Chromoanagenesis (CAG) is a catastrophic genomic event characterized by extensive chromosomal rearrangements and copy number alterations. It has been associated with complex karyotypes and poor outcomes in acute myeloid leukemia. However, its prevalence and associated clinicopathologic and cytogenomic features in myelodysplastic syndromes (MDSs) remain incompletely defined. In this study, we performed optical genome mapping in 332 patients with MDS, 205 newly diagnosed (ND), and 127 relapsed/refractory (R/R) cases. CAG was identified in 15.9% of cases overall and in 17.6% of ND MDS. Among ND patients, 97% of CAG cases harbored highly complex karyotypes versus 9% of non-CAG cases (P < .01). TP53 alterations were present in 97% of CAG cases, predominantly multihit events, whereas mutations in canonical MDS driver genes were uncommon in CAG cases. Recurrent oncogenic amplifications, including 11q23/KMT2A and 6p21/PIM1 and HMGA1, were enriched in CAG cases. Clinically, 80.6% of CAG cases were classified as very high risk by Molecular International Prognostic Scoring System. Median overall survival of patients with ND CAG was 9.9 months versus not reached in non-CAG cases (P < .01), and survival of CAG cases remained inferior compared with patients with non-CAG having very high Molecular International Prognostic Scoring System risk (20.4 months) or multihit TP53 alterations (18.3 months). We conclude that CAG defines a biologically distinct, ultra-high-risk subset of MDS characterized by TP53 disruption, extreme genomic complexity, and dismal prognosis. Optical genome mapping enables efficient CAG detection and may refine current risk stratification.

We summarize the technical principles of optical genome mapping (OGM) and evaluate its current clinical applications across myeloid neoplasms, with emphasis on acute myeloid leukemia and myelodysplastic syndromes. We highlight evidence demonstrating high concordance with standard cytogenetic methods, while also showing incremental diagnostic yield through detection of cryptic abnormalities and/or refinement complex rearrangements. We further discuss clinically relevant cytogenomic abnormalities that may be underrecognized by conventional cytogenetics, including chromoanagenesis, KMT2A partial tandem duplication and cryptic rearrangements involving genes such as NUP98 and MECOM. We also address practical considerations for implementation of OGM in a clinical cytogenetic laboratory, including pre-analytical DNA quality requirements, assay sensitivity, bioinformatic interpretation and workflow integration. Current data support OGM as a complementary component of genomic workups rather than a replacement for all existing assays. As analytical standards mature and outcome-linked evidence expands, OGM has strong potential to improve genomic risk stratification, refine disease classification and advance precision diagnostics in myeloid neoplasms.

Background: Specimens with low cell counts may fail to yield sufficient analyzable metaphases or are rejected for chromosomal analysis. Buffy coat enrichment (BCE) concentrates nucleated cells prior to culture; however, its impact on routine cancer cytogenetics has not been systematically evaluated. Methods: We first validated BCE in hypocellular specimens (<5 K/µL) and then conducted a prospective quality improvement study from November 2024 to October 2025, encompassing 12,088 specimens. A phased intervention strategy was implemented by performing BCE on specimens with cell counts of 2.0-4.9 K/µL (designated as phase I); followed by expanding BCE to specimens with cell counts of 1.0-4.9 K/µL (phase II). Outcomes were assessed by the rate of successful karyotypes, defined as ≥10 analyzable metaphases. Results: In the validation cohort (cell counts < 5 K/µL), BCE improved the success rate across all cell count strata. In the prospective study cohort, implementation of BCE increased the overall success rate from 78% at baseline to 83% in phase I, and further increased to 90% in phase II. Conclusions: BCE significantly improves the success rate of chromosomal analysis by increasing the yield of metaphases in hypocellular specimens. This simple and scalable intervention reduces specimen rejection and enhances diagnostic yield in routine cancer cytogenetics.

Background: KMT2A rearrangements occur in ~10% of acute myeloid leukemia (AML) cases and are critical for classification, risk stratification, and use of targeted therapy. However, insertions involving the KMT2A gene can evade detection using chromosomal analysis and/or fluorescence in situ hybridization (FISH). Methods: We present a case of a 22-year-old woman with acute monoblastic leukemia harboring a cryptic KMT2A::AFDN fusion identified by RNA sequencing. Initial FISH showed a 3′ KMT2A deletion, while conventional karyotyping and the automated bioinformatic pipeline for optical genome mapping (OGM) did not identify the canonical translocation. Results: To resolve these discrepancies, metaphase KMT2A FISH (break-apart fusion probe) was performed to assess whether KMT2A was translocated to another chromosome. However, the results did not support this possibility. As the fusion signal remained on the normal chromosome 11, with the 5′ KMT2A signal localized to the derivative chromosome 11. A subsequent manual review of the OGM data revealed a cryptic ~300 kb insertion of AFDN into the 3′ region of KMT2A, reconciling the discrepancies between chromosomal analysis, FISH, and RNA fusion results. Conclusions: This case highlights the importance of integrating multiple testing modalities with expert review when there is a discrepancy. Our findings emphasize the need for a comprehensive approach to genomic assessment to enhance diagnostic accuracy and guide therapeutic decision-making.

Background/Objectives: Gene rearrangements involving oncogenes are major drivers in acute leukemia, influencing disease classification, prognosis, and therapeutic decision-making. Targeted RNA sequencing (RNA-Seq) panels capable of detecting intergenic and intragenic fusions across multiple genes are increasingly used in diagnostic settings. However, comparative evaluation with orthogonal technologies remains limited. Material and Methods: We compared the performance of a 108-gene anchored multiplex PCR (AMP)-based RNA-Seq panel with that of Optical Genome Mapping (OGM) in 467 acute leukemia cases. The cohort included 360 cases of acute myeloid leukemia (AML), 89 B-lymphoblastic leukemia (B-ALL), 12 T-lymphoblastic leukemia (T-ALL), and 6 cases of mixed phenotype acute leukemia (MPAL). Results: Results of both methods were concordant in 175 (74.7%) of 234 detected gene/rearrangement fusions. The concordance rate varied significantly across different leukemia types, ranging from 80.2% in B-ALL to 41.7% in T-ALL (p < 0.001) OGM uniquely detected 37 of 234 (15.8%) clinically relevant rearrangements, whereas RNA-Seq exclusively identified 22 of 234 (9.4%). Enhancer-hijacking lesions, including MECOM and BCL11B rearrangements, CDK6::MNX1, and IGH rearrangements, had a markedly lower concordance (20.6%) compared with all other aberrations (93.1%) (p < 0.001). Conversely, some gene fusions arising from intrachromosomal deletions were interpreted by OGM as simple deletions rather than rearrangements or fusions. Conclusions: Targeted RNA-Seq was effective for detecting chimeric fusion transcripts and showed slightly better performance in identifying fusions resulting from deletions. However, OGM was effective for detecting enhancer-hijacking events that do not generate fusion transcripts. Both methods are complementary for the workup of acute leukemia cases.

Background and Objective: The primary objective of this study is to evaluate the added value of optical genome mapping (OGM) when integrated into the standard cytogenetic workup (SCGW) for hematological malignancies. Methods: The study cohort comprised 519 cases with different types of hematological malignancies. OGM and SCGW (including G-banded karyotyping and fluorescence in situ hybridization) were performed on blood and/or bone marrow. The analytical sensitivity of OGM, defined as the detection of all additional cytogenomic aberrations, and its clinical utility, referring to aberrations with diagnostic, prognostic, or therapeutic significance, were assessed. Results: OGM led to increased analytical sensitivity and clinical utility in 58% and 15% of the cases, respectively. The clinical utility varied across different malignancies, with the highest utility in T-lymphoblast leukemia (52%), followed by mixed phenotype acute leukemia (43%), B-lymphoblastic leukemia (37%), other B-cell lymphomas (22%), mature T-cell leukemia/lymphoma (20%), chronic lymphocytic leukemia (14%), acute myeloid leukemia (13%), multiple myeloma (13%), mantle cell lymphoma (8%), myelodysplastic/myeloproliferative neoplasms (6%), myelodysplastic syndrome (5%), and myeloproliferative neoplasms (0%). Conclusion: Compared to SCGW, OGM detects additional cytogenomic aberrations in approximately 58% of cases. OGM provides clinical utility at varying rates across different types of hematological malignancies. Given these differences, strategic triaging can help maximize the clinical value of OGM by focusing on diseases where it offers the most significant benefit.

Introduction KMT2A rearrangement defines a distinct class of acute myeloid leukemia (AML) and confers a poor prognosis and sensitivity to menin inhibitors. We report a case of AML with a KMT2A::MLLT10 fusion undetectable by karyotyping and fluorescence in situ hybridization (FISH) but identified by using a RNA-based next-generation sequencing (RNA-Seq) fusion panel. The discrepancy between RNA-Seq and traditional cytogenetic analysis was resolved by optical genome mapping (OGM), which identified an insertion of exons 9 to 62 of MLLT10 to the 3′ end of KMT2A exon 9 as the genomic alteration resulting in the fusion transcript. Case Presentation A 2-year-old boy with a history of AML diagnosed at another institution was reported to have a KMT2A rearrangement (no written report was available). He underwent multiple lines of therapy, including allogeneic stem cell transplant. He presented our institution with relapsed AML and has completed cycle 1 of venetoclax, azacitidine. He was subsequently screened for menin inhibitor therapy. Diagnostic Workup and Management Bone marrow evaluation:•Persistent AML, 86% blasts on aspirate smears.Flow Cytometry Immunophenotyping:•90% aberrant blast population with immunophenotype consistent with AMLRNA Sequencing for Fusion Detection:•KMT2A::MLL10 fusion involving exon 10 of KMT2A and exon 9 of MLL10.KMT2A FISH using breakapart probe:•nuc ish(KMT2Ax1)[11/200]Chromosomal analysis:•46,XY,del(14)(q24q32)[14]/46,idem,t(7;10)(p22;q24)[2]/46,XY,del(3)(q21q26.3),add(11)(p15)[cp2]/46,XY,add(2)(q33),add(5)(q22)[1]/46,XY[1]Optical genome mapping:•Insertion of exons 9 to 62 of MLLT10 to the 3′ end of KMT2A exon 9 . The detailed list of aberrations including other genomic alterations are listed below•ISCN:ogm[GRCh38]t(1;7)(p36.13;p15.2)(19283014;25615904),t(3;8)(p24.3;q24.21)(18410735;127868973),10p12.31(21626543_21906264)x1,t(10;19)(q26.13;p13.11)(123166285;18028922),ins(11;10)(q23.3;p12.31(118486504_118486505;21626543_21906264),14q24.1q32.2(69481965_96449359)x1∼2Management:•Patient was found to be eligible for a menin inhibitor trial targeting KMT2A rearrangement. Discussion and Conclusion Gene insertions are the primary culprit when RNA-based molecular results are positive, but FISH and karyotyping results are falsely negative. Discrepancies between molecular and cytogenetic findings in actionable biomarkers, such as KMT2A, can create confusion among clinicians.This case illustrates the power of OGM to provide a definitive explanation for the KMT2A::MLLT10 by identifying an insertion event, thereby resolving the discrepancy between RNA fusion and conventional cytogenetic findings. These findings underscore the importance of integrating complementary genomic testing approaches to enhance diagnostic accuracy, optimize treatment selection, and improve patient outcomes in AML.

Chromoanagenesis (CAG) encompasses a spectrum of catastrophic genomic events, including chromothripsis, chromoanasynthesis, and chromoplexy. We studied CAG in 410 patients with a diagnosis of acute myeloid leukemia (AML), 292 newly diagnosed (ND), and 118 refractory/relapsed, using optical genome mapping. CAG was identified by the presence of clusters (with 10 or more breakpoints) of structural abnormalities and/or segmental copy number alterations within one or more chromosomal regions. CAG was detected in 65 (16%) patients. Compared with patients without CAG, those with CAG showed significantly (p < 0.0001) higher frequencies of highly complex karyotype (92% vs. 11%), monosomal karyotype (88% vs. 12%), extensive clonal heterogeneity (75% vs. 7%), gene amplification (49% vs. 1%), and TP53 deletion/mutation (92% vs. 9%). Overall, CAG was detected in about two-thirds of AML patients who exhibited the abovementioned high-risk cytogenetic abnormalities/karyotype. Among the 42 patients with ND AML and CAG, 36 received treatments and follow-ups, and 28 (78%) had no or only partial response to therapy. Among patients with ND AML, those with CAG had a shorter overall survival than those without CAG (median survival: 5 vs. 14 months, p < 0.0001). However, in multivariate analysis, CAG did not appear to be an independent risk factor for survival. These results indicate that CAG is frequently associated with high-risk chromosomal alterations and genomic instability in AML and may contribute to treatment refractoriness and inferior survival in this subset of AML patients.

Identification of cytogenetic abnormalities is critical for the classification and risk stratification of myelodysplastic syndromes (MDS). Optical genome mapping (OGM) is an emerging cytogenomic platform that enables high-resolution genome-wide cytogenetic analysis. We analyzed bone marrow specimens of 236 MDS patients, 149 newly diagnosed and 87 with relapsed/refractory disease, using OGM, conventional karyotyping, and next-generation sequencing analysis. OGM and karyotyping showed concordant results in 68% of cases, including 34% with normal findings by both assays. OGM provided additional information in 27% of patients. Common abnormalities detected exclusively by OGM included chromoanagenesis (n = 33), KMT2A partial tandem duplication (n = 7), and MECOM rearrangement (n = 4). These OGM findings led to disease reclassification and/or changes in risk stratification in 14 patients (9.4%) with newly diagnosed MDS. In contrast, OGM failed to detect small clones or subclones in 5% of patients, resulting in risk group changes in 2% of newly diagnosed MDS patients. We conclude that OGM enhances the cytogenetic assessment of MDS in approximately 25% of patients and leads to a change in disease classification and/or risk stratification in approximately 10% of patients. However, low sensitivity for detecting small clones or subclones remains a limitation of OGM.

Introduction Gene rearrangements involving oncogenes are key drivers in acute leukemia, greatly influencing classification, prognosis and treatment decisions. Simultaneous testing for critical intergenic and intragenic fusions across multiple genes using targeted next generation sequencing (NGS)-based RNA fusion panels (RNA-Seq) is gaining prominence in diagnostic testing. However, a systematic assessment of the performance of targeted RNA-Seq panels compared with an orthogonal technology is currently lacking. In this study, we compare the performance of an in-house developed 108-gene panel utilizing Anchored Multiplex PCR (AMP) chemistry with that of Optical Genome Mapping (OGM). Methods A total of 467 acute leukemia cases were identified from our institutional database between October 1, 2023 and January 31, 2025. The cohort included 360 cases of acute myeloid leukemia (AML), 89 B-acute lymphoblastic leukemia (ALL), 12 T-ALL and 6 cases of Mixed Phenotype Acute Leukemia (MPAL). All cases underwent testing using the 108-gene targeted RNA-Seq panel and OGM. For these genes, information regarding DNA-level gene rearrangements (identified by OGM) and RNA-level gene fusions (identified by NGS) was extracted. The findings were categorized according to the Association for Molecular Pathology (AMP), American Society of Clinical Oncology (ASCO), and College of American Pathologists (CAP) guidelines. Tier 1 and Tier 2 findings were considered clinically significant. Results 192 of 467 (41%) cases had Tier 1 or Tier 2 findings detected by RNA-Seq and/or OGM. The five most frequent rearrangements identified were: KMT2A partial tandem duplication (KMT2A-PTD) (n=31), KMT2A rearrangements (n=26), BCR::ABL1 (n=23), MECOM (n=21), and CBFB (n=16). The overall concordance rate between RNA-Seq and OGM was 91%.OGM exclusively detected clinically significant findings in 34 cases. These included rearrangements involving: MECOM (n=16), BCL11B (n=4), IGH::CRLF2 (n=4), IGH::EPOR (n=2), KMT2A-PTD (n=2), PAX5 alt (n=2), IGH::BCL2 (n=1), CDK6::MNX1 (n=1), IGH::CEBPA (n=1), and ETV6 (n=1). Conversely, RNA-Seq exclusively identified clinically significant findings in 8 cases including: KMT2A-PTD (n=2), P2RY8::CRLF2 (n=2), SET::NUP214 (n=1), NU214::ABL1 (n=1), KMT2A::AFDN (n=1) and RUNX1::SENP1 (n=1). Discussion and Conclusion The 108-gene NGS RNA-Seq panel demonstrated high efficacy in detecting gene rearrangements that result in chimeric fusion transcripts. Additionally, NGS RNA-Seq was marginally more effective than OGM for identifying gene fusions caused by chromosomal deletions. However, RNA-Seq was less effective in detecting rearrangements that lead to oncogene overexpression via enhancer hijacking, as these aberrations do not produce chimeric fusion transcripts. Notably, OGM was more effective for detecting rearrangements involving MECOM in AML, IGH in B-ALL and BCL11B in T-ALL, Furthermore, acute leukemias with these abnormalities when assessed by RNA-Seq panels can be falsely negative.

Cytogenomic characterization is crucial for the classification and risk stratification of acute myeloid leukemia (AML), thereby facilitating therapeutic decision-making. We examined the clinical utility of optical genome mapping (OGM) in 159 AML patients (103 newly diagnosed and 56 refractory/relapsed), all of whom also underwent chromosomal banding analysis (CBA), fluorescence in situ hybridization, and targeted next-generation sequencing. OGM detected nearly all clinically relevant cytogenetic abnormalities that SCG identified with >99% sensitivity, provided the clonal burden was above 20%. OGM identified additional cytogenomic aberrations and/or provided information on fusion genes in 77 (48%) patients, including eight patients with normal karyotypes and four with failed karyotyping. The most common additional alterations identified by OGM included chromoanagenesis (n = 23), KMT2A partial tandem duplication (n = 11), rearrangements involving MECOM (n = 7), NUP98 (n = 2), KMT2A (n = 2), JAK2 (n = 2), and other gene fusions in 17 patients, with 10 showing novel fusion gene partners. OGM also pinpointed fusion genes in 17 (11%) patients where chromosomal rearrangements were concurrently detected by OGM and CBA. Overall, 24 (15%) aberrations were identified exclusively by OGM and had the potential to alter AML classification, risk stratification, and/or clinical trial eligibility. OGM emerges as a powerful tool for identifying fusion genes and detecting subtle or cryptic cytogenomic aberrations that may otherwise remain undetectable by CBA.

KMT2A partial tandem duplication (PTD) involves intragenic KMT2A duplications and has been associated with poorer prognosis. In this study, we evaluated KMT2A PTD in 1277 patients with hematological malignancies using optical genome mapping (OGM). KMT2A PTD was detected in 35 patients with acute myeloid leukemia (AML) (7%), 5 patients with myelodysplastic syndrome (MDS) (2.2%), and 5 patients with chronic myelomonocytic leukemia (CMML) (7.1%). The PTDs varied in size, region, and copy number. An Archer RNA fusion assay confirmed KMT2A PTD in all 25 patients tested: 15 spanning exons 2 to 8 and 10 spanning exons 2 to 10. Most patients exhibited a normal (n = 21) or non-complex (n = 20) karyotype. The most common chromosomal abnormalities included loss of 20q or 7q and trisomy 11/gain of 11q. All patients had gene mutations, with FLT3 ITD and DNMT3A prevalent in AML and DNMT3A and RUNX1 common in MDS and CMML. Among patients who received treatment and had at least one follow-up bone marrow evaluation, 82% of those with de novo AML achieved complete remission after initial induction chemotherapy, whereas 90% of patients with secondary or refractory/relapsed AML showed refractory or partial responses. All but one patient with MDS and CMML were refractory to therapy. We conclude that OGM is an effective tool for detecting KMT2A PTD. Neoplasms with KMT2A PTD frequently harbor gene mutations and display normal or non-complex karyotypes. Patients with KMT2A PTD are generally refractory to conventional therapy, except for de novo AML.

Background. Optical genome mapping (OGM) is a novel, non-sequencing-based technique for genome-wide detection of various types of chromosomal structural abnormalities (losses, gains, translocations, inversions, partial tandem duplications) at a very high (104X) resolution compared to chromosome banding analysis (CBA). We published our experience with OGM for cytogenomic profiling of a large retrospective cohort of newly diagnosed myelodysplastic syndrome/ neoplasm (MDS) patients using OGM (Yang et al., Leukemia 2022). Our findings revealed that OGM identified multiple cryptic abnormalities, with over half being undetectable by CBA across 34% of patients. Additionally, OGM results independently predicted overall survival. Subsequently, we implemented OGM as a clinical assay in our clinical cytogenetic laboratory. In this study, we present the results of an additional 105 MDS patients who underwent OGM prospectively as part of the routine diagnostic work-up in a clinical diagnostic laboratory. Methods. All patients diagnosed with MDS (2023-2024) who underwent OGM were identified. Clinicopathological data including standard-of-care cytogenetic studies were collected from medical records. All cases were classified using WHO (5th ed.) and International Consensus Classification (ICC) criteria. Results. The cohort included 105 MDS patients [67 (%) men, 38 (%) women] with age ranging from 40 to 92 and median age of 65 years. The median blast percentage was 7%. The distribution based on WHO (4th ed.) showed MDS with multilineage dysplasia (n=49), MDS excess blasts-1 (n=23), MDS with excess blasts-2 (n=27) and MDS with isolated del(5q) (n=6). Based on CBA, 26 (24%), 51 (48%) and 28 (26.7%) patients had normal, non-complex and complex karyotypes respectively. OGM was performed on 104 patients (in one patient, there were not enough cells). OGM allowed precise gene/exon-level mapping of clinically relevant biomarkers for application of AMP/ASCO/CAP classification tiers. Sixty-nine (66%) of MDS patients had at least one Tier 1 or 2 abnormality (clinically significant). The abnormalities detected in the remaining 35 (33%) patients did not meet the criteria for Tier 1/2. Due to inherent higher resolution compared to CBA, OGM showed additional clinically significant abnormalities in 33 (31%) of patients. Of these, 7 cases showed abnormalities that changed the WHO5/ICC classification (diagnostic) [additional del(17p) loss in 2 patients, del(5q) in 2 patients, MECOM rearrangement (2 patients), MLF::NPM1 and del(7q). In 5 patients had a change in the prognostication based on detection of biallelic TP53 alteration (n=2). or KMT2A-partial tandem duplication (n=3). Further, 18 cases showed chromoanagenesis, a genomic catastrophe resulting in multiple chromosomal rearrangements and copy number changes, only 15 of these patients showed complex karyotype by CBA. In contrary, even though limit of detection of OGM is similar to CBA (10-20%), OGM missed clonal (seen in at least 2 metaphases) abnormalities in 5 cases due to absence of proliferative bias, underscoring the necessity of performing CBA work-up in parallel. Conclusions. OGM is a powerful tool for identification of genome-wide chromosomal abnormalities and can be used to accurately apply WHO5 and ICC classification criteria.

Optical genome mapping (OGM) is a new DNA-based technology which provides comprehensive examination of the entire genome. We report two patients who presented with splenomegaly and leukocytosis with lymphocytosis including villous lymphocytes. Neither patient had lymphadenopathy. Bone marrow evaluation showed involvement by small B-cell lymphoma in a sinusoidal and interstitial distribution, and immunophenotypic analysis showed that the neoplastic cells were positive for B-cell markers and cyclin D1 but were negative for SOX11 and CD5. Initially, the clinicopathologic features in both patients were thought to be suspicious for hairy cell leukemia variant or splenic marginal zone lymphoma. However, OGM detected CCND1 rearrangement: t(2;11)/IGK::CCND1 in one case and t(11;14)/IGH::CCND1 in the other case. These cases illustrate the valuable role OGM can play in establishing the diagnosis of MCL. Case 1 also contributes to the paucity of literature on the rare occurrence of IGK::CCND1 in MCL.

Background. T-cell acute lymphoblastic leukemia (T-ALL) is caused by the accumulation of genomic alterations that disrupt the development and proliferation of T-cells. These include somatic mutations in multiple genes, with NOTCH1 seen in about 50% of patients. In addition to mutations, driver chromosomal rearrangements, such as those affecting BCL11B (14q32) form the basis of sub-classification per latest WHO (5th ed) and ICC classifications and treatment decisions. In addition, ICC has proposed multiple provisional entities based on the underlying driver genomic rearrangements, often affecting T-cell receptors (TRB/TRG) with genes such as TAL1/3, TLX1/3, LMO1/2, KMT2A rearrangements, PICALM::MLLT10, SET::NUP214 among others. Consequently, clinical genomic profiling and work-up of T-ALL cases requires the use of advanced, high-throughput genome-wide techniques for detecting both somatic mutations and chromosomal structural variants. NGS offers comprehensive mutational profiling, detecting point mutations, insertions, deletions, and copy number variations at a nucleotide level. Optical genome mapping (OGM) is a DNA-based technique, that enables genome-wide structural variant analysis at a high-resolution, including those alterations often missed by conventional cytogenetics. Integrating OGM and NGS in the diagnostic workflow allows for a more detailed and accurate genomic characterization of T-ALL, facilitating better classification and diagnosis. In this study, we present the results of targeted NGS and OGM data from our clinical lab in a large cohort of T-ALL patients. Methods. All patients diagnosed with T-ALL and who underwent bone marrow (BM) examination were identified from the medical records. Somatic mutation analysis was performed on BM aspirate samples using a clinical-grade 81-gene panel through next-generation sequencing (NGS). All patients underwent chromosome banding analysis (CBA). A subset of cases underwent OGM using ultra-high molecular weight DNA to identify driver genomic aberrations. Results. Our cohort included a total of 125 patients diagnosed with T-ALL [63 (50.4%) treatment-naïve; 62 (49.6%) previously treated]; 31% also met the criteria for early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). The median age of the patients was 36 years, with 94 men (75.2%) and 31 women (24.8%). By NGS, 98 (78.4%) patients had at least 1 mutation. The number of mutations per case ranged between 1 and 14. The most frequent mutations observed were in NOTCH1 (50.4%), PHF6 (24%), JAK3 (19%), DNMT3A (18%), NRAS (17%), and TP53 (17%). The majority of these mutations were missense, while frameshift and nonsense mutations occured less frequently. The highest median variant allele frequency (VAF) was seen in PHF6 (64%), followed by WT1, FBXW7, JAK3 and DNMT3A, all of which showed median >30%. The median NOTCH1 VAF was 22%. NOTCH1 mutations aggregated in the C-terminal PEST domain (nonsense or frameshift), and NOD/NODP domains (missense). CBA showed normal karyotype in 50 (40%) and abnormal karyotypes in 75 (60%) patients; complex karyotype was seen in 14 (11.2%) cases. Due to the limitations of CBA in T-ALL, primarily due to the poor growth of T-ALL cells under in vitro culture and the cryptic nature of most abnormalities, we performed OGM on 18 cases. Seventeen (94%) patients showed Tier 1/2 cytogenetic abnormalities. In 14 (78%) patient, a putative gene fusion implicated in T-ALL pathogenesis could be identified. These included known driver rearrangements involving BCL11B [TLX3::BCL11B (n=3), t(14;16) and t(2;14;10)], ETV6 [ETV6::MN1, CCDN2::ETV6, HOX transcription factors [TRB::HOXA13; fus(7;7), PICALM::MLL10, KMT2A::MLLT1, SPTAN1::NUP214], as well as novel fusions, such as NUP98::YY1, CBL [t(1;11)]. Among copy number changes, CDKN2A/B deletions were most frequent, seen in 5 patients. Four patients showed chromoanagenesis indicating a genomic catastrophe resulting in multiple chromosomal rearrangements and copy number changes. Conclusions. Combining data from both NGS and OGM, enabled accurate sub-classification of T-ALL cases, including those into the provisional entities defined by the ICC. This study demonstrates the feasibility and effectiveness of using genomic analysis (NGS and OGM) to fully understand the major regulators of leukemia cell growth and metabolism in treating T-ALL.

Background: Homozygous cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss is one of the parameters that support the designation of meningiomas as Central Nervous System (CNS) WHO grade 3 tumors. Evaluation of CDKN2A/B by sequencing or Fluorescence in situ hybridization (FISH) is costly and not always readily accessible. An immunohistochemistry (IHC)-based marker for the evaluation of CDKN2A/B loss would provide faster results at a lower cost. Methods: This retrospective study included patients diagnosed with meningioma at our institution between 2016 and 2019. Archival tumor tissue was used for analysis. MTAP immunohistochemistry (IHC) was performed at various dilutions (1:1200, 1:400, 1:200, 1:100) using two different antibodies, and p16 IHC was conducted simultaneously. These analyses were carried out at two different institutions. To determine the sensitivity and specificity of MTAP and p16 as surrogate markers for CDKN2A/B loss, CDKN2A FISH was utilized as the gold standard. Results: Overall, 46/49 tumors showed strong MTAP staining (94%) at institution 1, and 44/49 (90%) showed either faint positive or positive results at institution 2. One grade 3 meningioma that demonstrated homozygous CDKN2A loss by FISH also showed loss of MTAP expression by IHC. One grade 2 meningioma showed regional CDKN2A loss by FISH and variable MTAP expression under different IHC conditions. MTAP expression evaluation was superior at a dilution of 1:100 with the Abnova Anti-MTAP Monoclonal antibody. Conclusions: P16 expression was variable and did not correlate with either MTAP expression or CDKN2A FISH results. MTAP IHC is a promising surrogate marker for the evaluation of CDKN2A status in meningiomas.