly described pathological mutations found in the serious haematopoietic disorder paroxysmal nocturnal haemoglobinuria (PNH), though incidental cases of mutations in PIGT, PIGB and PIGV have been reported. Mutations in the family of 20 PIG (phosphatidylinositol glycan) genes can disrupt biosynthesis of glycosylphosphatidylinositol (GPI) anchors, critical for tethering many important proteins, including the vital complement regulators CD55 and CD59, to the cell membrane. Their absence in PNH leads to complement dysregulation and complications including potentially life-threatening haemolysis and thrombosis, which may be mitigated by lifelong anti-complement therapy. From the cohort of 273 PNH patients currently on complement inhibitor therapy at the Leeds National PNH Centre we have identified 37 patients whose diagnostic flow cytometry report or clinical course differs significantly from expectation, particularly with evidence of inflammatory presentations, which may be indicative of an alternative, non- PIGA, aetiology. Methods Patients consented to the Leeds PNH Research Tissue Bank with unusual clinical and laboratory presentations were identified, genomic DNA was extracted from peripheral blood and initially screened by targeted high sensitivity NGS of the PIGA gene. From the first ten patient samples sequenced, three lacked any evidence of PIGA mutations and so whole exome sequencing (WES) was carried out. Results Patient 1:An 80 year old man, diagnosed with MDS with rearrangement of chromosomes 3 and 7 resulting in a dicentric chromosome and loss of chromosome 20q, was diagnosed with PNH following an increase in transfusion requirement. In addition to classical symptoms of PNH he was diagnosed with seronegative rheumatoid arthritis. Flow cytometry showed an unusual PNH phenotype with a large population of partially GPI-anchor deficient cells only (Table 1). Symptoms improved on eculizumab but he remained transfusion dependent and the disease later transformed to acute myeloid leukaemia. WES: exonic frameshift insertion in PIGT (figure1), pathogenic variant rs760395465. Patient 2:A 34 year old woman presented with a 13 year history of intermittent episodes of generalized urticaria associated with nausea, vomiting, general malaise and myalgia, increasing in intensity and latterly being associated with haemoglobinuria, lethargy and jaundice leading to a PNH diagnosis. Episodes were triggered by pregnancy or infections, but she remained asymptomatic between attacks. Flow cytometry revealed a PNH clone with a majority of cells being partially GPI deficient. She responds well to C5 inhibition with no further symptoms, including urticaria. WES: exonic frameshift insertion in PIGT (figure 1), pathogenic variant rs760395465. Patient 3: A 14 year old girl presented with intermittent episodes of urticaria and severe lethargy and was diagnosed with symptomatic significant transfusion-dependent PNH, with completely GPI-deficient cells. Diagnosis followed an episode of massive haemolysis resulting in transient acute renal failure requiring haemodialysis. She commenced anticoagulation initially until eculizumab was approved 2 years later; despite occasional episodes of breakthrough haemolysis she displays no further urticaria. She has also had symptoms of diffuse arthralgia. WES: stopgain mutation in PIGV (figure1), pathogenic variant rs145160045. Conclusions These cases add to the emerging picture of non- PIGA associated PNH cases and suggest that the presence of joint problems and episodic urticaria may be sufficient indication to prompt screening for alternative causative mutations. Similar to the previously reported PIGB mutated case, the patients with PIGT mutations only had partially deficient cells. It is important to fully characterise these emerging non-canonical variants in order to improve our understanding of the pathophysiology of this disorder and to provide more individualised care.

Impaired Efficacy and Durability of Hematopoietic Stem Cell Engraftment Caused By Samd9l-Deficiency within Bone Marrow Niches

Germline mutations in the SAMD9 and SAMD9L (SAMD9/L) genes on chromosome 7q drive both childhood myelodysplastic syndrome (MDS)/secondary acute myeloid leukemia (sAML) associated with monosomy 7, and adult-onset MDS/sAML associated with distinct somatic genetic alterations. The ortholog for SAMD9/L in mice is Samd9l. Gain of Function (GoF) and Loss of Function (LoF) Samd9l alleles predispose to MDS at distinct ages. LoF models show that Samd9l-deficient mice, like humans, are more likely to develop MDS during adulthood, while GoF models result in early onset bone marrow (BM) dysfunction and MDS/sAML. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure (BMF), MDS, and AML arising from germline genetic syndromes. Our previous research demonstrated that deficits in host BM microenvironment (ME) niches contribute to hematopoietic failure and poor HSC engraftment in murine models of many MDS predisposition syndromes associated with BMF. As we have observed clinical cases where poor engraftment impaired outcomes in patients with SAMD9/L mutations, we recently hypothesized, BM niche functions critical to facilitating HSC engraftment may be impaired due to niche-intrinsic deficits caused by SAMD9/L mutations. Herein, we present data testing this hypothesis, demonstrating Samd9l deficiency alters BM ME composition, extracellular matrix (ECM) regulation, and proinflammatory cytokine signaling resulting in impaired donor HSC engraftment after HSCT. We performed primary (1 ry) and secondary (2 ry) HSCT to model impacts of SAMD9/L deficiency on BM niche function during HSCT using WT ( Samd9l+/+), Samd9l+/-, and Samd9l-/- mice as HSCT recipients of healthy WT GFP + donor BM to measure the capacity of Samd9l LoF niches to engraft donor HSCs. To assess changes in niche composition and gene expression during HSCT, flow cytometry, multiplex ELISA, and RNASeq analysis were performed on WT, Samd9+/- and Samd9-/- mice. Compared to WT control niches, Samd9+/- and Samd9-/- niches exhibited impaired efficiency of donor HSC engraftment at 3 weeks post-1 ry HSCT, as measured by competitive 2 ry-HSCT performed with 1 ry recipient BM harvested at this timepoint. Beyond 30 weeks post-2 ry-HSCT in this model, GFP + reconstitution of GR1 + myeloid, RBC's, and platelets was reduced by 25%, 18%, and 35% in 2 ry recipients of Samd9+/-1 ry recipient BM and by 26%, 28%, and 32% in 2 ry recipients of Samd9-/- 1 ry recipient BM. Durability of HSC engraftment by 8 months was even more significantly impaired in Samd9+/- and Samd9-/- niches, with Samd9-/- mice exhibiting low survival by this timepoint. Competitive 2 ry HSCT performed 8 months post-1 ry HSCT showed reduction in trilineage GFP + blood cells out to 24 weeks post 2 ry-HSCT, including reductions in GFP + Gr1 + myeloid cells by 50% and 83% in 2 ry recipients of Samd9l+/-and Samd9l-/-1 ry recipient BM, respectively, compared to control 1 ry recipient BM. Similar reductions in BM long-term HSC and Lin -Sca-1 +cKit + HSC/progenitor cells in 2 ry recipients of Samd9l-/-compared to Samd9l+/+ 1 ryrecipient BM 8 months post 1 ry-HSCT. Following TBI, Samd9l+/-and Samd9l-/- BM niches contained similar percentages of osteoprogenitors, mesenchymal stem cells, and endothelial cells compared to WT controls. Multiplex ELISA on BM plasma collected 48hrs post-TBI revealed that Samd9l+/-and Samd9l-/-deletion in the BM ME led to markedly reduced expression of IFN-γ (43% and 55% reduction in Samd9l+/-and Samd9l-/-), Eotaxin (33%/36%), GM-CSF (47%/47%), and IL-1a (48%/65%). In contrast, expression of niche cell extracts showed markedly increased levels of IL-17 (4 fold) and CXCL11 (1.4 fold) in Samd9l+/-and of IL-17 (5.3 fold) IL-12p70 (1.3 fold), and CXCL11 (1.5 fold) in Samd9l-/-, suggesting that aberrantly active Th17 responses caused by niche Samd9l deficiency may be responsible for impairing HSC function and maintenance post-HSCT. RNASeq analysis showed downregulation of ECM genes in both Samd9l-/-and Samd9l+/-models. Our data demonstrates BM niche function is impaired by Samd9l deficiency in a gene dosage-dependent manner. These deficits are associated with increased expression of a specific inflammatory cytokine pathway, suggesting that aberrant growth factor receptor recycling due to Samd9l deficiency may drive stress hematopoiesis and alter HSC cell fate decisions leading to failure of durable donor HSC maintenance post-HSCT.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

Overexpression of the Signaling Integrator Gab2 Accelerates AML Development in Mice with Dnmt3a R878Hand Npm1 cA Mutations

NPM1 and DNMT3A are among the most commonly mutated genes in Acute Myeloid Leukemia (AML), affecting 25-30% of patients. Importantly, these mutations co-occur much more frequently than expected by chance, with ~15% of AML samples containing mutations in both genes.This synergy is recapitulated in mouse models, where mice with either Dnmt3aR878H or Npm1cA mutations develop leukemia with a long latency and low penetrance, whereas mice with both Dnmt3aR878H and Npm1cA mutations develop leukemia in 8-12 months with nearly 100% penetrance (Dovey et al., Blood, 2017; Loberg et al., Leukemia, 2019).To better understand the mechanisms underlying this synergy, and the process of leukemic transformation, we have characterized pre-leukemic bone marrow and 9 independent spontaneous AMLs that have arisen in these mice. These spontaneous AMLs rapidly cause fatal leukemias in secondary recipients; whole genome sequencing of all tumors revealed one or more human AML-like cooperating mutations in each, including canonical mutations in Ptpn11 (E69K, A72V and S506L), an activating KitD818Y mutation, a Cbl exon 9 deletion, a CblY369H mutation,and an amplification of Flt3 associated with >20 fold overexpression of WT Flt3. Characterization of the pre-leukemic transcriptomes and methylomes demonstrated little change in the pre-leukemic state for several months, followed by dramatic and canonical shifts in transcription and methylation after transformation. Remarkably, 8 of the 9 AMLs contained an amplification of the entirety of murine chromosome 7 as the sole structural variant in each genome. We analyzed panel sequencing data from a set of published AMLs arising in this model (Loberg et al., Leukemia, 2019), and also exome sequencing data from a different Npm1 cA driven tumor model (Dovey et al., Blood, 2017); these AMLs also developed complete or partial amplification of chromosome 7 in 7 of 8 and 10 of 15 cases, respectively. Through integration of these datasets, we identified a minimally amplified 8.9 Mbp region on chromosome 7, containing 209 genes, including two candidate genes ( Gab2, Pak1) that show overexpression in both murine and human AMLs with DNMT3A and NPM1 mutations. Retroviral constructs expressing each cDNA (with an IRES-GFP tag) were transduced into lineage depleted bone marrow cells from preleukemic WT, Dnmt3aR878H, Npm1cA or Dnmt3aR878H x Npm1cA mice, and transplanted into recipient mice. Overexpression of GAB2 (but not “Empty Vector” or PAK1) induced significant expansion of hematopoietic cells from the Dnmt3aR878Hx Npm1cAmice (and to a lesser extent, from Npm1cA mice),but not Dnmt3aR878H mice or WT mice ( Figure 1A), suggesting that the mutations in Npm1 and Dnmt3a synergize to account for this phenotype. Strikingly, overexpression of GAB2 in Dnmt3aR878Hx Npm1cAbone marrow led to development of AML in 5 of 5 engrafted mice in less than 5 months (p<10 -6 compared to untransduced marrow; Figure 1B). 0 of 3 engrafted mice with “Empty Vector” (GFP only) in Dnmt3aR878Hx Npm1cAbone marrow and 0 of 3 engrafted mice with overexpression of PAK1 in Dnmt3aR878Hx Npm1cAbone marrow developed leukemia during this 5 month period. Taken together, these data suggest that AML-associated mutations in DNMT3A and NPM1 allow for the selective expansion of cells overexpressing GAB2 as an important step towards leukemic transformation. In human myeloid neoplasms, amplification of chromosome 11q (containing human GAB2) is a recurrent cytogenetic event, observed in 1.5% of cases (Papaemmanuil, NEJM, 2016), and expression of GAB2 across AML samples containing NPM1 mutations is increased ~50% compared to healthy CD34+ cells or promyelocytes. GAB2 is known to bind to GRB2 and SHP2/PTPN11 to facilitate signaling from receptor tyrosine kinases (including FLT3) to downstream pathways (including RAS/RAF/MEK/ERK, PI3K and AKT, in some contexts). These data nominate GAB2 overexpression as a factor that may be relevant for the progression of founding clones with DNMT3A and NPM1 mutations to overt AML. Additional studies to define its protein interactome in preleukemic cells, its mechanism of action, and how leukemia-causing mutations in DNMT3A and NPM1 shape the fitness landscape to select for GAB2 overexpression are underway.

ABIN1 (Q478) is Required to Prevent Hematopoietic Deficiencies through Regulating Type I IFNs Expression.

A20-binding inhibitor of NF-κB activation (ABIN1) is a polyubiquitin-binding protein that regulates cell death and immune responses. Although Abin1 is located on chromosome 5q in the region commonly deleted in patients with 5q minus syndrome, the most distinct of the myelodysplastic syndromes (MDSs), the precise role of ABIN1 in MDSs remains unknown. In this study, mice with a mutation disrupting the polyubiquitin-binding site (Abin1Q478H/Q478H ) is generated. These mice develop MDS-like diseases characterized by anemia, thrombocytopenia, and megakaryocyte dysplasia. Extramedullary hematopoiesis and bone marrow failure are also observed in Abin1Q478H/Q478H mice. Although Abin1Q478H/Q478H cells are sensitive to RIPK1 kinase-RIPK3-MLKL-dependent necroptosis, only anemia and splenomegaly are alleviated by RIPK3 deficiency but not by MLKL deficiency or the RIPK1 kinase-dead mutation. This indicates that the necroptosis-independent function of RIPK3 is critical for anemia development in Abin1Q478H/Q478H mice. Notably, Abin1Q478H/Q478H mice exhibit higher levels of type I interferon (IFN-I) expression in bone marrow cells compared towild-type mice. Consistently, blocking type I IFN signaling through the co-deletion of Ifnar1 greatly ameliorated anemia, thrombocytopenia, and splenomegaly in Abin1Q478H/Q478H mice. Together, these results demonstrates that ABIN1(Q478) prevents the development of hematopoietic deficiencies by regulating type I IFN expression.

Exome and genome sequencing to unravel the precise breakpoints of partial trisomy 6q and partial Monosomy 2q

BackgroundPatients with complex phenotypes and a chromosomal translocation are particularly challenging, since several potentially pathogenic mechanisms need to be investigated.Case presentationHere, we combined exome and genome sequencing techniques to identify the precise breakpoints of heterozygous microduplications in the 6q25.3-q27 region and microdeletions in the 2q37.1-q37.3 region in a proband. The 5-year-old girl exhibited a severe form of congenital cranial dysinnervation disorder (CCDD) in addition to skeletal dysmorphism anomalies and severe intellectual disability. This is the second case affecting chromosomes 2q and 6q. The individual’s karyotype showed an unbalanced translocation 46,XX,del(2)t(2;6)(q37.1;q25.3), which was inherited from her unaffected father [46,XY,t(2;6)(q37.1;q25.3)]. We also obtained the precise breakpoints of a de novo heterozygous copy number deletion [del(2)(q37.1q37.3)chr2:g.232963568_24305260del] and a copy number duplication [dup(6)(q25.3q27)chr6:g.158730978_170930050dup]. The parental origin of the observed balanced translocation was not clear because the parents declined genetic testing.ConclusionPatients with a 2q37 deletion and 6q25.3 duplication may exhibit severe significant neurological and skeletal dysmorphisms, and the utilization of exome and genome sequencing techniques has the potential to unveil the entire translocation of the CNV and the precise breakpoint.

Head and neck INI1-deficient carcinoma without primary: a case report

BackgroundSMARCB1, also known as INI1, is a member of a large protein complex involved in chromatin remodeling and thus the regulation of gene expression. It is located on chromosome 22q11.2. SMARCB1 tumors have been found in various locations, including the sinonasal region, gastrointestinal tract, central nervous system (in atypical teratoid and rhabdoid tumors), and perirenal region (in malignant rhabdoid tumors) in both adults and children.Case presentationWe describe here the first case in the literature of an INI1-deficient neck carcinoma without a primary tumor managed with surgical therapy and neck dissection in a young Caucasian woman of 29 years old, followed by chemotherapy before radiotherapy, with regional control after 18 months of follow-up. Histologic analysis showed an undifferentiated carcinoma without glandular or epidermoid differentiation. Biomolecular analysis of the tumor revealed a homozygous deletion of the SMARCB1 gene on RNA sequencing.ConclusionResearch of INI1 deletion should be performed for undifferentiated carcinoma of young patients because of possibilities of molecular therapies such as autophagy inhibitors or proteasome inhibitors could be used in clinical trials.

EPCO-22. LONGITUDINAL MOLECULAR CHARACTERIZATION IDENTIFIES TREATMENT DRIVEN EVOLUTION OF OLIGODENDROGLIOMA

Abstract Oligodendroglioma is characterized by oligodendrocyte-like histology, mutation in the IDH genes, along with codeletion of chromosome arms 1p and 19q. Treatment for oligodendrogliomas combines surgery, followed by observation, and chemotherapy and/or radiotherapy when tumors progress. Most oligodendrogliomas eventually recur with a more aggressive phenotype, leading to patient mortality but molecular mechanisms remain poorly understood. To investigate the molecular trajectory of oligodendroglioma, we collected more than 270 longitudinal samples from over 130 oligodendroglioma patients, to perform whole genome or whole exome sequencing, bulk RNA, and single-nucleus RNA sequencing (n = 30 samples). Common mutations in IDH1/2, TERT promoter, CIC, and FUBP1 were observed. About half of the CIC and FUBP1 mutations occurred in the initial sample and persisted stably throughout recurrence. The majority of PIK3CA and PIK3R1 mutations were either shared or present in recurrences only implicating the PI3K pathway in oligodendroglioma recurrence. Mutational signature analysis revealed 34.5% of tumors treated with alkylating agents acquired SBS11/SBS31 associated hypermutation. By integrating signatures with clonality estimates, we approximated the timing of mutational processes and found that clonal timings of treatment related mutations correlate with the treatment administration date. We found that aging signature-related mutations were mostly clonal, and treatment signature-related mutations were more likely to be sub-clonal, supporting the late emergence of these mutations following treatment. We observed increased somatic copy number alterations in oligodendroglioma recurrences, reflected in notable chromosome 4 losses. Associating treatment with snRNAseq derived cellular states, we observed a significant increase in the fraction of proliferating stem-like cells in the hypermutator versus non-hypermutator recurrent samples. These findings shed light on the molecular changes occurring in oligodendrogliomas following treatment and provide insights that may aid in targeting treatment resistance.

PATH-50. GENETIC AND EPIGENETIC PROFILING OF MENINGIOMAS TO GAIN INSIGHTS INTO MOLECULAR HETEROGENEITY AND RISK STRATIFICATION.

Abstract BACKGROUND Meningiomas are slow-growing CNS tumors, with most being benign (WHO grade 1) and having indolent behavior. Limited data on molecular profiling of meningiomas and its clinical implications have hindered the implementation of effective therapies and new treatments. Aims & Objectives: Perform DNA methylation profiling, copy number analysis, and targeted sequencing in meningiomas. MATERIALS & METHODS 208 meningiomas, grade 1 (n=109), grade 2 (n=83), and grade 3 (n=16)were assessed for pTERT mutation, CDKN2A homozygous deletion, and H3K27me3 expression. Additionally, 35 diagnostically challenging meningiomas underwent DNA methylation profiling, copy number analysis, and targeted sequencing. RESULTS there were 199 adult and 9 pediatric cases, (mean age 44.5 years). The tumors were located in basal (53%) ,convexity (42%),and 5% in other locations.pTERT C228T mutation was detected in 4.8% and CDKN2A deletion in 2.4% cases. Loss of H3K27me3 expression was found in a significant proportion of tumors across all three grades. Methylation profiling identified four distinct molecular subgroups: Malignant, Intermediate, Benign A, and Benign B. Intermediate, Benign A, and Benign B subgroups included both WHO grade 1 and grade 2 cases. Mutations showed a strong association with specific methylation subgroups (NF2, AKT1, SMO, TRAF7, and pTERT). Loss of chromosome 22q was observed in all subgroups. 1p/14q co-deletion was predominantly seen in the malignant and intermediate subgroups (50%). CDKN2A loss was mainly observed in the malignant subgroup (50%). The DKFZ classifier successfully classified 71% of cases with an acceptable score. Methylation profiling significantly influenced PFS in WHO grade 1 and 2 meningiomas (p=0.0051). CONCLUSIONS Genome-wide DNA methylation profiling reveals clinically distinct molecular subgroups and heterogeneity within the same grade of meningiomas and is particularly valuable for identifying high-risk cases and predicting prognosis in WHO grade 1 and 2 meningiomas. The pTERT mutation serves as a significant and cost-effective marker for risk stratification in meningiomas.

BIOM-53. MENINGIOMA COPY NUMBER VARIANT THRESHOLDS REVEAL OPTIMAL SIZES FOR GAINS OR LOSSES THAT PREDICT LOCAL FREEDOM FROM RECURRENCE OR OVERALL SURVIVA

Abstract INTRODUCTION Meningiomas demonstrate heterogeneous rates of postoperative local freedom from recurrence (LFFR) and overall survival (OS). Risk-stratification models incorporating copy number variants (CNVs) improve outcome prediction, but optimal size thresholds for defining CNVs in meningiomas are unknown, and previous models have used thresholds ranging from 5-80% that are applied uniformly across all chromosome arms. Here we show that optimizing chromosome-specific CNV size thresholds improves meningioma risk-stratification. METHODS 565 retrospective meningiomas with clinical data and DNA methylation profiles from 2 international institutions were included. CNVs were defined from DNA methylation profiles using iterative thresholds ranging from 5-95% for each chromosome arm. The area under the curve (AUC) for 5-year LFFR or OS was calculated using CNVs across iterative thresholds for each chromosome arm. LASSO and Elastic Net Cox regressions were used to generate multi-CNV models for LFFR or OS. RESULTS AUC for LFFR or OS peaked with CNV thresholds that were unique to each chromosome arm. Chromosome 1p deletion had optimal AUCs for LFFR and OS of 0.72 and 0.70 at thresholds of 40% and 30%, respectively. Chromosome 1q amplification had optimal AUCs for LFFR and OS of 0.58 and 0.60 at thresholds of 25% and 20%, respectively. Two previously published CNV-based risk-stratification models (integrated score and integrated grade, optimal LFFR thresholds of 5% and 20%, AUCs of 0.77 and 0.78, respectively) demonstrated heterogeneous sensitivity to CNV thresholds. LASSO and Elastic Net Cox regression identified size-dependent risk-stratification models incorporating loss of 1p, 3q, 4p, 5p/q, 6p/q, 9p, 10q, 11p, 12q, 13q, 14q, 17q, and 18p/q, and gain of 1q, 9q, 10p, and 17q. The optimal AUCs for LFFR and OS from multi-CNV models were 0.77 and 0.77 at thresholds of 25% and 35%, respectively. CONCLUSIONS Interrogating CNV thresholds reveals optimal chromosome-specific CNV sizes for predicting LFFR and OS.

Modeling familial sinus node dysfunction with a large intergenic deletion between PITX2 and ANK2 using iPS cell-derived sinoatrial nodal-like cardiomyocytes

Abstract Background Pituitary homeobox 2 (PITX2) encoded by PITX2, is one of the transcription factors and plays an important role in establishing the left-right axis during development. In the heart, PITX2 inhibits the development of the sinoatrial node (SAN) in the left atrium, whereas the absence of PITX2 allows the development of SAN in the right atrium. In a multicenter study, we recently identified a sizeable intergenic deletion between PITX2 and ANK2 on chromosome 4q25 in familial sinus node dysfunction (SND). The deletion contains a CTCF-binding motif (19 bps) which forms a topologically associating domain (TAD) involved in PITX2, and putatively disrupts the TAD. In order to investigate how the intergenic deletion causes SND, we analyzed iPS cells (iPSCs) by specifically differentiating them into SAN cardiomyocytes. Methods and results We identified an intergenic heterozygous deletion of 30k bps in a Japanese family with SND by whole genome sequencing. In the SND family, all 11 genetically affected patients developed SND since childhood, and seven of them underwent pacemaker implantation (the mean age was 26). To elucidate the pathology, we utilized 3 iPSC lines: an iPSC line from a patient, a homozygous CTCF-binding motif knockout line by gene editing, and a control line from a healthy volunteer. We modified the previously reported SAN differentiation protocol1 and generated SAN-like iPSC-derived cardiomyocytes (SANLCMs). SANLCMs showed a higher beating rate than ventricular-like cardiomyocytes (VLCMs), and exhibited SAN-like action potential morphology recorded using a patch-clamp technique. SANLCMs contained lesser NKX2-5 positive cells than VLCMs by immunostaining analysis and their gene expression patterns were compatible with SAN cardiomyocytes: higher expression levels of SHOX2, TBX18, TBX3, and HCN4, and lower expression levels of PITX2, NKX2-5, and MYL2 compared with VLCMs. Then, we analyzed the gene expression profiles during the SAN differentiation. SANLCMs from CTCF-binding motif knockout iPSC lines showed lower beating rates than controls. We also found that the levels of PITX2 expression in SANLCMs from patient-derived and CTCF-binding motif knockout iPSC lines were significantly higher compared to those from the control. Conclusion Our findings suggest that the lack of CTCF-binding motif involved in a large intergenic deletion on chromosome 4q25 might be associated with a hypoplasia of SAN via PITX2 overexpression during cardiac development.

A genetic polymorphism on chromosome 4q25 (rs17570669_T) may predict atrial fibrillation recurrence after successful electrical cardioversion

Abstract Background Direct current electrical cardioversion (DCCV), is an effective treatment method in patients with symptomatic persistent atrial fibrillation (AF). However, AF recurrence may occur following successful DCCV in nearly half of the patients. There is not enough data investigating the association between single nucleotide polymorphisms (SNP) and AF recurrence after DCCV. Purpose We aimed to investigate whether 11 SNPs previously associated with AF (rs2200733, rs10033464, rs6838973, rs3853445 and rs17570669 SNPs in PITX2 gene; rs2106261 SNP in ZFHX3 gene; rs751141 SNP on chromosome 8q21; rs3807989 SNP in CAV1 gene; rs10570248 SNP in TBX5 gene; rs1800469 SNP in TGF-1 gene; and rs6795970 SNP in SCN10A gene) were predictive for recurrence after successful DCCV in patients with persistent AF in Turkish society. Methods We included 75 patients with sinus rhythm after direct current electrical cardioversion (DCCV). Baseline clinical features and 11 AF-related SNPs were compared between patients with and without AF recurrence during the follow-up. The association between SNPs and AF recurrence was evaluated with the additive model (wild type vs heterozygous variant vs homozygous variant), the dominant model (wild type/heterozygous vs homozygous variant), and the recessive model (wild type vs heterozygous variant/homozygous variant). Results The mean age of the patients was 61.9 ± 11.5 years. At a mean follow-up of 17.0 (11.0-25.0) months, 38 patients (50.7%) developed AF recurrence. The frequency of the female gender was higher (55.3% vs. 32.4%), AF duration before DCCV was longer [9.2 (5.5-11.6) vs 5.0 (2.7-11.0) months], and left atrial volume index before DCCV was higher (43.6 ± 8.8 ml/m2 vs. 38.2 ± 12.9 ml/m2) in patients who developed AF recurrence than those without it (p=0.046, 0.029 and 0.036, respectively) (Figure 1a). In the additive model, a SNP at the PITX2 gene (rs17570669_T: OR 9.00, 95% CI 1.28-63.02, p=0.027) and a SNP at the ZFHX3 gene (rs2106261_T: OR 8.96, 95% CI 1.03-77.66, p=0.047) were significantly associated with AF recurrence. Female gender (HR: 1.99, 95% CI: 1.05-3.79, p=0.035) and the rs17570669_T SNP (HR: 4.16, 95% CI: 1.22-14.11, p=0.022) were predictors for AF recurrence in univariate analysis. A multivariate Cox regression analysis showed that the rs17570669_T SNP was an independent predictor of AF recurrence following successful DCCV in the additive model (HR: 3.59, 95% CI: 1.05-12.21, p=0.04) (Figure 1b). A Kaplan-Meier curve showed that AF-free survival was significantly lower in patients who had rs17570669_T SNP in the additive model (Figure 2). Conclusion A SNP at the PITX2 locus (rs17570669_T) may predict AF recurrence after successful DCCV in patients with persistent AF in Turkish society. Such individuals may be closely monitored for the development of AF recurrence after DCCV. In addition, reversible risk factors associated with AF recurrence may be more tightly controlled in such patients.Baseline features and Cox analysisKaplan-Meier curve

Age of diagnosis for children with chromosome 15q syndromes

ObjectiveThe objective of this study was to identify the age of diagnosis for children with one of three neurogenetic conditions resulting from changes in chromosome 15 (Angelman syndrome [AS], Prader-Willi syndrome [PWS], and duplication 15q syndrome [Dup15q]).MethodsData about the diagnostic process for each condition were contributed by the advocacy organizations. Median and interquartile ranges were calculated for each condition by molecular subtype and year. Comparison tests were run to explore group differences.ResultsThe median age of diagnosis was 1.8 years for both AS and Dup15q. PWS was diagnosed significantly younger at a median age of 1 month. Deletion subtypes for both PWS and AS were diagnosed earlier than nondeletion subtypes, and children with isodicentric duplications in Dup15q were diagnosed earlier than those with interstitial duplications.ConclusionUnderstanding variability in the age of diagnosis for chromosome 15 disorders is an important step in reducing the diagnostic odyssey and improving access to interventions for these populations. Results from this study provide a baseline by which to evaluate efforts to reduce the age of diagnosis for individuals with these conditions.

Identification of a Complex Karyotype Signature with Clinical Implications in AML and MDS-EB Using Gene Expression Profiling.

Complex karyotype (CK) is associated with a poor prognosis in both acute myeloid leukemia (AML) and myelodysplastic syndrome with excess blasts (MDS-EB). Transcriptomic analyses have improved our understanding of the disease and risk stratification of myeloid neoplasms; however, CK-specific gene expression signatures have been rarely investigated. In this study, we developed and validated a CK-specific gene expression signature. Differential gene expression analysis between the CK and non-CK groups using data from 348 patients with AML and MDS-EB from four cohorts revealed enrichment of the downregulated genes localized on chromosome 5q or 7q, suggesting that haploinsufficiency due to the deletion of these chromosomes possibly underlies CK pathogenesis. We built a robust transcriptional model for CK prediction using LASSO regression for gene subset selection and validated it using the leave-one-out cross-validation method for fitting the logistic regression model. We established a 10-gene CK signature (CKS) predictive of CK with high predictive accuracy (accuracy 94.22%; AUC 0.977). CKS was significantly associated with shorter overall survival in three independent cohorts, and was comparable to that of previously established risk stratification models for AML. Furthermore, we explored of therapeutic targets among the genes comprising CKS and identified the dysregulated expression of superoxide dismutase 1 (SOD1) gene, which is potentially amenable to SOD1 inhibitors.

WILLIAMS SYNDROME IN PEDIATRIC PRACTICE

This clinical case presents our observations of a 5-month-old boy diagnosed with Williams’s syndrome, highlighting the complexities of recognizing this condition, especially in the early stages of life, where paediatricians and neonatologists play a crucial role in the diagnostic process. Williams’s syndrome is a rare genetic disorder, affecting approximately 1 in 10,000 newborns across different ethnic groups and genders. The condition is primarily caused by a mutation in a group of genes located on the long arm of the 7th chromosome (7q11.23), leading to microdeletion and the loss of specific genes, including elastin, LIM-kinase-1, and replication factor C2 (RFC), while some "lost" genes remain unidentified. In rarer cases, deletions are found on the 11th and 12th chromosomes (11q13-q14 and 22q). This syndrome often presents sporadically, complicating early diagnosis due to its multisystem nature, affecting various organs and systems, such as the cardiovascular, nervous, muscular, urinary, and facial structures. The absence of timely diagnosis and treatment can adversely impact physical and neuropsychological development in affected children. In our case, the final diagnosis was confirmed through molecular genetic examination. Our conclusion highlights the challenges of diagnosing Williams’s syndrome due to its multisystem clinical manifestations. Early diagnosis is essential for parents to gain insights into the disease, its prognosis, and to make informed decisions about raising the child within the family. We recommend a comprehensive approach to the care and rehabilitation of patients with Williams’s syndrome. This necessitates the coordinated efforts of various specialists including paediatricians, geneticists, cardiologists, surgeons, orthopaedists, orthodontists, endocrinologists, ophthalmologists, neurologists, and psychologists throughout the patient's follow-up care.

Cell-Free DNA from Blood or Bone Marrow as Alternatives to Bone Marrow Cells for Molecular Diagnostics and Monitoring of Multiple Myeloma

Introduction: Molecular profiling of bone marrow (BM) tumor cells in multiple myeloma (MM) can be limited by low tumor cell content for analysis by multiple assays. We therefore evaluated the concordance of molecular profiles of cancer cells enriched from BM versus cell-free DNA (cfDNA) derived from peripheral blood (PB) and BM plasma collected at diagnosis. Use of cfDNA has the potential to enable the prioritization of scarce cancer cells for single-cell RNA-sequencing or other applications that require intact cells. Methods: The MM Molecular Monitoring (M4) Study is a prospective cohort study of newly diagnosed MM patients (pts) receiving standard-of-care treatment at 12 sites across Canada. From 27 pts in this study, we analyzed diagnostic BM-derived CD138+ enriched plasma cell (PC) DNA (n=27, 15-20 mL BM per pt), BM plasma cfDNA (n=26 same tube as BM sampling), PB cfDNA (n=27, 15-20 mL), and PB buffy coat DNA (n=27, 4-6mL) using a combination of a targeted next generation sequencing (NGS) panel (10,000X coverage) and shallow (0.1-1X) whole-genome sequencing (sWGS). PB and BM samples collected in STRECK and EDTA tubes, respectively incurred overnight shipping to a central lab for initial processing. The targeted panel includes exons from 37 genes of potential clinical relevance, all immunoglobulin (Ig) loci, and 5 MM-specific translocation hotspots. CD138+ PCs were profiled by clinical FISH for recurrent MM translocations and copy number variations (CNVs). Results: All PB and BM cfDNA samples yielded sufficient DNA quantity for analyses (>20ng), yet only 19/26 BM cfDNA samples passed quality control and were sequenced. Various strategies such as shearing, dilution, and the use of heparinase were not effective in salvaging the failed BM cfDNA. However, the high success rate in a separate local study (24/25 BM cfDNA samples) suggests shipping in non-cell stabilizing tubes may have contributed to failures. The average DNA yield for PB and BM cfDNA samples was 1408ng (sd = 2752ng) and 2462ng (sd = 4301ng), respectively. We estimated tumor fraction (TF) by examining the proportion of genome altered by CNVs using ichorCNA in samples with sWGS. 14/24 (58%) of PB cfDNA samples and 10/18 (56%) of BM cfDNA samples had a high TF (>5%). TF in PB and BM cfDNA were significantly correlated (rho = 0.85, p < 0.001, n = 18 pairs), but were consistently higher in PB cfDNA (p = 0.03, n = 18 pairs). We first compared mutations between BM-derived PCs and cfDNA. We found 12 distinct somatic mutations (variant allele fraction >1%) that were previously reported to have a pathogenic effect in 11/27 (41%) PC samples. This consisted of 10 single nucleotide variants (SNVs) and 2 deletions across NRAS, BRAF, KRAS, CYLD and PRDM1. We found 9/12 mutations in matched PB cfDNA samples [sensitivity (SN)=75%] and 4/8 mutations in matched BM cfDNA (SN = 50%). We next searched for FISH-validated chromosome rearrangements among enriched BM PCs. From these, we correctly detected 5/8 (63%) fusions using the targeted panel that were called out by clinical FISH. These were further detected in matched PB cfDNA (4/5) and BM cfDNA (2/4) samples. Recurrent MM CNVs (amplification at chromosome 1q or deletions at chromosomes 1p, 13p or 17p) were identified in 18/27 (67%) PC samples by sWGS. These CNVs were successfully detected in 8/9 matched PB cfDNA samples with high TF (by sWGS) and in 2/7 samples with low TF. In BM cfDNA, CNVs were successfully detected in 7/8 high TF samples and 1/6 low TF samples. Lastly, we evaluated BCR Ig rearrangements. We detected a clonal Ig rearrangement in 26/27 (96%) BM-derived PC samples. These clones were detected in 25/26 (96%) matched PC to PB cfDNA pairs and in 16/18 (89%) PC to BM cfDNA pairs. The proportion of fragments at Ig loci which corresponded to productive Ig rearrangements was significantly correlated with TF in PB cfDNA (rho = 0.62, p = 0.002, n = 23) but not in BM cfDNA (rho = 0.37, p = 0.14, n = 17). Conclusions: PB cfDNA yielded 78% of the aberrations detected in BM-derived PCs, whereas BM cfDNA yielded 62%. The lower rate of detection in BM cfDNA may reflect DNA contamination from dying white blood cells as a result of shipping BM samples in non-cell stabilizing preservative tubes. BM and PB cfDNA contain highly concordant CNV calls to PCs when cfDNA TF estimates were >5%. Tumor Ig rearrangements were more readily detected in cfDNA compared to other sources of variation and may provide a less-invasive alternative for disease monitoring.

Performance of Newer Staging Systems for Myeloma in a Contemporary, Large Cohort of Patients in the United States

CONCLUSION The most widely used multiple myeloma (MM) staging system, the Revised International Staging System (R-ISS), is based on lactate dehydrogenase (LDH), albumin, high-risk genetic markers by FISH, and beta-2 microglobulin (B2M). However, it does not account for additional risk impacted by multiple genetic abnormalities, or the risk imparted by presence of gain of chromosome 1q, and most patients are staged as R-ISS Stage 2. Two new staging systems, the Mayo Additive Staging System (MASS), and the Second Revision of the International Staging System (R2-ISS) have been recently proposed. A comparison of the performance of these two systems has not previously been done. We aimed to externally demonstrate the application of these staging systems and compare their performances in a large, contemporary cohort of patients in the United States. We conducted a retrospective cohort study using the nationwide Flatiron Health electronic health record (EHR)-derived de-identified database, comprising de-identified patient-level structured and unstructured data, curated via technology-enabled abstraction and originating from approximately 280 US cancer clinics (~800 sites of care). The study included newly diagnosed MM patients initiating treatment between January 1, 2016 and October 1, 2022. We used the Kaplan-Meier method to evaluate real-world overall survival (rwOS) and patients were followed from first-line treatment initiation (index date) until the first of death, end of the study period or last recorded activity. The log-rank test to compare survival curves across derived stages. Multivariable analysis was conducted using stratified Cox models to account for non-proportional hazards by age with covariate adjustment for race/ethnicity, sex, practice type (academic or community) and diagnosis year (2016-2019 vs 2020-2022). Discrimination and calibration were evaluated in crude models using Harrell's c-index and the Hosmer-Lemeshow goodness-of-fit test, respectively. There were 497 patients with MM included. Patient characteristics are listed in Panel A. The distribution of patients across R-ISS stages were as follows, 24%, 63% and 13% for Stage 1, 2 and 3 respectively. rwOS differed across R-ISS stage (log-rank p = 0.0006) and the median OS (OS) was not reached (NR), 63, and 37 months for R-ISS stage 1, 2, and 3, respectively. 34%, 35% and 31% for MASS 1, 2, and 3, respectively. The corresponding survival curves were significantly different (p < 0.0001), with associated mOS of 77, 61 and 45 months for MASS stages 1, 2, and 3, respectively. R2-ISS includes four risk categories (stages I-IV) and in our cohort, 20% (n = 100) were stage I (low), 126 (25%) were stage II (low-intermediate), 229 (46%) were stage III (intermediate), and 42 (9%) were stage IV (high). Survival across stages were significantly different (p<0.0001), though the survival curves for low and low-intermediate, and intermediate-high and high, were clustered, suggesting comparable outcomes in stages I-II and stages III-IV. mOS for R2-ISS stages I, II, III, and IV was NR, 69, 50 and 51 months respectively (Panel B). In fully adjusted analyses, as compared to MASS I, the hazards of death were 2.0 (95% CI 1.3-3.2) for MASS II and 2.7 (1.7-4.2) for MASS III. The hazards of death were similar for R2-ISS low and low-int (HR for low-int vs low: 1.2 [0.7, 2.3]). As compared to R2-ISS low, the hazards of death were similarly higher for int-high and high (HRs: 2.4 [1.4, 4.1] and 2.6 [1.3, 5.2], respectively). Discrimination and calibration were similar across all staging systems (c = 0.6, and Hosmer-Lemeshow p>0.05 for all three staging systems). Application of contemporary MM staging systems in a large cohort of US patients shows that they are prognostic for OS in MM. MASS demonstrated a greater separation for OS across stages, whereas R2-ISS intermediate-high and high were largely overlapping. Although our results demonstrate similar accuracy and reliability of MASS and R2-ISS, the discrimination of patients across different stages seen with the newer staging systems, as opposed to R-ISS, calls for greater use of these newer staging systems in routine clinical practice.

STAG2 Somatic Mutations Are Associated with Specific Dysplastic Megakaryocytic and Myeloid Cell Features in Myelodysplastic Syndrome

Introduction: Although myelodysplastic neoplasms/myelodysplastic syndromes (MDS) are driven by genetic mutations, their diagnosis relies on morphologic evaluation of abnormal hematopoiesis in the bone marrow. Only a small number of genetic abnormalities define bone marrow morphologic features in MDS, such as those harboring SF3B1 mutations and deletions of chromosome 5q. We hypothesized that there are additional genetic alterations and dysplastic morphologic features in MDS that have not been well-described in the literature. We assessed genetic morphologic associations between 50 commonly mutated genes and 10 morphologic features in a cohort of MDS bone marrows with a high degree of dysplasia. We validated and extended these findings in an independent cohort of myeloid neoplasms. STAG2 mutations were strongly associated with specific dysplastic megakaryocytic and myeloid cell features in both cohorts. Methods: We reviewed bone marrow core biopsies and aspirates from 272 MDS clinical cases (2013-2014) and selected 89 for further study based on the presence of prominent dysplastic features. Each case was independently evaluated by two pathologists for the following dysplastic features: small hypolobated megakaryocytes, widely separated megakaryocyte nuclei; abnormal myeloid nuclear segmentation, myeloid hypogranulation, Auer rods; ring sideroblasts, erythroid binucleation, irregular erythrocyte nuclei, erythroid nuclear cytoplasmic dyssynchrony, and erythroid karyorrhexis. Targeted next generation sequencing was performed using a 50 gene panel comprising the most common somatic myeloid mutations. For each dysplastic feature, univariate risk analysis was used to identify genetic mutations for use in multivariable analysis. Significant associations were validated through blinded morphologic review in an independent cohort of 155 bone marrow biopsies of myeloid neoplasms, comprised of 83 myelodysplastic syndromes, 33 myelodysplastic/myeloproliferative neoplasms and 39 acute myeloid leukemias, diagnosed between 2014 and 2019 and selected for mutations identified in the initial discovery cohort ( STAG2, RUNX1, SRSF2, ASXL1 and SETBP1) . Results: Pairwise analyses demonstrated broad patterns of genetic associations with myeloid/megakaryocytic dysplasia versus erythroid dysplasia (Panel 1). In multivariable analysis, STAG2 and SRSF2 mutations were significantly associated with megakaryocytes with widely separated nuclei ( STAG2, OR = 25.5, 95% CI [4.17, 493.5], P = 0.003; SRSF2, OR = 10.1, 95% CI [2.07, 75.0], P = 0.008). Mutations in STAG2 and SETBP1 were significantly associated with abnormal myeloid nuclear segmentation ( STAG2, OR = 7.08, 95% CI [1.94, 28.0], P = 0.003; SETBP1, OR = 12.2, 95% CI [1.29, 267.4], P = 0.04). Furthermore, STAG2 mutations were significantly associated with myeloid cell hypogranulation (OR = 12.7, 95% CI [3.10, 86.3], P = 0.002). Because this initial cohort was relatively small, which led to wide confidence intervals suggesting estimation instability, we validated and extended these findings in an independent cohort of myeloid neoplasms. In the validation cohort (enriched for mutations in STAG2, SRSF2, and SETBP1), STAG2 mutations were significantly associated with separated megakaryocyte nuclei (univariate OR = 4.18, 95% CI [1.98, 8.80], P = 0.0002), abnormal myeloid nuclear segmentation (univariate OR = 3.47, 95% CI [1.47, 8.22], P = 0.008), and myeloid cell hypogranulation (multivariable OR = 5.28, 95% CI [2.18, 13.4], P = 0.0003) (Panel 2). In contrast, morphologic associations with SRSF2 and SETBP1 mutations were not validated. Conclusion: STAG2 genetic alterations are strongly associated with the presence of separated megakaryocyte nuclei, abnormal myeloid nuclear segmentation and myeloid cell hypogranulation in MDS. Our data suggest that STAG2 may affect pathways involved in nuclear shape control and secondary granule formation. Panel 1: Pairwise associations between gene mutation and bone marrow morphology in MDS. N, number. Panel 2: Multivariable (black) and univariate (white) analysis in two independent cohorts showing significant gene associations with megakaryocytes with separated nuclei, abnormal myeloid nuclear segmentation, and myeloid cell hypogranulation. CI, confidence interval.

An In-Depth Analysis of Ependymoma: Neuropathological Insights, Therapeutic Strategies, and Prognostic Implications in Intracranial Ependymal Tumors

Ependymomas are primary central nervous system (CNS) neoplasms arising from ependymal cells lining the ventricular system and spinal cord. They represent a heterogeneous group of tumors with distinct histological and molecular subtypes, making their diagnosis, management, and prognosis a complex and evolving challenge in the field of neuro-oncology. This comprehensive review delves into the intricate landscape of ependymomas, elucidating their pathogenesis, classification, and molecular underpinnings. Histologically, they can manifest as myxopapillary, subependymoma, and anaplastic ependymomas, each carrying distinct clinical implications and therapeutic strategies. Moreover, recent advancements in molecular profiling have unveiled significant genetic alterations such as amplifications in RELA, YAP1, and C11orf95-MAML2 fusion, which have profound implications for prognosis and therapeutic decision-making. We explore the intricacies of clinical management, encompassing surgical resection, radiation therapy, and chemotherapy regimens tailored to the tumor's location, grade, and molecular profile. Despite their location within the CNS, ependymomas are notorious for their diverse clinical presentations, including headache, neurological deficits, and intracranial hypertension, necessitating multidisciplinary care and vigilant surveillance. Additionally, this article investigates the latest developments in targeted therapies, immunotherapeutic approaches, and ongoing clinical trials in an attempt to provide a glimpse into the future of ependymoma treatment, as precision medicine begins to play a more significant role in guiding therapeutic decisions. Furthermore, the abstract examines the critical issue of ependymoma prognosis, emphasizing the importance of integrating histopathological and molecular data in order to stratify patients into risk categories more accurately. The role of genetic markers, such as chromosome 1q gain and chromosome 6q loss, in predicting outcomes is thoroughly explored. In summary, this article aims to offer a comprehensive perspective on ependymomas, from their cellular origins to the latest breakthroughs in their management, offering insights into their clinical course, and underscoring the critical role of multidisciplinary collaboration in advancing the understanding and treatment of these complex intracranial tumors.