Rearrangement Partner Research Articles

Unbalanced MYC break-apart FISH patterns indicate the presence of a MYC rearrangement in HGBCL-DH-BCL2

AbstractFluorescence in situ hybridization (FISH) using break-apart probes is recommended for identifying high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). Unbalanced MYC break-apart patterns, in which the red or green signal is lost, are commonly reported as an equivocal result by clinical laboratories. In a cohort of 297 HGBCL-DH-BCL2, 13% of tumors had unbalanced MYC break-apart patterns with loss of red (LR; 2%) or loss of green (LG; 11%) signal. To determine the significance of these patterns, MYC rearrangements were characterized by sequencing in 130 HGBCL-DH-BCL2, including 3 LR and 14 LG tumors. A MYC rearrangement was identified for 71% of tumors with LR or LG patterns, with the majority involving immunoglobulin loci or other recurrent MYC rearrangement partners. The architecture of these rearrangements consistently preserved the rearranged MYC allele, with the MYC gene predicted to be on the derivative chromosome containing the signal that is still present in nearly all cases. MYC protein expression, MYC messenger RNA expression, and the proportion of tumors expressing the dark-zone signature was not significantly different between balanced and unbalanced groups. These results support a recommendation that unbalanced MYC break-apart FISH patterns be reported as positive for MYC rearrangement in the context of diagnosing HGBCL-DH-BCL2.

Abstract LB_B04: Deciphering FGFR3-TACC3 oncogenic fusions

Abstract Chromosomal rearrangements of the fibroblast growth factor receptor (FGFR) genes that give rise to fusions are one of several mechanisms by which the FGF/FGFR signaling axis can become deregulated and result in enhanced signaling in cancer. We have previously identified truncation of exon (E) 18 of FGFR2 as a potent single-driver alteration in cancer, independently of the rearrangement (RE) partner. In contrast, the same does not appear to hold true for its E18-truncated ortholog FGFR3. We mined human oncogenomic datasets from Hartwig Medical Foundation (>2,500 WGS profiles) and Foundation Medicine (>200,000 hybrid-capture panel-seq profiles) for alterations affecting FGFR3. Examination of the structural variants affecting FGFR3 uncovered that 85% of all E18-truncating FGFR3 REs involved transforming acidic coiled-coil-containing protein 3 (TACC3) as the downstream fusion partner gene, while REs with intergenic regions and other known protein-encoding genes were much less frequent. Additionally, there was a clear predominance of self-interacting domains (95%) among all FGFR3 RE partners, with the coiled-coil domain being the most recurrent, suggesting enhanced receptor dimerization and downstream signaling capacity for the majority of the FGFR3 fusions. Our findings led to the generation of a compendium of FGFR3 structural variants which we then functionally tested. In vitro testing of mouse mammary epithelial cells expressing Fgfr3ΔE18-Tacc3E7-E16 fusion variants showed that both E18-truncation and a fusion partner were required for 3D outgrowth and signaling induction. In vivo evaluation of the oncogenic capacity of Fgfr3 variants using somatic delivery of lentiviruses to the mouse mammary gland via intraductal injection and lung via intratracheal injection found that Fgfr3ΔE18-Tacc3E7-E16 fusion variants rapidly induced mammary and lung tumor formation in wild-type and Wap-Cre;Cdh1F/F, and in wild-type and Trp53F/F mice, respectively. In contrast, Fgfr3full-length(FL), Fgfr3ΔE18 and Fgfr3FL–Tacc3E7-E16 were non-tumorigenic. Noteworthy, mammary tumors driven by Fgfr3ΔE18-Tacc3E7-E16 fusions were sensitive to the FGFR inhibitor AZD4547 during a drug intervention study. In summary, our findings show that E18-truncating FGFR3 alterations are recurrent across human cancers and, as opposed to FGFR2 E18-truncating alterations, FGFR3 appears to depend both on E18-truncation and an additional fusion partner with dimerizing capacity. Notably, TACC3 is the most recurrent 3’ fusion partner across all FGFR3 E18-truncating alterations, and somatic tumor modeling has shown that Fgfr3ΔE18-Tacc3 fusions drive mouse mammary and lung tumorigenesis, and are sensitive to FGFR targeted therapy. Citation Format: Julia Yemelyanenko, Jinhyuk Bhin, Sjoerd Klarenbeek, Ji-Ying Song, Catrin Lutz, Marieke van de Ven, Shridar Ganesan, Lodewyk F. A. Wessels, Daniel Zingg, Jos Jonkers. Deciphering FGFR3-TACC3 oncogenic fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B04.

Epithelioid inflammatory myofibroblastic sarcoma: a pitfall in the differential diagnosis of ALK-positive anaplastic large cell lymphoma.

An 18-year-old female presented with a 4.5 cm abdominal mass. Biopsy showed sheet-like growth of large tumor cells with round to oval nuclei, 1-2 nucleoli, and abundant cytoplasm. Immunohistochemistry showed strong, uniform CD30 staining and cytoplasmic ALK staining. B-cell markers (CD20, CD79a, PAX5, kappa/lambda) and T-cell markers (CD2, CD3, CD4, CD5, CD43, granzyme B, T-cell receptor-β) were negative. Other hematopoietic markers (CD45, CD34, CD117, CD56, CD163, EBV) were negative, but CD138 was positive. Non-hematopoietic markers showed desmin positivity and negativity for S100, melan A, HBM45, PAX8, PAX2, WT1, MYO-D1, myogenin, pancytokeratin, and CAM5.2. Sequencing identified PRRC2B::ALK fusion. A diagnosis of epithelioid inflammatory myofibroblastic sarcoma (EIMS) was made. EIMS is a rare, aggressive form of inflammatory myofibroblastic tumor typically presenting in children and young adults. The tumor comprises large epithelioid cells that express ALK and often CD30. ALK-positive ALCL has a similar age range and also is a large-cell tumor expressing CD30 and ALK. Other ALK-positive neoplasms (e.g., carcinomas, ALK-positive large B-cell lymphoma, ALK-positive histiocytosis) typically lack CD30 and have distinct clinicopathologic features that aid diagnosis. Hematopathologists need to distinguish EIMS from ALK-positive ALCL, which frequently shows loss of pan-T-cell antigens. Careful morphologic evaluation for the hallmark cells of ALCL and comprehensive phenotyping are critical to avoid this diagnostic pitfall. If known, the ALK rearrangement partner gene may also provide diagnostic clues; for example PRRC2B::ALK and RANBP2::ALK occur in EIMS but not ALCL.

A novel VCP::TFE3 gene fusion resulting from t(X;9)(p11.23;p13.3) chromosome translocation in TFE3 rearranged renal cancer cell carcinoma.

Renal cell carcinoma (RCC) with rearrangement of transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3; TFE3-rearranged RCC) at Xp11.2 is a rare tumor entity but the most frequent among the microphthalmia transcription factor family translocation RCCs. Here, we report the identification of a new VCP::TFE3 fusion gene as the result of a t(X;9)(p11.23;p13.3) translocation identified by whole transcriptome sequencing. No other relevant molecular alteration was identified by whole exome sequencing. This case showed typical morphological features of TFE3-rearranged RCC with positive TFE3 immunostaining and positive TFE3 break-apart fluorescence in situ hybridization. MET was also overexpressed on immunohistochemistry. The patient had metastatic disease and was treated by surgery and five lines of therapy, including 24 months of stable disease on the mesenchymal epithelial transition (MET) inhibitor cabozantinib, with an overall survival of 7 years. In addition to expanding the spectrum of TFE3 rearrangement partners, this report highlights the complexity of these tumors and supports the development of translational programs in renal cancer.

Functional Profiling Identifies Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Diffuse Large B-Cell Lymphoma

Strong expression of MYC and MYC-regulated genes defines the molecular high-grade / double-hit signature subgroup of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) which shows poor clinical outcomes with current therapy. About half of such cases show large-scale chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, but there is little understanding of the specific regulatory elements that activate MYC in absence of a rearrangement, or in the setting of non-immunoglobulin MYC rearrangement partners (RPs). We used high-throughput CRISPR-interference functional profiling (CRISPRi) to pinpoint cis-regulatory element dependencies in MYC-intact and MYC-rearranged lymphoma cell lines at the level of individual nucleosome-free regions (NFRs). Custom sgRNA libraries targeted 233 individual NFRs (median 42 sgRNA / NFR after stringent quality filtering) across the MYC topological domain (TAD) and MYC RP loci in six GCB-DLBCL cell lines and two comparator mantle cell lymphoma (MCL) cell lines. Promoter-targeting sgRNAs against 587 sequence-specific transcription factor (TF) genes and 65 essential gene controls allowed for interrogation of trans-factor dependencies in the same assay. CRISPRi profiling in one GCB-DLBCL and one MCL cell line with intra-chromosomal rearrangements of chromosome 8 showed strong, unique dependencies on enhancers within the cell-line-specific RPs, supporting an "enhancer-hijacking" function of these rearrangements. Three profiled cell lines showed structurally distinct variants of a recurrent t(3;8)(q27;q24) rearrangement that links the MYC gene to the distal super-enhancer region 150-240 kB upstream of BCL6. Although the distal BCL6 super-enhancers have been shown to sustain the essential BCL6 oncogene itself, we surprisingly found only weak dependency on the distal BCL6 enhancers for the three GCB-DLBCL cell lines lacking t(3;8) rearrangements. In contrast, all three GCB-DLBCL cell lines with t(3;8) rearrangements showed strong dependency on specific NFRs within the distal BCL6 super-enhancer. Validation assays showed that concurrent targeting of two BCL6 enhancer NFRs lead to even greater growth suppression and loss of MYC expression in t(3;8)-bearing cell lines. In both GCB-DLBCL cell lines lacking MYC rearrangements, we identified a strong, novel dependency on a specific enhancer ("GCB-ME1") in the 3' portion of the MYC TAD. This enhancer was acetylated in MYC-intact GCB-DLBCL patient samples and normal centroblasts, but not in MCL or MYC-rearranged DLBCL samples. We confirmed that sustained MYC expression in MYC-intact cell lines requires activity of GCB-ME1. GCB-ME1 showed little or no essentiality in the four MYC-rearranged DLBCL cell lines or in the MYC-intact MCL cell line SP-49, which is dependent on previously identified Notch/RBPJ-regulated 5' MYC enhancers (Ryan et al., 2017). ChIP-Seq showed that both GCB-ME1 and all of the most strongly essential BCL6 enhancers in t(3;8) cell lines were strongly bound by a triad of TFs, MEF2B, POU2F2, and POU2AF1, that were previously implicated in BCL6 activation. These genes were required to sustain MYC expression in both MYC-intact and t(3;8) GCB-DLBCL, and were highly essential in those cell lines, but POU2F2 and MEF2B were not essential in some cell lines with alternate MYC rearrangements. Our data show that DLBCL is highly sensitive to selective disruption of discrete MYC-activating enhancers. Transcriptional regulatory factor dependencies in DLBCL may be shaped by the requirement to sustain MYC activation via the native GCB-ME1 enhancer or alternately via enhancers in specific RP loci, a finding with implications for therapeutic targeting of transcriptional regulatory mechanisms in DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recurrent Translocations in Topoisomerase Inhibitor-Related Leukemia Are Determined by the Features of DNA Breaks Rather Than by the Proximity of the Translocating Genes.

Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility—but not spatial predisposition of the rearrangement partners—plays a major role in the formation of these translocations.

Revisiting Aromatic Claisen Rearrangement Using Unstable Aryl Sulfonium/Iodonium Species: The Strategy of Breaking Up the Whole into Parts.

Since Ludwig Claisen's discovery of the sigmatropic rearrangement of allyl aryl ethers in 1912, aromatic Claisen rearrangement has continuously attracted the attention of both experimental and theoretical chemists. Over more than a century of growth, this protocol has proven to be a practical and powerful synthetic tool in many aspects. However, the reaction scope has long been limited to aryl ethers and their S or N analogs until the serendipitous discovery of aromatic iodonium-Claisen rearrangement by Oh et al. in 1988 and the development of aromatic sulfonium-Claisen rearrangement by Kita et al. in 2004. Unlike traditional Claisen rearrangements, these hypervalent-bonding-based Claisen-type rearrangements can be performed by simply mixing electrophilically activated aryl sulfoxides/iodanes with certain nucleophiles to directly deliver rearrangement products. In addition to the simple operation, remarkable features, such as readily available substrates, valuable products and intriguing rearrangement patterns, have led to a dramatic resurgence of this rearrangement chemistry.In this Account, we summarize our recent works on developing new aromatic rearrangement modes using sulfonium/iodonium reagents. Interestingly, the program started with an accidental discovery that aryl sulfoxides could be coupled with alkyl nitriles in the presence of Tf2O and base. Mechanistic studies reveal that the reaction proceeds in three major steps, including the Tf2O-triggered assembly of both coupling partners, base-promoted deprotonation of in situ-generated aryl sulfonium-imine species leading to a key rearrangement precursor called aryl sulfonium-ketenimine species, and subsequent facile and rapid [3,3]-rearrangement. On the basis of the mechanistic underpinning, we divided the one-step operation into two steps called the "assembly/deprotonation" protocol for constructing unstable rearrangement precursors. Most notably, the switch from the commonly used one-step to mechanism-based multiple-step manipulation, which can be termed "breaking up the whole into parts", not only enables the independent control of each step of the reaction, thus significantly expanding the accessible synthetic scope, but also raises opportunities for developing new rearrangement patterns. For example, the "assembly/deprotonation" protocol has also been applied to the development of [5,5]-rearrangement of aryl sulfoxides and the asymmetric rearrangement of aryl iodanes, thus enabling the unprecedented regio- and stereocontrol of the rearrangement process. Furthermore, the "breaking up the whole into parts" thinking triggered us to merge the Morita-Baylis-Hillman (MBH) reaction into the rearrangement process to accomplish Z-selective MBH-type [3,3]-rearrangement of α,β-unsaturated nitriles and E-selective MBH-type [3,3]-rearrangement of α,β-unsaturated 2-oxazolines, which expands the scope of rearrangement partners to include α,β-unsaturated carbonyls. In addition, the impressive rapidity of the rearrangement process found in our initial discovery has also been recognized as a congestion-acceleration effect, which was further utilized to forge the rapid ortho-cyanoalkylative rearrangement of aryl iodanes, and thus leading to the first dearomatization of aryl iodanes. We anticipate that our protocols and ideas behind the methods will be complementary to the traditional thinking of the aromatic Claisen rearrangement.

The Genomic Landscape of Plasmablastic Lymphoma (PBL) - an L.L.M.P.P. Project

The Genomic Landscape of Plasmablastic Lymphoma (PBL) - an L.L.M.P.P. Project

Myeloid/Lymphoid Neoplasm with Eosinophilia and a Novel RUFY1-Pdgfrb Rearrangement

▪Myeloid/lymphoid neoplasms associated with eosinophilia (MLN-Eo) and tyrosine kinase fusion genes include rearrangements of PDGFRB with over 30 different partner genes, most commonly ETV6-PDGFRB. Rearrangements are evident by cytogenetic analysis and fluorescence in situ hybridization (FISH) break-apart probes. Herein, we describe a novel rearrangement partner for PDGFRB.A 25-year-old man with a medical history significant for gout and hypertension presented to medical care after several days of progressive dyspnea, chest pain, fatigue, epistaxis, and rash. Physical exam revealed palpable cervical, axillary, and inguinal lymphadenopathy and massive splenomegaly. By CT scan the spleen measured 25 cm and diffuse lymphadenopathy was identified in the chest, abdomen and pelvis with the largest lymph node mass measuring 8.9 x 2.5 cm. Transthoracic echocardiogram showed normal heart function and no evidence of myocardial injury. The patient subsequently developed respiratory failure requiring intubation and mechanical ventilation, shock requiring vasopressors, and renal failure requiring renal replacement therapy. Initial laboratory evaluation demonstrated a leukocytosis to 83.3 x 10 9/L with neutrophilic predominance with left shift and eosinophilia (10% of WBCs, absolute eosinophil count of 8.2 x 10 9/L); hemoglobin of 8.3 g/dL and hematocrit of 25%; and thrombocytopenia to 50 x 10 9/L. Ferritin was 660 ng/mL, lactate dehydrogenase 384 U/L, and uric acid 10.4 mg/dL. Secondary causes of eosinophilia including infection, immunodeficiency, atopy, medications, and rheumatologic disease were ruled out. The patient was positive for strongyloides IgG antibodies and received ivermectin.Core biopsy of a lymph node was non-diagnostic and could not repeated safely due to progressive severe thrombocytopenia. Skin biopsy demonstrated a perivascular leukocytoclasis, but no abnormal B or T cell population. Bone marrow aspirate and biopsy revealed a hypercellular marrow (90%) with mild trilineage dysplasia, granulocytic hyperplasia, moderate reticulin fibrosis, and slightly increased mast cells. Blasts were not increased and no hemophagocytic histiocytes were identified. Eosinophils accounted for 6.9% of myeloid cells. Flow cytometry did not reveal an abnormal B or T lymphoid, blast or myeloid population.Peripheral blood flow cytometry did not reveal an abnormal B or T-cell population and T-cell rearrangement studies showed an oligoclonal population. No clonal B cell population was identified.FISH, which identified a rearrangement of PDGFRB in 28% of cells, confirmed the diagnosis. Karyotype analysis revealed an abnormal male karyotype with a paracentric inversion of 5q with breakpoints at 5q32 and 5q35. FusionPlex® Solid Tumor Panel (Clinical Genomics Laboratory, University of Washington SOM) identified a novel fusion between RUFY1 and PDGFRB. RUFY1 is expressed in most tissues and encodes a protein that contains a RUN domain and a FYVE-type zinc finger domain and plays a role in early endosomal trafficking (Figure). We are currently demonstrating the fusion is oncogenic and sensitive to tyrosine kinase inhibition in Ba/F3 cells. The patient's remarkable clinical response, however, supports imatinib sensitivity and is in keeping with other published reports of MLN-Eo with PDGFRB rearrangements. Initial treatment included high dose steroids for 7 days and hydroxyurea briefly. Imatinib 400 mg daily was started, then decreased to 200 mg daily due to gastrointestinal toxicity. Follow-up is limited (8 weeks). However, the patient has recovered clinically. Six weeks after starting imatinib he had renal recovery and has achieved a complete hematologic response except for mild anemia due to resolving acute kidney injury. Splenomegaly and peripheral lymphadenopathy have resolved. PDGFRB FISH has declined to 9%.Inversions of chromosome 5 have very rarely been described in association with eosinophilia, but only one report has specifically identified a rearrangement of PDGFRB. This report described a novel rearrangement involving exons 12 to 23 of PDGFRB at 5q32 and the 5′ (UTR) of G3BP1 at 5q33.1. Notably, this specific rearrangement was undetectable by PDGFRB break-apart FISH. These reports and ours highlight the clinical relevance of NGS in identifying novel rearrangement partners. Furthermore, patient-specific ddPCR assays can be designed for sensitive monitoring. [Display omitted] DisclosuresOehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy.

Single-Cell Genomic Analysis Identifies Prognostically Significant Gene Expression Programs in Infant Acute Lymphoblastic Leukemia

Introduction:Infant acute lymphoblastic leukemia (ALL) is an aggressive subtype of leukemia with low rates of survival. Rearrangements involving the KMT2A gene are associated with poor prognosis for infants with this cancer. Although many patients with KMT2A rearrangements (KMT2A-r) ultimately relapse, some do not. The molecular basis for this distinction has not yet been determined. There is evidence to suggest that cancers with distinct KMT2A fusion partners show distinct patterns of gene expression, though these differences have not previously been linked to prognostic outcomes. Here we utilize single-cell genomic analysis for infant ALL samples with and without KMT2A-r taken at diagnosis to explore transcriptional dynamics and identify gene expression programs with potential prognostic value.Methods:We performed 10x Chromium single-cell multiome sequencing to obtain both gene expression (scRNA-seq) and chromatin accessibility (scATAC-seq) data for blood and/or bone marrow samples obtained from a total of 34 infant ALL patients at time of diagnosis. Of these, 19 KMT2A-r patients later relapsed, 6 KMT2A-r patients did not relapse, and 9 patients did not show KMT2A rearrangement. Sequencing data were processed and aggregated with cellranger-arc, with normalization and differential gene expression performed using the Seurat package for gene expression analysis. Differential gene expression was performed between the three groups described above, (KMT2A-r + relapse, KMT2A-r no relapse, no KMT2A-r), then further subdivided for KMT2A-r cases based on the KMT2A partner gene (MLLT1 n=13 or AFF1 n=12).Results:In preliminary scRNA- and scATAC-seq in infant ALL patients, we had observed that cancer cells from individual patients tend to cluster separately, while non-blast cell populations showed similar interindividual patterns, suggesting a high degree of divergence among blast cell transcriptional programs. We replicated this finding here in larger patient samples, demonstrating no overt similarity based on KMT2A-r status or whether patients later relapsed. Strikingly, samples did broadly cluster according to KMT2A rearrangement partner gene, indicating that this appears to be a major driver of the transcriptional program in infant ALL patients. Differential expression analysis within the KMT2A-MLLT1 and KMT2A-AFF1 samples separately revealed expression of other genes associated with particular KMT2A fusion genes. Specifically, we observed that expression of HOXA genes was mostly restricted to KMT2A-MLLT1 samples. Consistent with previous results, we observed that within KMT2A-AFF1 samples there were two distinct groups of cells with mutually exclusive expression of HOXA9 or IRX1. Furthermore, the limited HOX gene expression observed in KMT2A-AFF1 samples was enriched in patients who did not relapse, supporting previous observations that the KMT2A-AFF1 samples expressing IRX1 comprise a more aggressive leukemia.Conclusions:Utilizing a single-cell transcriptomic approach enabled us to identify gene expression programs associated with good and poor prognosis in KMT2A-r infant ALL cases. We found that the KMT2A fusion partner gene appears to drive a large portion of the transcriptional heterogeneity observed across KMT2A-r samples. By treating the KMT2A-MLLT1 and KMT2A-AFF1 samples separately and exploring transcriptional dynamics within these groups, we identified transcriptional differences between patients who did and did not relapse in the presence of a KMT2A-AFF1 fusion. We are continuing to explore these data to identify prognostic markers in KMT2A-MLLT1 patients.Figure 1. Mutually exclusive gene expression programs distinguish subsets of infant ALL samples. A) Uniform manifold approximation and projection (UMAP) visualization of gene expression in individual cells across all patient samples at diagnosis, each color corresponding to a single patient (n=34). B) Cells colored according to KMT2A-r status and relapse or lack thereof. C) Cells colored according to KMT2A fusion partner gene. D) HOXA9 expression among KMT2A-AFF1 patients is strongly biased to patients that do not relapse. E) In KMT2A-AFF1 samples, HOXA9 and IRX1 show mutually exclusive expression patterns. [Display omitted] DisclosuresBrown: Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Kura: Membership on an entity's Board of Directors or advisory committees; KIte: Membership on an entity's Board of Directors or advisory committees.

Oligoblastic (

Introduction: The current World Health Organization (WHO) classification considers acute myeloid leukemia (AML) with PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 to be AML regardless of blast count. Myeloid neoplasms with KMT2A rearrangement present with high blast percentages and are classified as AML in the vast majority of cases; however, rare myeloid neoplasms with KMT2A rearrangement may present with <20% blasts and these are currently considered to be myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML). We questioned whether these oligoblastic KMT2A rearranged neoplasms shared similar biology with AML, and if this genetic finding should be considered to be AML-defining.Methods: We searched the archives of three large institutions in the United States for untreated adult with oligoblastic (<20%) myeloid neoplasms with KMT2A rearrangement and untreated adult AML patients with KMT2A rearrangement and compared their clinical and laboratory characteristics. KMT2A rearrangement was detected by chromosomal analysis and/or fluorescence in-situ hybridization (FISH) using KMT2A breakapart probe. KMT2A rearrangement partners were assessed based on a conventional karyotype results and/or RNA based fusion assay.Results: Between 2010 and 2021 we identified 22 patients with MDS or CMML and KMT2A rearrangement and 88 patients with AML and KMT2A rearrangement. The clinical and laboratory information are summarized below. MDS/CMML with KMT2A-rearrangement showed a similar age and sex distribution to AML, but had more patients with preceding history of chemotherapy for unrelated malignancies (68% vs 37%, p=0.012). Aside from a low blast count in PB and BM, patients with MDS/CMML had a lower WBC and were less anemic and thrombocytopenic. MDS/CMML patients also showed a lower percentage of BM cells with KMT2A rearrangement by FISH (58% vs. 92%, p<0.0001), and significantly less likely to have MLLT3 as the partner gene (23% vs. 58%, p=0.0005). With a similar mutation frequency among patients tested (42% versus 34%, p=0.22), MDS/CMML patients had significantly fewer mutations involving the RAS pathway (KRAS, NRAS, FLT3, PTPN11) than AML patients (p=0.0035).Seventeen of 22 MDS/CMML patients (77%) progressed to AML with a median time of progression being 4 months (range, 1-27 months). Of the 5 patients who did not progress to AML, 3 received allogeneic stem cell transplantation (SCT) and remain in complete remission, while 2 remain AML-free 3 and 16 months after the initial diagnosis.With a median follow-up of 12 months (range, <1-117), 15 MDS/CMML patients and 58 AML patients died. There was no difference in use of SCT to treat MDS/CMML versus AML (p=0.47). MDS/CMML and AML patients displayed similar overall survival (OS) (p=0.9059, Figure 1). There was also no statistical difference in OS between MDS/CMML and AML when therapy-related (p=0.569) and de novo (p=0.962) neoplasms were analyzed separately. On multivariable analysis, therapy-related disease (HR 1.774 [1.058-2.975], p=0.03), SCT (HR 0.166 [0.000-0.291], p<0.0001), and platelet count (HR 0.992 [0.000-0.998], p=0.013) were independently associated with shorter OS. Neither AML versus MDS/CMML nor blast percentage (whether dichotomized as <5% or >=5% or as a continuous variable) were associated with shorter OS.Conclusion: Oligoblastic myeloid neoplasms with KMT2A rearrangement tend to progress rapidly to overt AML and demonstrate similar aggressive behavior to AML patients with KMT2A rearrangement, irrespective of therapy relatedness. Patients with KMT2A rearrangement and <20% blasts are currently classified as MDS or CMML, and may be inadequately treated. Our findings suggest that all myeloid neoplasms with KMT2A (MLL) rearrangement should be classified as AML, irrespective of the blast count. [Display omitted] DisclosuresRavandi: AbbVie: Honoraria, Research Funding; Prelude: Research Funding; Taiho: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Issa: Novartis: Consultancy, Research Funding; Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding. Pozdnyakova: Scopio Labs: Consultancy. Wang: Stemline Therapeutics: Honoraria.

Clinicopathologic and prognostic features of TdT-negative pediatric B-lymphoblastic leukemia

Little is known about B-lymphoblastic leukemia (B-ALL) that lacks expression of terminal deoxynucleotidyl transferase (TdT). To address this, we performed the largest study to date of TdT-negative B-ALL using data from St. Jude Total XV and XVI clinical trials. Compared to TdT-positive B-ALL (n = 896), TdT-negative B-ALL (n = 21) was associated with younger age (median, 1.4 versus 6.8 years, P < 0.001), higher white blood cell count (median, 52.8 versus 9.9 × 109/L, P < 0.001), absence of hyperdiploidy (0 versus 27.8%, P = 0.002), KMT2A rearrangement (100 versus 1.9%, P < 0.001), and inferior 5-year event-free survival (EFS) (76.2 versus 90.3%, P = 0.047). In the context of KMT2A-rearranged B-ALL (n = 38), TdT-negativity was significantly associated with the MLLT1 rearrangement partner (P = 0.026) but was not independently predictive of survival, suggesting that the high-risk features of TdT-negative B-ALL are secondary to underlying KMT2A rearrangements. Finally, we compared the sensitivity of TdT-negativity to neuron-glial antigen 2 (NG.2) expression for the detection of KMT2A rearrangements and found that 63% of KMT2A-rearranged B-ALL cases not identified by NG.2 were TdT-negative. The results of this study expand the spectrum of immunophenotypic features that are specific for high-risk KMT2A rearrangements in pediatric B-ALL and can be readily implemented using existing standard acute leukemia flow cytometry panels.

Epigenetic and Transcriptomic Profiling of MLL Rearranged Infant Acute Lymphoblastic Leukemia

[Background] Infant acute lymphoblastic leukemia (ALL) accounts for less than 5% of childhood leukemia, and is characterized by Mixed lineage leukemia (MLL also called KMT2A ) rearrangements found in approximately 80% of patients. Also found in 5-10% of ALL in older children and adults, MLL -rearrangement positivity is highly associated with poor outcome in ALL; there is an urgent need for molecular findings of potential therapeutic targets and/or risk stratification markers, which can be translated into the clinical practice. Recently, the mechanisms that MLL fusion proteins recruit epigenetic regulators, including histone H3 lysine 79 methyltransferase DOT1L and BET family histone readers, to activate the transcription of target genes have been reported, suggesting epigenetic dysregulation should play a central role in the leukemogenesis. However, DNA-level methylation profiles of MLL -rearranged leukemia and their associations with gene expression profiles are not fully characterized.[Materials and Methods] Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. Both methylation array and RNA sequencing were performed on the diagnostic leukemia samples from 38 infants with ALL, consisting of 30 cases with MLL -rearrangements identified by fluorescence in situ hybridization or Southern blotting, and 8 MLL -germline cases. Methylation beta values were normalized with beta-mixture quantile normalization method. For RNA sequencing data analysis, we used publicly available Genomon Pipeline.[Results] RNA sequencing confirmed MLL -rearrangements in 30 cases, whose partner genes comprised AFF1 (n=15), MLLT1 (n=6), MLLT3 (n=3), MLLT10 (n=2), EPS15 (n=1), AFF2 (n=1), ABI1 (n=1), and SARNP (n=1). As expected, DNA methylation-based hierarchical clustering divided the cases into 2 clusters with highly distinctive methylation profiles: MLL -rearranged cluster (MLL -R cluster) and MLL -germline cluster (MLL -G cluster). Intriguingly, 3 out of 30 (10%) MLL -rearranged cases were classified into MLL -G cluster; these cases revealed uncommon partner genes, such as ABI1 , SARNP , and MLLT10, respectively. ABI1, an Eps8 SH3 domain-binding protein, and SARNP, a SAP domain containing ribonucleoprotein, are rare fusion partners of MLL. The MLL -germline leukemia-like methylation profiles suggested different leukemogenic mechanisms from common fusion partners, such as AFF1 and MLLT1, which directly or indirectly interact with DOT1L and positive transcription elongation factor b. Because DOT1L-binding domain of MLLT10 was retained in both of the 2 cases with MLL-MLLT10 in our cohort, additional mechanisms causing MLL-germline leukemia-like methylation in a single case may be present. As a result of inter-cluster comparison, MLL -R clusterexhibited global hypermethylation compared to MLL -G cluster (P -value < 2.2 × 10−16, Kolmogorov-Smirnov test). Methylation-based gene set enrichment analysis demonstrated significant promoter hypermethylation on cell development/differentiation-related gene sets and hypomethylation on DNA replication/cell proliferation-related gene sets (P -value < 0.01, hypergeometric test) in MLL -G cluster. Consistently, RNA sequencing-based gene expression analysis demonstrated overexpression of cell cycle regulators, such as cyclins/cyclin-dependent kinases, in MLL -R cluster, which may be associated with more aggressively proliferating diseases.[Conclusion] Corresponding to known aberrant histone modifications in MLL -rearranged leukemia, largely different DNA methylation profiles between MLL -R and MLL -G clusters have been demonstrated. Because DNA-level methylation on CpG dinucleotides interactively affects histone modifications, these results implicated aberrant DNA methylation in the pathogenesis of MLL -rearranged leukemia as a part of epigenetic dysregulation. We also identified a subset of MLL -rearranged cases with MLL -germline leukemia-like methylation/expression profiles, indicating that not only MLL -rearrangement positivity, but also the types of MLL -rearrangement partners may affect a biological feature of leukemic cells. These cases may possibly be therapeutically stratified as relatively lower risk group compared to other MLL -rearranged cases. Further detailed analyses using clinical information are planned. DisclosuresNo relevant conflicts of interest to declare.

High-Resolution Architecture and Partner Genes of MYC Rearrangements in Lymphoma with DLBCL Morphology

High-Resolution Architecture and Partner Genes of MYC Rearrangements in Lymphoma with DLBCL Morphology

Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Morita-Baylis-Hillman-Type [3,3]-Rearrangement: Switching from Z- to E-Selective α-Arylation by New Rearrangement Partners.

α-aryl α,β-unsaturated carbonyls represent an important class of derivatizable synthetic intermediates, however, the synthesis of such compounds still remains a challenge. Recently, we showcased a novel Z-selective α-arylation of α,β-unsaturated nitriles with aryl sulfoxides via [3,3]-rearrangement involving an Morita-Baylis-Hillman (MBH) process. Herein, we demonstrate the feasibility of reversing the stereoselectivity of such MBH-type [3,3]-rearrangement by switching to a new pair of rearrangement partners consisting of aryl iodanes and α,β-unsaturated oxazolines. As a result, the two protocols complement each other in approaching E- or Z-α-aryl α,β-unsaturated carbonyl derivatives. Mechanistic studies reveal a possible reaction pathway and provide an explanation for the opposite stereoselectivities.

Morita–Baylis–Hillman‐Type [3,3]‐Rearrangement: Switching fromZ‐ toE‐Selective α‐Arylation by New Rearrangement Partners

Abstractα‐aryl α,β‐unsaturated carbonyls represent an important class of derivatizable synthetic intermediates, however, the synthesis of such compounds still remains a challenge. Recently, we showcased a novelZ‐selective α‐arylation of α,β‐unsaturated nitriles with aryl sulfoxides via [3,3]‐rearrangement involving an Morita–Baylis–Hillman (MBH) process. Herein, we demonstrate the feasibility of reversing the stereoselectivity of such MBH‐type [3,3]‐rearrangement by switching to a new pair of rearrangement partners consisting of aryl iodanes and α,β‐unsaturated oxazolines. As a result, the two protocols complement each other in approachingE‐ orZ‐α‐aryl α,β‐unsaturated carbonyl derivatives. Mechanistic studies reveal a possible reaction pathway and provide an explanation for the opposite stereoselectivities.

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In This Issue

RNA-Based next generation sequencing complements but does not replace fluorescence in situ hybridization studies for the classification of aggressive B-Cell lymphomas

Aggressive B-cell lymphomas are currently classified based in part upon the presence or absence of translocations involving BCL2, BCL6, and MYC. Most clinical laboratories employ fluorescence in situ hybridization (FISH) analysis for the detection of these rearrangements. The potential role of RNA-based sequencing approaches in the evaluation of malignant lymphoma is currently unclear. In this study, we performed RNA sequencing (RNAseq) in 37 cases of aggressive B-cell lymphomas using a commercially available next generation sequencing assay and compared results to previously performed FISH studies. RNAseq detected 1/7 MYC (14%), 3/8 BCL2 (38%) and 4/8 BCL6 (50%) translocations identified by FISH. RNAseq also detected 1 MYC/IGH fusion in a case not initially tested by FISH due to low MYC protein expression and 2 BCL6 translocations that were not detected by FISH. RNAseq identified the partner gene in each detected rearrangement, including a novel EIF4G1-BCL6 rearrangement. In summary, RNAseq complements FISH for the detection of rearrangements of BCL2, BCL6 and MYC in the evaluation and classification of aggressive B-cell lymphomas by detecting rearrangements that may be cryptic by FISH methods and by identifying the rearrangement partner genes. Detection of these clinically important translocations may be optimized by combined use of FISH and RNAseq.

Outcomes of Patients with Down Syndrome and CRLF2-Overexpressing Acute Lymphoblastic Leukemia (ALL): A Report from the Children's Oncology Group (COG)

Background: Patients with Down syndrome (DS) have an approximately 10-fold increased risk of developing ALL, and the spectrum of genetic alterations differs from that of non-DS ALL. Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of DS ALL, compared to only 5-10% CRLF2+ cases in non-DS children and adolescents. JAK2 or JAK1 mutations co-occur in about half of CRLF2+ cases in both DS and non-DS ALL. The prognostic significance of CRLF2+ ALL also appears to differ in the limited data reported to date, with less adverse impact in patients with DS compared to non-DS ALL. Here, we report the clinical characteristics and prognostic significance of B-ALL with CRLF2 overexpression and JAK alterations in children and adolescents/young adults (AYA) with DS who were treated on Children's Oncology Group (COG) clinical trials from 2003-2016. Methods: We analyzed clinical, laboratory, and outcome data for 317 patients with DS B-ALL treated on standard risk (SR) trials AALL0331 and AALL0932 and high risk (HR) trials AALL0232 and AALL1131, for whom CRLF2 status and rearrangement partners (IGH or P2RY8) were ascertained by flow cytometric assessment of surface expression; fluorescence in situ hybridization; and/or polymerase chain reaction (PCR) testing. JAK mutations were ascertained in a subset by PCR and sequencing. Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at end of consolidation (EOC) for a subset of EOI MRD+ patients. Results: We identified 168/317 (53.0%) CRLF2+ cases, and among those assessed for CRLF2 partner, 17/73 (23.3%) were IGH-CRLF2 and 56/73 (76.7%) were P2RY8-CRLF2. In the subset of 165 cases tested for JAK mutations (85 CRLF2- and 80 CRLF2+), 42/165 (25.4%) had JAK mutations, all of which co-occurred in CRLF2+ cases. CRLF2 positivity was significantly associated with younger age at diagnosis: 140/168 (83.3%) of CRLF2+ cases were under 10 years old, versus 106/149 (71.1%) of CRLF2- cases, p&amp;lt;0.01. P2RY8-CRLF2 cases were significantly more likely than IGH-CRLF2 cases to be associated with age under 10 years at diagnosis (92.9% vs 29.4%, p&amp;lt;0.0001), initial white blood count (WBC) &amp;lt;50x103 (80.4% vs 52.9%, p&amp;lt;0.024), National Cancer Institute (NCI) standard risk status (73.2% vs 23.5%, p&amp;lt;0.0002), and day 29 MRD &amp;lt;0.01% (67.9% vs 37.5%, p&amp;lt;0.028). There were no significant associations between JAK mutation status and any clinical features assessed (sex, initial WBC, central nervous system or testicular involvement, NCI risk group, or EOI/EOC MRD). Among patients with neutral cytogenetics (defined as neither favorable [ETV6-RUNX1, double trisomies of chromosomes 4 and 10] nor unfavorable [BCR-ABL1, KMT2A-rearranged, hypodiploid, iAMP21]), survival did not significantly differ between CRLF2+ and CRLF2- patients (Figure 1A,B), either for 5-year event-free survival (EFS) (79.1 +3.6% vs 77.6 +4.7%, p=0.856) or 5-year overall survival (OS) (89.5 ±2.8% vs 84.9 ±4.1%, p=0.674). There were also no significant differences in EFS or OS based on JAK mutation status or CRLF2 partner, although there were trends toward poorer outcomes in the CRLF2+/JAK+ and IGH-CRLF2 subgroups (Figure 1C). Finally, there was no significant difference in 5-year cumulative incidence of relapse (CIR) according to CRLF2 status (CRLF2+ 16.0 +3.0%, CRLF2- 10.6 +2.6%, p=0.179), although there were non-significant trends toward more relapses in CRLF2+ cases overall and in the NCI HR subgroup analysis. Discussion: Whereas CRLF2 and JAK alterations are associated with higher MRD, poorer survival, and increased CIR in patients with high-risk ALL without DS, these alterations do not demonstrate strong adverse prognostic impact in children and AYAs with DS-ALL treated on recent frontline COG trials, although larger sample sizes are needed to adequately assess for possible poorer prognoses associated with the CRLF2+/JAK+ and IGH-CRLF2 subgroups. Regardless, given the frequency of these targetable lesions and the increased risk of relapse and chemotherapy-associated toxicities in patients with DS-ALL, targeted therapies currently under investigation for these genetic lesions may be beneficial to replace some intensive blocks of therapy in DS-ALL with CRLF2 and/or JAK alterations, both to enhance anti-leukemic efficacy and decrease intensive chemotherapy-associated toxicities. Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau. Raetz:Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria.