Recurrent Chromosomal Rearrangements Research Articles

Novel Biomarkers and Molecular Targets in ALL.

Acute lymphoblastic leukemia (ALL) is a widely heterogeneous disease in terms of genomic alterations, treatment options, and prognosis. While ALL is considered largely curable in children, adults tend to have higher risk disease subtypes and do not respond as favorably to conventional chemotherapy. Identifying genomic drivers of leukemogenesis and applying targeted therapies in an effort to improve disease outcomes is an exciting focus of current ALL research. Here, we review recent updates in ALL targeted therapy and present promising opportunities for future research. With the utilization of next-generation sequencing techniques, the genomic landscape of ALL has greatly expanded to encompass novel subtypes characterized by recurrent chromosomal rearrangements, gene fusions, sequence mutations, and distinct gene expression profiles. The evolution of small molecule inhibitors and immunotherapies, and the exploration of unique therapy combinations are some examples of recent advancements in the field. Targeted therapies are becoming increasingly important in the treatment landscape of ALL to improve outcomes and minimize toxicity. Significant recent advancements have been made in the detection of susceptible genomic drivers and the use of novel therapies to target them.

Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3–FOXO1 in rhabdomyosarcoma

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.

Therapy-related core binding factor acute myeloid leukemia.

Therapy-related acute myeloid leukemia (t-AML) usually stems from exposure of the bone marrow to cytotoxic chemotherapy and/or radiation therapy. t-AML is usually associated with poor overall survival, but occasionally t-AML can involve favorable-risk cytogenetics, including core binding factor AML (CBF-AML), which shows a recurrent chromosomal rearrangement with t(8;21) (q22;22) and 'inv(16) (p13.1;q22)/t(16;16)(p13.1;q22)', leading to 'RUNX1::RUNX1T1 and CBFB::MYH11' fusion genes, respectively. Therapy-related CBF-AML (t-CBF-AML) accounts for 5-15% of CBF-AML cases and tends to have better outcomes than t-AML with unfavorable cytogenetics. Although CBF-AML is sensitive to high-dose cytarabine, t-CBF-AML has worse overall survival than de novo CBF- AML. The objective of this review is to discuss the available data on the pathogenesis, mutations, and therapeutic options in patients with t-CBF-AML.

TP63 Fusions Drive Enhancer Rewiring, Lymphomagenesis, and Dependence on EZH2

Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas, which is highly unique among chromosomal translocations. Specifically, TP63 rearrangements were found in 5-10% of ALK-negative anaplastic large cell lymphoma (ALK- ALCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and primary cutaneous ALCL, 7% of mycosis fungoides with large cell transformation (MF-LCT), and 1% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. This contrasts with rearrangements of DUSP22, which are also observed in ALK- ALCLs and confer a good prognosis. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients. Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 rearrangements suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell development and lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice, which results in expression beginning during early lymphoid development. Transgenic mice had both cytokine and transcriptional evidence of immune activation as well as T cell repertoire narrowing, consistent with clonal selection. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma that mimicked ALCL, PTCL-NOS and MYC-expressing DLBCL. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR screening, transcriptomic, epigenomic, and proteomic analyses. We also performed a domain-focused CRISPR-Cas9 knockout scan using 452 sgRNAs tiling the entire coding regions of TBL1XR1 and TP63 to clarify essential oncogenic domains within TBL1XR1-TP63. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions (TBL1XR1, FOXK2, etc.) interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 fusions (which recapitulates the deltaNp63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of EZH2, which encodes the histone H3K27 methylase. Knockdown of the TP63 fusion in TCL cell lines led to complete loss of H3K27me3. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 inhibitor tazemetostat and the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, was highly effective in three different in vivo models of TP63-rearranged TCL. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2.

Rare Deleterious TCF3 Germline Variants and Predisposition to Acute Lymphoblastic Leukemia in Children

Introduction TCF3 is a hematopoietic transcription factor essential for lymphoid differentiation. The role of TCF3 in the pathogenesis of B-cell acute lymphoblastic leukemia (B-ALL) is well documented because of recurrent chromosomal rearrangements leading to TCF3 fusion with PBX1 or HLF1. In contrast, less is known about the occurrence and significance of germline TCF3 variants in this blood cancer. Here we present a comprehensive screen and characterization of TCF3 germline variants in children with B-ALL. Methods First, we performed targeted sequencing of TCF3 coding regions in 4,183 children with newly diagnosed B-ALL enrolled in Children's Oncology Group (COG) clinical trials AALL0232, P9904/5/6, and AALL0331 as well as St. Jude Total Therapy XIII and XV clinical trials. Rare variant analysis was performed using the CoCoRV pipeline (Chen W et al., Nat Commun. 2022, PMID: 35545612), where the GnomAD v2 genome-based summary counts (n=15,708) were used as the non-ALL control cohort. Rare deleterious TCF3 variants were defined as follows: allele frequency ≤5x10-4 in GnomAD, protein-truncating variants (frameshift and nonsense) or missense variants with a REVEL score ≥0.65. For transcription factor activity measurement, we used a dual-luciferase reporter assay in HEK 293T cells. Finally, we compared the clinical characteristics of B-ALL patients with rare deleterious TCF3 germline variants to B-ALL patients without these variants enrolled in COG AALL0232 (n=2,180) and COG P9904/5/6 (n=1,619) clinical trials. Results TCF3 alternative splicing gives rise to two distinct isoforms, E47 and E12, of which we found TCF3-E12 to be the predominant transcript expressed in B-ALL. Focusing our analysis on the E12 isoform (NM_003200), we identified 12 rare deleterious TCF3 variants in 12 B-ALL patients, significantly and strikingly more frequent than what is seen in non-ALL controls (p=1.24×10-5, OR=19.9) (Figure 1A). Nine TCF3 variants lead to protein truncation with the loss of the basic helix-loop-helix (b-HLH) domain and three missense variants cluster within TCF3 exon 18a, which encodes the b-HLH domain that is responsible for TCF3 dimerization and DNA binding. Functional characterization by luciferase reporter assay revealed a severe reduction of TCF3 transcriptional activity for two B-ALL-related missense variants. TCF3 E12-p.N554S and E12-p.A568D showed a 6.2- (p<0.0001) and 6.9-fold (p<0.0001) reduction in transcription factor activity compared to wildtype TCF3 E12 (Figure 1B). TCF3 E47-p.E555K is a known loss of function variant and therefore was included as a positive control. As expected, its transcription factor activity was reduced by 11.8-fold compared to wildtype TCF3 E47 (p<0.0001). These results are consistent with the notion that single nucleotide variations within the b-HLH domain impair TCF3 dimerization and DNA binding. Nonsense and frameshift variants result in the loss of the b-HLH domain and were presumed to be deleterious. Finally, we compared clinical features of 10 TCF3 germline variant carriers to 3,799 B-ALL patients without TCF3 variation enrolled in COG clinical trials. We did not observe any difference in age, sex, ancestry, or white blood count at diagnosis. Further, none of the 10 patients with TCF3 germline variant had somatic fusion genes involving TCF3 (i.e., TCF3-PBX1 or TCF3-HLF1). Conclusion We systematically examined germline genetic variants in the TCF3 gene in B-ALL patients, reporting 12 rare deleterious TCF3 variants with a cumulative frequency of 0.29%. These variants directly affect the TCF3 b-HLH domain, which is critical for TCF3 transcription factor activity. Taken together, our findings provide new insights into genetic predisposition to pediatric B-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular cytogenetics in acute myeloid leukemia in adult patients: practical implications.

Throughout the last 50 years, cytogenetic analyses of pretreatment bone marrow and / or blood samples from patients diagnosed with acute myeloid leukemia (AML) revealed a large number of recurring chromosome aberrations, both structural and numerical. Using standard banding methods and, more recently, molecular cytogenetic techniques, such as fluorescence in situ hybridization, spectral karyotyping, multiplex fluorescence in situ hybridization and comparative genomic hybridization, cytogenetic investigations detect acquired abnormalities that, together with submicroscopic gene mutations and changes in gene expression, strongly influence the clinical features and prognosis of patients with AML. Selected reciprocal translocations and inversions and their molecular counterparts, as well as a number of unbalanced chromosome abnormalities are used, together with bone marrow morphology, immunophenotype, and clinical characteristics, to define separate AML entities in the World Health Organization Classification of Haematolymphoid Tumours. Moreover, cytogenetic findings (and specific gene mutations) are being used in geneticrisk classifications, such as the 2022 European LeukemiaNet classification. Such classifications divide patients into broad prognostic categories: favorable, intermediate, and adverse, which are useful in the management of adults with AML. In this article, I review the present data on recurrent chromosome rearrangements in AML and on correlations between cytogenetic findings and clinical features and treatment outcomes of adult patients diagnosed with AML.

16p13.11p11.2 triplication syndrome: a new recognizable genomic disorder characterized by optical genome mapping and whole genome sequencing.

Highly identical segmental duplications (SDs) account for over 5% of the human genome and are enriched in the short arm of the chromosome 16. These SDs are susceptibility factors for recurrent chromosomal rearrangements mediated by non-allelic homologous recombination (NAHR). Chromosomal microarray analysis (CMA) has been widely used as the first-tier test for individuals with developmental disabilities and/or congenital anomalies and several genomic disorders involving the 16p-arm have been identified with this technique. However, the resolution of CMA and the limitations of short-reads whole genome sequencing (WGS) technology do not allow the full characterization of the most complex chromosomal rearrangements. Herein, we report on two unrelated patients with a de novo 16p13.11p11.2 triplication associated with a 16p11.2 duplication, detected by CMA. These patients share a similar phenotype including hypotonia, severe neurodevelopmental delay with profound speech impairment, hyperkinetic behavior, conductive hearing loss, and distinctive facial features. Short-reads WGS could not map precisely any of the rearrangement's breakpoints that lie within SDs. We used optical genome mapping (OGM) to determine the relative orientation of the triplicated and duplicated segments as well as the genomic positions of the breakpoints, allowing us to propose a mechanism involving recombination between allelic SDs and a NAHR event. In conclusion, we report a new clinically recognizable genomic disorder. In addition, the mechanism of these complex chromosomal rearrangements involving SDs could be unraveled by OGM.

Neurodevelopmental phenotype in 36 new patients with 8p inverted duplication-deletion: Genotype-phenotype correlation for anomalies of the corpus callosum.

Inverted duplication deletion 8p [invdupdel(8p)] is a complex and rare chromosomal rearrangement that combines a distal deletion and an inverted interstitial duplication of the short arm of chromosome 8. Carrier patients usually have developmental delay and intellectual disability (ID), associated with various cerebral and extra-cerebral malformations. Invdupdel(8p) is the most common recurrent chromosomal rearrangement in ID patients with anomalies of the corpus callosum (AnCC). Only a minority of invdupdel(8p) cases reported in the literature to date had both brain cerebral imaging and chromosomal microarray (CMA) with precise breakpoints of the rearrangements, making genotype-phenotype correlation studies for AnCC difficult. In this study, we report the clinical, radiological, and molecular data from 36 new invdupdel(8p) cases including three fetuses and five individuals from the same family, with breakpoints characterized by CMA. Among those, 97% (n=32/33) of patients presented with mild to severe developmental delay/ID and 34% had seizures with mean age of onset of 3.9 years (2 months-9 years). Moreover, out of the 24 patients with brain MRI and 3 fetuses with neuropathology analysis, 63% (n=17/27) had AnCC. We review additional data from 99 previously published patients with invdupdel(8p) and compare data of 17 patients from the literature with both CMA analysis and brain imaging to refine genotype-phenotype correlations for AnCC. This led us to refine a region of 5.1 Mb common to duplications of patients with AnCC and discuss potential candidate genes within this region.

Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation

SummaryPIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.

Incidences of Submicroscopic Deletions Are Variable According to Disease Entities and Chromosomal Translocations in Hematologic Malignancies: Investigation by FISH.

Submicroscopic deletions of gene in recurrent chromosomal rearrangement are known to occur frequently in hematologic malignanacies. With wide application of FISH technique in diagnosis of hematologic malignanices, it has become easy to assess the submicroscopic deletions. We investigated the incidence of submicroscopic deletion and analyzed their association with specific genetic rearrangements in various hematologic malignancies. FISH study was conducted with bone marrow cells in 336 patients with acute lymphoblastic leukemia (ALL), 223 patients with acute myeloid leukemia (AML) and 103 patients with chronic myelogenous leukemia (CML). We performed extra signal (ES)-FISH BCR/ABL, ES-FISH TEL/AML1 rearrangement and dual color split MLL rearrangement for ALL and Dual fusion-FISH (D-FISH) AML1/ETO, D-FISH PML/RARA and dual color break apart-MLL rearrangement for AML. For CML, D-FISH BCR/ABL were done. The incidence of submicroscopic deletion in the patients with chromosomal rearrangement was highest in TEL/AML1 rearrangement (65.0%), followed by BCR/ABL (10.9%), MLL (5.6%), AML1/ETO (4.0%), and PML/RARA (0.0%). Fifteen (19.0%) of 79 patients with CML showed either BCR and/or ABL deletion on derivative chromosome 9. One (5.6%) of 18 patients with MLL+ acute leukemia (5 ALL, 13 AML) showed submicroscopic deletion of telomeric portion of MLL gene. One (4.0%) of 25 patients with AML/ETO+ AML showed deletion of AML1 gene on derivative chromosome 21. None (0%) of 22 patients with PML/RARA+ AML and 59 patients with BCR/ABL+ ALL showed deletion in chromosomal rearrangement. Of note, none of 59 patients with BCR/ABL+ ALL (including major and minor rearrangement) showed submicroscopic deletion, in contrast to that of patients with BCR/ABL+ CML. In conclusion, the incidences of submicroscopic deletion was quite different among specific genetic changes and disease entities. Careful FISH study should be included in the work-up for hematologic malignancies, and their association with clinical prognosis need to be further studied.

TRIM63 is a sensitive and specific biomarker for MiT family aberration-associated renal cell carcinoma.

Microphthalmia-associated transcription factor (MiT) family aberration-associated renal cell carcinoma (MiTF-RCC) is a subtype of renal cell carcinoma harboring recurrent chromosomal rearrangements involving TFE3 or TFEB genes. MiTF-RCC is morphologically diverse, can histologically resemble common RCC subtypes like clear cell RCC and papillary RCC, and often poses a diagnostic challenge in genitourinary clinical and pathology practice. To characterize the MiTF-RCC at the molecular level and identify biomarker signatures associated with MiTF-RCC, we analyzed RNAseq data from MiTF-RCC, other RCC subtypes and benign kidney. Upon identifying TRIM63 as a cancer-specific biomarker in MiTF-RCC, we evaluated its expression independently by RNA in situ hybridization (RNA-ISH) in whole tissue sections from 177 RCC cases. We specifically included 31 cytogenetically confirmed MiTF-RCC cases and 70 RCC cases suspicious for MiTF-RCC in terms of clinical and morphological features, to evaluate and compare TRIM63 RNA-ISH results with theresults from TFE3/TFEB fluorescence in situ hybridization (FISH), which is the current clinical standard. We confirmed that TRIM63 mRNA was highly expressed in all classes of MiTF-RCC compared to other renal tumor categories, where it was mostly absent to low. While the TRIM63 RNA-ISH and TFE3/TFEB FISH results were largely concordant, importantly, TRIM63 RNA-ISH was strongly positive in TFE3 FISH false-negative cases with RBM10-TFE3 inversion. In conclusion, TRIM63 can serve as a diagnostic marker to distinguish MiTF-RCC from other renal tumor subtypes with overlapping morphology. We suggest a combination of TFE3/TFEB FISH and TRIM63 RNA-ISH assays to improve the accuracy and efficiency of MiTF-RCC diagnosis. Accurate diagnosis of MiTF-RCC and other RCC subtypes would enable effective targeted therapy and avoid poor therapeutic response due to tumor misclassification.

NR4A3 (NOR-1) Immunostaining Shows Better Performance than DOG1 Immunostaining in Acinic Cell Carcinoma of Salivary Gland: a Preliminary Study.

ABSTRACTObjectivesAcinic cell carcinoma of salivary gland harbours recurrent and specific chromosomal rearrangement [t(4;9)(q13;q31)], resulting in the translocation of secretory calcium-binding phosphoprotein gene cluster at 4q13 to nuclear receptor subfamily 4 group a member 3 at 9q31. This upregulates the transcription factor nuclear receptor subfamily 4 group A member 3, which can be detected by immunohistochemistry. The purpose of this pilot study is to evaluate the performance of nuclear receptor subfamily 4 group A member 3 immunostaining on whole-slide acinic cell carcinoma tissue, in comparison with discovered on GIST-1 immunostaining.Material and MethodsWe retrieved 6 cases of acinic cell carcinoma (AciCC), including 5 conventional low-grade and 1 dedifferentiated high-grade. Immunohistochemistry (IHC) for nuclear receptor subfamily 4 group A member 3 (NR4A3) and discovered on GIST-1 (DOG1) were performed at the University of Pittsburgh Medical Centre in Pittsburgh, Pennsylvania on all retrieved cases.ResultsThe result shows that NR4A3 IHC shows better performance than DOG1 IHC: 5 of the 6 (83.3%) AciCC cases (including the dedifferentiated high-grade) demonstrated strong diffuse nuclear staining for NR4A3, also five AciCC cases (including the dedifferentiated high-grade) demonstrated weak to moderate membranous staining with variable distribution for DOG1. Moreover, only 3 (50%) cases showed complete membranous staining with DOG1.ConclusionsThis pilot study showed that nuclear receptor subfamily 4 group A member 3 immunostaining is a sensitive marker for acinic cell carcinoma and of better utility than discovered on GIST-1 immunostaining in making a diagnosis of acinic cell carcinoma.

Abstract IA08: The role of gene expression in the classification of aggressive B-cell lymphoma

Abstract Aggressive B-cell lymphomas collectively make up half of all lymphoma diagnoses. The current classification system, the 2017 revision of the WHO 4th edition, assigns these tumors into groups based on morphology, immunophenotype, site of disease, and the presence of recurrent chromosomal rearrangements. Accurate and reproducible diagnosis is required for selection of optimal treatment, prognostication, and ongoing basic research and clinical trials aimed at improving outcomes. Ideally, the taxonomy would continue to evolve towards further defining homogeneous groups of tumors sharing targetable biology. Gene expression (GE) profiling of tumors supports the biologic validity of the entities in the current classification and, along with genomic sequencing, is driving the identification of new lymphoma subtypes. In the mid-2000s GE profiling studies identified specific signatures that distinguish aggressive B-cell entities from each other—namely, primary mediastinal large B-cell lymphoma (PMBL) from diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) from DLBCL. The increasingly divergent treatment of these entities makes reliable diagnosis important. Prior to those studies, GE profiling identified 2 distinct subtypes of DLBCL. This binary division of DLBCL into the cell-of-origin groups of germinal center B-cell-like (GCB) and activated B-cell-like (ABC) has been foundational to our understanding of the pathology of DLBCL. However, recent failure of clinical trials to improve outcomes by adding targeted agents to R-CHOP in upfront treatment of ABC-DLBCL highlights that this binary division may not be sufficiently granular to support precision medicine. These signatures have been translated onto tractable technology platforms, including nuclease protection assay, RT-MLPA, and NanoString, allowing potential integration into diagnostic workflows. More recently, GE signatures have been described that identify distinct molecular subtypes within GCB-DLBCL. Working from the standpoint of tumors that have GE profiles sitting between BL and DLBCL (the “molecular high grade” signature [MHG]) or the GE signature of tumors with rearrangement of MYC and BCL2 (the “double hit” signature [DHITsig]), a sizeable group of GCB-DLBCL can be identified with poor prognosis. Finally, a group of DLBCL without mediastinal involvement have been shown to display the PMBL GE signature. These tumors share perturbation of the hallmark pathways of PMBL but arrive at this biology through different genetic mechanisms. These subtypes map onto, and complement, the newly minted genetics-based classifications of DLBCL. In order to arrive at a tractable unified molecular classification for aggressive B-cell lymphoma, ongoing efforts are needed to integrate GE and genetic aberrations across the disease spectrum. Such a classification framework holds the promise of improved diagnostic accuracy and reliability while providing the foundation for improving patient outcomes through precision medicine. Citation Format: David W. Scott. The role of gene expression in the classification of aggressive B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA08.

Diagnosis of FOXG1 syndrome caused by recurrent balanced chromosomal rearrangements: case study and literature review

BackgroundThe FOXG1 gene plays a vital role in mammalian brain differentiation and development. Intra- and intergenic mutations resulting in loss of function or altered expression of the FOXG1 gene cause FOXG1 syndrome. The hallmarks of this syndrome are severe developmental delay with absent verbal language, post-natal growth restriction, post-natal microcephaly, and a recognizable movement disorder characterized by chorea and dystonia.Case presentationHere we describe a case of a 7-year-old male patient found to have a de novo balanced translocation between chromosome 3 at band 3q14.1 and chromosome 14 at band 14q12 via G-banding chromosome and Fluorescence In Situ Hybridization (FISH) analyses. This rearrangement disrupts the proximity of FOXG1 to a previously described smallest region of deletion overlap (SRO), likely resulting in haploinsufficiency.ConclusionsThis case adds to the growing body of literature implicating chromosomal structural variants in the manifestation of this disorder and highlights the vital role of cis-acting regulatory elements in the normal expression of this gene. Finally, we propose a protocol for reflex FISH analysis to improve diagnostic efficiency for patients with suspected FOXG1 syndrome.

Activation of PLAG1 and HMGA2 by gene fusions involving the transcriptional regulator gene NFIB.

The pleomorphic adenoma (PA), which is the most common salivary gland neoplasm, is a benign tumor characterized by recurrent chromosome rearrangements involving 8q12 and 12q14-15. We have previously shown that the PLAG1 and HMGA2 oncogenes are the targets of these rearrangements. Here, we have identified previously unrecognized subsets of PAs with ins(9;8)/t(8;9) (n = 5) and ins(9;12)/t(9;12) (n = 8) and breakpoints located in the vicinity of the PLAG1 and HMGA2 loci. RNA-sequencing and reverse transcriptase (RT)-PCR analyses of a case with an ins(9;8) revealed a novel NFIB-PLAG1 fusion in which NFIB exon 4 is linked to PLAG1 exon 3. In contrast to the developmentally regulated PLAG1 gene, NFIB was highly expressed in normal salivary gland, indicating that PLAG1 in this case, as in other variant fusions, is activated by promoter swapping. RT-PCR analysis of three PAs with t(9;12) revealed two tumors with chimeric transcripts consisting of HMGA2 exon 4 linked to NFIB exons 9 or 3 and one case with a fusion linking HMGA2 exon 3 to NFIB exon 9. The NFIB fusion events resulted in potent activation of PLAG1 and HMGA2. Analysis of the chromatin landscape surrounding NFIB revealed several super-enhancers in the 5'- and 3'-parts of the NFIB locus and its flanking sequences. These findings indicate that PLAG1 and HMGA2, similar to MYB in adenoid cystic carcinoma, may be activated by enhancer-hijacking events, in which super-enhancers in NFIB are translocated upstream of PLAG1 or downstream of HMGA2. Our results further emphasize the role of NFIB as a fusion partner to multiple oncogenes in histopathologically different types of salivary gland tumors.

NOR-1 distinguishes acinic cell carcinoma from its mimics on fine-needle aspiration biopsy specimens

Acinic cell carcinoma of the salivary gland (ACC-SG) is characterized by a recurrent chromosomal rearrangement (t(4; 9)(q13; q31)) that upregulates the transcription factor NR4A3. Studies conducted on formalin-fixed paraffin-embedded (FFPE) tissue have found that nuclear expression of a monoclonal antibody NR4A3 (NOR-1) is a sensitive and specific diagnostic marker for ACC-SG. The aims of this study were to evaluate the performance of the NOR-1 antibody and to compare its utility in separating ACC-SG from its mimics on cytology cell block specimens. Cell blocks were obtained from 70 fine-needle aspiration specimens from multiple institutional archives over a 7-year period (2013-2019). These included 10 cases of conventional low-grade ACC-SG, 1 case of dedifferentiated high-grade ACC-SG, and 59 cases of non-ACC-SG. An automated immunohistochemistry system (Bond-III, Leica) was used for the detection of NR4A3, using the commercially available antibody NOR-1 (sc-393902 [H-7], Santa Cruz Biotechnology Inc.). Optimization of the antibody on the cell blocks was successfully completed by increasing the titer from 1:100 (suggested titer for FFPE specimens) to 1:30. Distinct nuclear reactivity was observed in all 11 cases of ACC-SG (10 of 11 with 3+ diffuse nuclear positivity and 1 case with 2+ focal reactivity). Expression of NR4A3 was absent in all non-ACC-SG cases in the cell blocks. Application of the NOR-1 immunohistochemical staining in fine-needle aspirates of salivary gland tumors for which ACC-SG is a diagnostic consideration successfully distinguishes ACC-SG from its cytologic mimics and provides an early opportunity for oncologic intervention.

Abstract GMM-050: IDENTIFICATION OF NOVEL CHROMOSOMAL REARRANGEMENTS IN HIGH-GRADE SEROUS OVARIAN CANCER

Abstract High-grade serous ovarian cancer (HGS-OVCA) is the most common type of ovarian cancer. HGS-OVCA genomes are usually very complex and unstable. Many studies have addressed cytogenetic changes, and with the advent of high-throughput sequencing, complex chromosomal rearrangements. However, little is known about how gene fusions, potentially causing aberrant transcripts and protein products drive tumorigenesis, cell growth and/or metastasis in HGS-OVCA. Patients with the same histological or morphological phenotype of cancer have tumors with diverse genetic backgrounds or molecular phenotypes. There is thus an urgent need to better stratify patients and their tumors and to understand the chemotherapy and other treatment responses at the molecular level. The purpose of this study is to find and charaterize novel recurrent chromosomal rearrangements from HGS-OVCA patient samples. We aim to identify rearrangement hotspots from genomic data. However, not only do we need to identify, characterize and stratify the genomic alterations, but also to find those changes with potentially tumorigenic functions at the RNA and protein levels. We utilise datasets of The Cancer Genome Atlas (https://cancergenome.nih.gov/) and HERCULES Project (http://www.project-hercules.eu/) to identify rearrangement-prone chromosomal regions (putative “breakpoint hotspots”) and complex structural aberrations and variations. We concentrate on exonic aberrations seen in both datasets in at least 2 patients. The findings will be validated using two HGS-OVCA fresh-frozen tumor sample collections from Helsinki and Turku University Hospitals. Clinical data and homologous recombination (HR) score of the tumor samples (HR proficient or HR deficient; Tumiati et al. 2018) will also be analyzed. Preliminary results of this work will be presented. We believe that combining sequencing and clinical data with state-of-the-art molecular level analyses and functional tests will deepen our understanding of HGS-OVCA biology and help to develop new anti-cancer treatment options for this “silent killer”. Citation Format: Nanna Sarvilinna, Barun Pradhan, Imrul Faisal, Manuela Tumiati, Amjad Alkodsi, Liisa Kauppi. IDENTIFICATION OF NOVEL CHROMOSOMAL REARRANGEMENTS IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-050.

‘Distal 16p12.2 microdeletion’ in a patient with autosomal recessive deafness-22

The 16p12.2 chromosome band contains three large segmental duplications: BP1, BP2 and BP3, providing a substrate for recombination and recurrent chromosomal rearrangements. The '16p12.2 microdeletion' is a recurrent deletion comprised between BP2 and BP3, associated with variable clinical findings. We identified a heterozygous 16p12.2 microdeletion spanning between BP1 and BP2 in a child evaluated for short stature and mild dyslexia. Unexpectedly, the mother carried the same deletion in the homozygous state and suffered from severe hearing loss. Detailed family history revealed consanguinity of the maternal grandparents. The 16p12.2 microdeletion is a rare condition and contains only three genes: METTL9, IGSF6 and OTOA of which the OTOA is considered responsible for DFNB22 hearing loss (MIM: 607039) under its homozygous condition. A number of OTOA mutations have been described, whereas very few cases of a 16p12.2 microdeletion similar to that observed in our family have been reported. In conclusion, we describe a rare 'distal 16p12.2microdeletion' widening the phenotypic spectrum associated with the recurrent 16p12.2 microdeletion and support the causative role of OTOA microdeletion in hearing impairment.

Characterization of a cryptic KMT2A/AFF1 gene fusion by mate-pair sequencing (MPseq) in a young adult with newly diagnosed B-lymphoblastic leukemia

The detection of recurrent chromosomal rearrangements in B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is critical for patient management decisions. We present a newly diagnosed case of B-ALL in a young adult with a cryptic KMT2A/AFF1 fusion that was unappreciable by conventional chromosome and fluorescence in situ hybridization (FISH) KMT2A break-apart probe studies. To further characterize this abnormality, a next-generation sequencing strategy, mate-pair sequencing (MPseq) was performed and characterized a cryptic, insertional rearrangement that created KMT2A/AFF1 gene fusion. This case highlights the superior precision and resolution capabilities of NGS when compared to traditional cytogenetic methodologies, including conventional chromosome and FISH studies.

Targeting chromatin complexes in fusion protein-driven malignancies.

Recurrent chromosomal rearrangements leading to the generation of oncogenic fusion proteins are a common feature of many cancers. These aberrations are particularly prevalent in sarcomas and haematopoietic malignancies and frequently involve genes required for chromatin regulation and transcriptional control. In many cases, these fusion proteins are thought to be the primary driver of cancer development, altering chromatin dynamics to initiate oncogenic gene expression programmes. In recent years, mechanistic insights into the underlying molecular functions of a number of these oncogenic fusion proteins have been discovered. These insights have allowed the design of mechanistically anchored therapeutic approaches promising substantial treatment advances. In this Review, we discuss how our understanding of fusion protein function is informing therapeutic innovations and illuminating mechanisms of chromatin and transcriptional regulation in cancer and normal cells.