Recurrent Chromosomal Abnormalities Research Articles

Comparative Analysis of Two NGS-Based Platforms for Product-of-Conception Karyotyping.

Cytogenetic information about the product of conception (POC) is important to determine the presence of recurrent chromosomal abnormalities that are an indication for preimplantation genetic testing for aneuploidy or structural rearrangements. Although microscopic examination by G-staining has long been used for such an evaluation, detection failures are relatively common with this method, due to cell-culture-related issues. The utility of low-coverage whole-genome sequencing (lcWGS) using short-read next-generation sequencing (NGS) has been highlighted recently as an alternative cytogenomic approach for POC analysis. We, here, performed comparative analysis of two NGS-based protocols for this purpose based on different short-read sequencers (the Illumina VeriSeq system using a MiSeq sequencer and the Thermo Fisher ReproSeq system using an Ion S5 sequencer). The cytogenomic diagnosis obtained with each NGS method was equivalent in each of 20 POC samples analyzed. Notably, X chromosome sequence reads were reduced in some female samples with both systems. The possibility of low-level mosaicism for monosomy X as an explanation for this was excluded by FISH analysis. Additional data from samples with various degrees of X chromosome aneuploidy suggested that it was a technical artifact related to X chromosome inactivation. Indeed, subsequent nanopore sequencing indicated that the DNA in the samples showing the artifact was predominantly unmethylated. Our current findings indicate that although X chromosome data must be interpreted with caution, both the systems we tested for NGS-based lcWGS are useful alternatives for the karyotyping of POC samples.

The genomic and molecular landscape of splenic marginal zone lymphoma, biological and clinical implications.

Splenic marginal zone lymphoma (SMZL) is a rare, predominantly indolent B-cell lymphoma constituting fewer than 2% of lymphoid neoplasms. However, around 30% of patients have a shorter survival despite currently available treatments and the prognosis is especially poor for the 5-15% of cases that transform to a large cell lymphoma. Mounting evidence suggests that the molecular pathogenesis of SMZL is critically shaped by microenvironmental triggering and cell-intrinsic aberrations. Immunogenetic investigations have revealed biases in the immunoglobulin gene repertoire, indicating a role of antigen selection. Furthermore, cytogenetic studies have identified recurrent chromosomal abnormalities such as deletion of the long arm of chromosome 7, though specific disease-associated genes remain elusive. Our knowledge of SMZL's mutational landscape, based on a limited number of cases, has identified recurring mutations in KLF2, NOTCH2, and TP53, as well as genes clustering within vital B-cell differentiation pathways. These mutations can be clustered within patient subgroups with different patterns of chromosomal lesions, immunogenetic features, transcriptional signatures, immune microenvironments, and clinical outcomes. Regarding SMZL epigenetics, initial DNA methylation profiling has unveiled epigenetically distinct patient subgroups, including one characterized by elevated expression of Polycomb repressor complex 2 (PRC2) components. Furthermore, it has also demonstrated that patients with evidence of high historical cell division, inferred from methylation data, exhibit inferior treatment-free survival. This review provides an overview of our current understanding of SMZL's molecular basis and its implications for patient outcomes. Additionally, it addresses existing knowledge gaps, proposes future research directions, and discusses how a comprehensive molecular understanding of the disease will lead to improved management and treatment choices for patients.

Genetic and clinical characteristics of acute B-cell lymphoblastic leukemia with MEF2D fusions and report of two novel MEF2D rearrangements.

The MEF2D rearrangement is a recurrent chromosomal abnormality detected in approximately 2.4-5.3% of patients with acute B-cell lymphoblastic leukemia (B-ALL). Currently, MEF2D-rearranged B-ALL is not classified as an independent subtype in the WHO classification. Consequently, the clinical significance of MEF2D rearrangement in B-ALL remains largely unexplored. In this study, we retrospectively screened 260 B-ALL patients with RNA sequencing data collected between November 2018 and December 2022. Among these, 10 patients were identified with MEF2D rearrangements (4 with MEF2D::HNRNPUL1, 3 with MEF2D::BCL9, 1 with MEF2D::ARID1B, 1 with MEF2D::DAZAP1 and 1 with MEF2D::HNRNPM). Notably, HNRNPM and ARID1B are reported as MEF2D fusion partners for the first time. The patient with the MEF2D::HNRNPM fusion was resistant to chemotherapy and chimeric antigen receptor T-cell therapy and relapsed early after allogenic stem cell transplantation. The patient with MEF2D::ARID1B experienced early extramedullary relapse after diagnosis. All 10 patients achieved complete remission after induction chemotherapy. However, 9/10 (90%) of whom experienced relapse. Three of the 9 patients relapsed with aberrant expression of myeloid antigens. The median overall survival of these patients was only 11 months. This small cohort showed a high incidence of early relapse and short survival in patients with MEF2D rearrangements.

Chromosomal Abnormalities in Chromosome 5 and Chromosome 8 in Iraqi Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the mostly diagnosed leukemia in adults, resulting from accumulate immature blasts in the bone marrow that are replaced instead of normal blasts that cannot function like normal blood cells. AML resulting from genetic abnormalities, including multiple gene mutations and chromosomal aberrations, are found in approximately 80% of children with AML. These shown correlations between specific recurrent chromosomal abnormalities and clinical-biological characteristics and outcomes, also, novel therapies are being developed that target some of the identified genetic defects. The aim of this study was to investigate and record the role of chromosome 5 and 8 aberrations in the development of newly diagnosed and relapsed cases of AML in Iraqi patients. Chromosomal changes were studied in peripheral blood samples from 30 Iraqi patient samples diagnosed with acute myeloid leukemia and divided into 9 newly diagnosed and 13 chemotherapy-treated subjects who were incomplete remission and 8 relapsed subjects. The results of the chromosomal analysis of this study revealed numerical and structural abnormalities of both chromosomes: 5 and 8 in 16 (53.33%) and 12 (40%), respectively, within a complex karyotype and high frequencies in numerical abnormalities of chromosomes for chromosome 5 and structural abnormalities for chromosome 8. Chromosomal aberrations involving chromosomes 5 and 8 are linked to AML development and prognosis, and their presence in a complex karyotype can lead to disease progression and the worst prognosis, especially for abnormalities on chromosome 5, which were detected the most frequently. Future molecular genetic tests and complete karyotype analysis should enhance AML diagnosis and treatment.

Synovial Chondromatosis of Right Knee: A Case Report

Abstract Synovial chondromatosis is a rare cartilaginous neoplasm that most commonly affects the knee joint. Histologically, it presents as a multinodular cartilaginous proliferation showing clusters of chondrocytes. These can be quite atypical, simulating chondrosarcoma. The lesion is usually self-limited but sometimes may exhibit a more aggressive clinical course characterized by multiple local recurrences. Chondrosarcomatous transformation in synovial chondromatosis is exceedingly rare but has been documented. Until recently, synovial chondromatosis was considered a reactive process. Recent cytogenetic studies have shown recurrent chromosomal abnormalities, especially involving chromosome6, therefore supporting a clonal, neoplastic nature of this intriguing lesion.

Association of Latino Ethnicity with Cytogenetic Subtypes in Pediatric Acute Myeloid Leukemia

Background: While advances in molecular cytogenetic assessment and incorporation of recurrent chromosomal abnormalities with prognostic significance into risk stratification have improved outcomes for children with acute myeloid leukemia (AML), outcome disparities persist. Specifically, Latino children are noted to have inferior outcomes compared with their non-Latino peers. These disparities may be related in part to ethnic-based differences in cytogenetic features, but the prevalence of AML cytomolecular subtypes within specific demographic populations is a relatively understudied area. Our aim is to evaluate the association between self-reported race/ethnicity and recurrent prognostic cytogenetic features in pediatric AML. Methods: We analyzed data from the multi-institutional Reducing Ethnic Disparities in Acute Leukemia (REDIAL) Consortium between 2007 to 2022. REDIAL includes children newly diagnosed with AML at six major pediatric cancer centers in the Southwestern U.S. Demographic and clinical characteristics include self-reported race/ethnicity, sex, and age at diagnosis. Cytogenetic subtypes evaluated included KMT2A rearrangements, FLT3 mutation, PML::RARA, RUNX1::RUNX1T1, CBFβ::MYH11, CEBPA, DEK::NUP21, RBM15::MKL1, RPN::1EV11, del5q, monosomy 5, monosomy 7, and BCR::ABL1. Features were dichotomized as present or absent in those that were tested, and subtypes categorized into favorable, unfavorable, and neutral groups. Multivariate logistic regression models were used to estimate adjusted odds ratios (aORs) and 95% confidence intervals (CIs), adjusting for sex and age at diagnosis. Results: Among the 665 AML patients included in this analysis, 46.3% self-reported as Latino, 32.6% as non-Latino White (NLW), 9.4% as Black, 6.4% as Asian/Pacific Islander (API), and 5.1% as other/unknown. The mean age at diagnosis was 7.9 years (standard deviation 6.3); a majority were male (51.6%). Among all patients that were tested, 20.8% (134/644) had at least one favorable subtype, and 27.3% (161/590) had at least one unfavorable subtype. Adjusted logistic regression models showed Latino children had 1.59 times greater odds of having at least one favorable subtype compared to NLW children (aOR 1.59, 95% CI 1.01-2.51) but were not at increased odds of having at least one unfavorable subtype (aOR 1.03, 95% CI 0.68-1.56) or a neutral subtype (aOR 0.84, 95% CI 0.-57-1.23) compared to patients of other race/ethnicity. While multivariable analyses of specific subtypes did not meet the threshold for statistical significance, there were some notable trends. For instance, compared with NLW children, Latino and Black children had a higher prevalence of the favorable RUNX1::RUNX1T1 translocation (aOR: 1.40, 95% CI: 0.70-2.83; 1.90, 0.69-5.20, respectively). Compared with NLW children, Latino and Black children also had a higher prevalence of the favorable CEBPA mutation (aOR 1.02, 95% CI 0.15-6.56; 1.36, 0.11-16.6, respectively), and were more likely to have unfavorable KMT2A rearrangements (aOR 1.18, 95% CI 0.67-2.08; 1.23, 0.47-3.1, respectively). Additionally, Latino children were more likely to have the favorable translocation CBFβ::MYH11 compared with Black and NLW children (aOR 2.2, 95% CI 0.27-18.1; 1.20, 0.42-3.37, respectively), and less likely to carry the unfavorable FLT3 activating mutation compared with their NLW peers (aOR 0.56, 95% CI 0.26-1.21). Conclusions: Our findings suggest Latino children are more likely to have a favorable cytogenetic subtype than NLW children, which suggests disparities in outcomes are not primarily due to underlying differences in prognostic subtypes. Additional analyses of pediatric AML as it relates to cytogenetic subtypes and race/ethnicity is ongoing, including the incorporation of disease response and treatment outcomes, to understand how these factors may impact disparities in outcomes.

Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion.

In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co-occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

H3 G34-mutant high-grade gliomas: integrated clinical, imaging and pathological characterisation of a single-centre case series.

Diffuse hemispheric glioma, H3 G34-mutant, is a novel paediatric tumour type in the fifth edition of the WHO classification of CNS tumours associated with an invariably poor outcome. We present a comprehensive clinical, imaging and pathological review of this entity. Patients with confirmed H3 G34R-mutant high-grade glioma were included in a single-centre retrospective cohort study and examined for clinical, radiological and histo-molecular data. Twelve patients were enrolled in the study - 7 males/5 females; the mean age was 17.5years (10-57years). Most patients presented with signs of raised intracranial pressure (8/12). The frontal lobe (60%) was the prevalent location, with a mixed cystic-nodular appearance (10/12) and presence of vascular flow voids coursing through/being encased by the mass (8/12), and all tumours showed cortical invasion. Nine patients had subtotal resection limited by functional margins, two patients underwent supra-total resection, and one patient had biopsy only. 5-ALA was administered to 6 patients, all of whom showed positive fluorescence. Histologically, the tumours showed a marked heterogeneity and aggressive spread along pre-existing brain structures and leptomeninges. In addition to the diagnostic H3 G34R/V mutation, pathogenic variants in TP53 and ATRX genes were found in most cases. Potential targetable mutations in PDGFRA and PIK3CA genes were detected in five cases. The MGMT promoter was highly methylated in half of the samples. Methylation profiling was a useful diagnostic tool and highlighted recurrent structural chromosome abnormalities, such as PDGFRA amplification, CDKN2A/B deletion, PTEN loss and various copy number changes in the cyclin D-CDK4/Rb pathway. Radiochemotherapy was the most common adjuvant treatment (9/12), and the average survival was 19.3months. H3 G34R-mutant hemispheric glioma is a distinct entity with characteristic imaging and pathological features. Genomic landscaping of individual tumours can offer an opportunity to adapt individual therapies and improve patient management.

Evaluating the Effects of Targeted Drug Therapies for 8q24.3 Amplified Breast Cancer

Background/Objective: Cancer studies have helped us understand recurrent chromosome abnormalities leading to tumor progression. One such recurrent genomic aberration found in breast cancer is chromosome 8q24.3 (Chr. 8q24.3) amplification. We identified Tonsoku Like, DNA Repair Protein (TONSL) located within this amplicon as an immortalizing oncogene, with TONSL-overexpressing cells exhibiting distinctively upregulated homologous recombination (HR). Further experiments have shown that cancer cells with TONSL amplification are sensitive to the FACT inhibitor CBL0137, which is in early phase of clinical development. Based on known functions of TONSL in promoting dsDNA repair, we hypothesized that drug combinations targeting multiple pathways of DNA repair would synergize to kill chromosome 8q24.3 amplified breast cancer cell lines. Methods: Chr.8q24.3-amplified breast adenocarcinoma cell line TMD436 was utilized in this study. Cells were treated with various drugs targeting DNA repair pathways such as ATR inhibitor (VE-822), PARP inhibitor (Talazoparib), and PI3K inhibitor (BYL719). Cell proliferation rates were measured using bromodeoxyuridine incorporation ELISA. Results: Thus far, the use of PI3Ki and PARPi combination has had an additive effect - the combined effect of the two drugs is equal to the sum of the effect of each agent given alone. The effect of ATRi and PARPi combination was antagonistic. Conclusion/Potential Impact: This study establishes the potential feasibility of using DNA repair signaling inhibitors in the treatment of TONSL-overexpressing breast cancer. Future studies extending the range of drug concentrations and newer combinations may ultimately lead to translation of these drugs to in vivo models and clinical trials. By targeting multiple DNA repair pathways, similar approaches may sensitize patients to lower doses of chemotherapeutics, thus decreasing unwanted side effects. Moreover, negative data concerning the ATRi/PARPi combination helps to further refine which drugs may be used to treat Chr. 8q24.3 amplified tumors and to understand signaling pathways active in TONSL-overexpressing breast cancer.

Identification of COL1A1/2 Mutations and Fusions With Noncoding RNA Genes in Bizarre Parosteal Osteochondromatous Proliferation (Nora Lesion)

Bizarre parosteal osteochondromatous proliferation (BPOP) (Nora lesion) is a benign bone surface lesion, which most commonly occurs in the digits of young patients and has a high rate of recurrence. Histologically, it is composed of a mixture of disorganized bone, cartilage, and spindle cells in variable proportions and characterized by amorphous “blue bone” mineralization. Recurrent chromosomal abnormalities, including t(1;17)(q32-42;q21-23) and inv(7)(q21.1-22q31.3-32), have been reported in BPOP. However, the exact genes involved in the rearrangements remain unknown. In this study, we analyzed 8 BPOP cases affecting the fingers, toe, ulna, radius, and fibula of 5 female and 3 male patients, aged 5 to 68 years. RNA sequencing of 5 cases identified genetic fusions between COL1A2 and LINC-PINT in 3 cases and COL1A1::MIR29B2CHG fusion in 1, both validated using fluorescence in situ hybridization and reverse transcription (RT)-PCR. The remaining fusion-negative case harbored 3 COL1A1 mutations as revealed by whole-exome sequencing and confirmed using Sanger sequencing. All these genetic alterations were predicted to cause frameshift and/or truncation of COL1A1/2. The chromosomal locations of COL1A2 (7q21.3), LINC-PINT (7q32.3), COL1A1 (17q21.33), and MIR29B2CHG (1q32.2) were consistent with the breakpoints identified in the previous cytogenetic studies. Subsequent screening of 3 BPOPs using fluorescence in situ hybridization identified 1 additional case each with COL1A1 or COL1A2 rearrangement. Our findings are consistent with reported chromosomal abnormalities and implicate the disruption of type I collagen, and perhaps of either noncoding RNA gene as a tumor suppressor, in the tumorigenesis of BPOP. The prevalence and tumorigenic mechanisms of these COL1A1/2 alterations in BPOP require further investigation.

Pre-leukemic Cell Detection and Leukemic Transformation of a Normal Marrow Cell: A Mini review

The hematopoietic cells are multipotent primitive cells, which differentiate into either common myeloid and lymphoid progenitor. However, if there an abnormality in this process of differentiation, condition of leukemia arises, which is the 11th leading cause of cancer-related mortality worldwide in the year 2018. These abnormalities are brought about by array of mutations occurring at cellular level. According to the two-hit model hypothesis, key oncogenic events are classified into two classes: class I mutations and class II mutations. Class I mutations are those that causes activation of the receptor tyrosine kinase (RTK), FLT3, c-kit (KIT), and Ras signaling pathways thereby increasing proliferation rate of progenitor cells. Class II mutations include recurrent chromosomal abnormalities such as t(8; 21), inv(16), and t(15; 17), which result in fusion transcripts of RUNX1/ETO, CBF/MYH11, and PML/RAR, respectively that eventually impair hematopoietic differentiation. The factors associated with leukemia can be biological, chemical or socio-economical. The advancement in the researches on the topic have aided to the development of various technologies such as detection of DNMT3A and xenografts assays, in order to detect these mutations in pre-leukemic cells. This review aims to provide an introduction to the condition, its types and provide brief summary on genes and mutations responsible for the condition. The factors associated with leukemia and technologies involved in the detection of leukemia are also reviewed.

GenIDA: an international participatory database to gain knowledge on health issues related to genetic forms of neurodevelopmental disorders.

Intellectual disability with or without manifestations of autism and/or epilepsy affects 1-2% of the population, and it is estimated that more than 30-50% of these cases have a single genetic cause. More than 1000 genes and recurrent chromosomal abnormalities are involved in these genetic forms of neurodevelopmental disorders, which often remain insufficiently described in terms of clinical spectrum, associated medical problems, etc., due to their rarity and the often-limited number of patients' phenotypes reported. GenIDA is an international online participatory database that aims to better characterise the clinical manifestations and natural histories of these rare diseases. Clinical information is reported by parents of affected individuals using a structured questionnaire exploring physical parameters, cognitive and behavioural aspects, the presence or absence of neurological disorders or problems affecting major physiological functions, as well as autonomy and quality of life. This strengthens the implication in research of the concerned families. GenIDA aims to construct international cohorts of significant size of individuals affected by a given condition. As of July 2022, GenIDA counts some 1545 documented patient records from over 60 nationalities and collaborates with clinicians and researchers around the world who have access to the anonymized data collected to generate new, medically meaningful information to improve patient care. We present the GenIDA database here, together with an overview of the possibilities it offers to affected individuals, their families, and professionals in charge of the management of genetic forms of neurodevelopmental disorders. Finally, case studies of cohorts will illustrate the usefulness of GenIDA.

The Co-Occurrence of 1q21+/1q23+ and t(4;14) Abnormalities with Specific Clone Size Is Predictive of Shorter Response to Transplant in Patients with Multiple Myeloma

Introduction: The presence or absence of recurrent chromosomal abnormalities (CA) as detected by karyotype and/or fluorescence in-situ hybridization (FISH) has emerged as one of the most clinically relevant prognostic factors for patients with newly diagnosed multiple myeloma (MM). The Revised-International Staging System (R-ISS) defined high-risk disease as the presence of at least one of the following: deletion(17p), translocation t(4;14)(p16;q32), or translocation t(14;16)(q32;q23). Amplification or gain of chromosome 1q (1q21+/1q23+) has also been growing in its clinical relevance, with 1q21+ found in approximately 35-40% of patients. As a result, this study evaluated differences in survival outcomes and disease characteristics in patients with both 1q+ (1q21+ or 1q23+) and t(4;14), compared to those patients with only 1q+ or t(4;14). Methods: A retrospective chart review was performed on 243 newly diagnosed MM patients with the cytogenetic abnormalities of interest obtained from established FISH data from bone marrow aspirate samples at diagnosis. Primary endpoints were Progression-Free Survival (PFS) and Overall Survival (OS) after autologous stem cell transplant (ASCT), and Kaplan Meier methods were used to calculate PFS and OS. Secondary endpoints included the identification of disease characteristics between single CA and combined abnormalities. To specifically analyze the quantitative CA cutoff values and the correlation of clone size to survival outcomes, we established subgroupings with positivity defined as 1q21+ or 1q23+ present in more than 20% of cells and t(4;14) present in more than 30% of cells. Patients who satisfied both cutoff criteria were labeled as 'double-hit', and patients with CA present, but clone sizes less than the prespecified cutoffs for both CA, were denoted as 'CA(low)'. All tests were two-sided with p-values <0.05 considered statistically significant. Results: The median age at diagnosis was 61.5 years, 54% were male, and 84% of patients identified as non-Hispanic White. Of the total cohort and independent of quantitative CA cutoff, 1q+ (1q21+ or 1q23+) only was the most prevalent CA in 198 (81%) of patients, t(4;14) only in 13 (5%) patients, and both 1q+ (1q21+ or 1q23+) and t(4;14) in 32 (13%) patients. One hundred seventy (70%) patients underwent ASCT, followed by maintenance therapy in 85% of them. Pre-transplant best hematologic responses did not differ amongst CA groups. There was no difference in baseline hemoglobin, serum creatinine, calcium, albumin, lactate dehydrogenase, or beta-2 microglobulin amongst the CA groups. No differences in outcomes were noted without including size clone cutoffs. There was a statistically significant difference in univariable analysis in PFS and OS from ASCT in patients with 1q21+ and t(4;14) 'CA(low)’ (n=76, 31%) having better survival outcomes compared to the corresponding 'double-hit’ group (n=25, 10%) (PFS: HR=0.52, 95% CI: 0.30-0.92, p=0.025; OS: HR=0.41, 95% CI: 0.21-0.81, p=0.010). This pattern in PFS and OS after ASCT was also seen in patients with 1q23+ and t(4;14) 'CA(low)’ (n=119, 49%) faring better compared to its 'double-hit’ group (n=19, 8%) (PFS: HR=0.47, 95% CI:0.26-0.83, p=0.009; OS: HR=0.34, 95% CI:0.18-0.66, p=0.001). Patients with t(4;14) only (>30%) (n=19, 8%) within the 1q23+ cohort also demonstrated longer PFS and OS after ASCT as compared to its 'double-hit’ group (PFS: HR=0.42, 95% CI:0.20-0.91, p=0.027; OS: HR=0.38, 95% CI:0.15-0.94, p=0.037). After adjusting for age and R-ISS staging, the difference between 1q23+ and t(4;14) 'CA(low)’ vs 'double-hit’ and t(4;14) only within the 1q23+ cohort vs 'double-hit’ remained significant in multivariable analysis in PFS after ASCT ('CA(low)’ group: HR=0.50, 95% CI:0.25-0.99, p=0.048; and t(4;14) only group: HR=0.41, 95% CI:0.18-0.98, p=0.045). Conclusion: Our findings reveal the differences seen in MM patients with single CA compared to the combined high-risk aberrations with both 1q21+/1q23+ and t(4;14). The effects of quantitative clone sizes on survival outcomes were displayed as patients with smaller clone sizes (i.e. 'CA(low)’ subsets) had superior survival outcomes than those patients with 'double-hit’ 1q21+ (or 1q23+) >20% and t(4;14) >30%. It is thus imperative for clinicians to be mindful of not only the presence of high-risk CA but also the size of the aberrant clone influencing subsequent MM disease characteristics.

A Novel Integrated Approach for Cytogenomic Evaluation of Plasma Cell Neoplasms

Plasma cell neoplasm (PCN) is associated with characteristic chromosomal aberrations of diagnostic and prognostic significance. The presence of a small percentage of neoplastic cells is a drawback in the application of karyotyping and fluorescence in situ hybridization for the evaluation of bone marrow aspirate. The analysis of samples enriched for CD138+ cells has improved the detection rate. However, fluorescence in situ hybridization requires several probes and may not be completed due to a limited number of isolated cells. To address the issues experienced with the conventional approach, a novel integrated protocol that consists of whole-genome amplification of DNA isolated from CD138+ cells, followed by microarray as well as one fluorescence in situ hybridization assay for balanced IGH gene rearrangements, has been developed. In the present study in a cohort of 56 patients with clinical suspicion for PCN, compared to conventional cytogenetic analysis, this approach provided higher yield in the detection of PCN-related abnormalities, irrespective of the initial percentage of plasma cells. Whole-genome profiling uncovered recurrent chromosomal abnormalities of prognostic value, including unbalanced alterations within the MYC locus, 16q loss, and hypodiploidy, that were not otherwise detectable by conventional methods. The proposed approach is cost-efficient and provides a superior detection rate, required for proper risk stratification and differential diagnosis of PCN regardless of initial plasma cell percentage.

Molecular genetic and clinical characterization of acute myeloid leukemia with trisomy 8 as the sole chromosome abnormality

ABSTRACT Introduction The aim of the study was to determine molecular genetic and clinical characterization of acute myeloid leukemia (AML) with trisomy 8 as the sole chromosome abnormality, a recurrent but rare chromosomal abnormality in AML. Methods Interphase fluorescence in situ hybridization, reverse transcriptase-quantitative polymerase chain reaction for gene rearrangement and next-generation sequencing (NGS) were performed on sole trisomy 8 AML patients. Results A total of 35 AML patients with trisomy 8 as the sole chromosome abnormality were screened. The most frequently mutated genes were DNMT3A(37.1%), RUNX1(28.6%), FLT3-ITD(28.6%), IDH2(22.9%), NPM1(17.1%), and ASXL1 (14.3%). The sole +8 AML patients exhibited more mutations in RUNX1 (28.6% vs. 4.8%, P = 0.001) and ASXL1 (14.3% vs. 4.8%, P = 0.039) by comparing with normal karyotype AML (NK AML) patients(n = 63). The sole +8 AML patients(n = 35) with RUNX1 or IDH2 mutations showed significantly lower WBC counts, while FLT3-ITD showed higher white blood cell (WBC) counts as compared to the corresponding wild-type groups. Total of 45.7% patients achieved complete remission (CR) after the first induction therapy. The CR rate of patients with FLT3-ITD or IDH1 mutation was significantly lower than that in the corresponding wild-type cases (P = 0.047, 0.005, respectively). The median overall survival (OS) and disease-free survival (PFS) were 18.0 (95% CI: 10.8–25.2) and 10 (95% CI: 6.7–13.3) months, respectively. FLT3-ITD mutations and allogeneic hematopoietic stem cell transplantation (allo-HSCT) were independent prognostic markers for OS in multivariable analysis. Conclusion The results suggest a possible association between trisomy 8 and additional mutations that may influence clinical feature and prognosis.

56. Optical genome mapping reveals genomic complexity and detects novel genetic abnormalities in T-Lymphoblastic Leukemia

Background: Few recurrent chromosomal abnormalities have been recognized in T-lymphoblastic leukemia (T-ALL) by conventional chromosomal analysis (CCA), largely due to the poor growth of T-ALL cells under in vitro culture and/or abnormalities are cryptic by CCA. Optical Genome Mapping (OGM) enable us to map the whole genome and identify numerical and structural chromosomal abnormalities with high resolution.

False Positive Conventional Cytogenetic Findings that Mimic the Recurrent Chromosome Abnormalities Associated with Hematolymphoid Disorders

False Positive Conventional Cytogenetic Findings that Mimic the Recurrent Chromosome Abnormalities Associated with Hematolymphoid Disorders

Targeting PRMT9 Suppresses Acute Myeloid Leukemia Maintenance

AML is a heterogenous disease in which prognosis and treatment is determined by recurrent genetic mutations and chromosomal abnormalities. Some genetic alterations result in aberrant RNA translation, which is exploited by leukemia stem cells (LSCs) to produce short-lived oncoproteins (c-Myc, Mcl-1) to promote cell survival. Since LSCs are considered as the source of treatment failure and relapse, it is critical to understand how LSCs hijack the translational machinery in order to develop effective therapeutics in AML.Arginine methylation catalyzed by protein arginine methyltransferases 1-9 (PRMT1-9) regulates various activities, including RNA translation. We revealed that PRMT1 over activation contributes to leukemia maintenance (Blood, 2019; Blood, 2019). Unlike PRMT1, the recently described PRMT9, is not defined in leukemia. Follow up of AML patients from existing datasets (GSE12417, TARGET) confirms that patients with higher levels of PRMT9 correlates with decreased overall survival. In our AML cohort, we observed significantly increased PRMT9 mRNA levels in AML CD34 + cells relative to normal counterparts (Fig. 1A), spurring our interest in evaluating its function in AML. We thus developed a Prmt9 conditional KO mouse (Mx1-Cre/Prmt9 f/f) and crossed it with an MLL-AF9 (MA9) knock-in mouse to generate Prmt9-KO/MA9 mice for further transplant assay. As a result, Prmt9-KO significantly delayed leukemogenesis in recipient mice carrying MA9 transplants evidenced by prolonged survival (Fig. 1B). Prmt9-KO impaired leukemia-initiating activity evidenced by secondary transplantation that significant less residual CD45.2 + MA9 leukemia cells were found in BM of secondary recipients receiving Prmt9-KO cells relative to those of Prmt9-WT controls. We next validated PRMT9 function in human AML. PRMT9-KD potentially impaired survival of primary AML CD34 + cells, whereas large sparing normal counterparts (Fig. 1C). Moreover, we designed a WT PRMT9 construct and corresponding catalytically dead mutant, both resistant to shPRMT9 (WT-R and MUT-R). Unlike MUT-R, WT-R expression rescued survival effects seen following shPRMT9 treatment, indicating that PRMT9 catalytic activity is required for AML survival (Fig. 1D). To define PRMT9 downstream effectors, we searched for methylated substrates in Molm13 with inducible PRMT9-KD or corresponding controls by performing SILAC analysis coupled with quantitative Mass-Spec (MS) (Fig. 1E). Complete analyses of normalized methyl peptides SILAC ratios revealed more prominent down-regulation of R-methylation in PRMT9-KD cells, with 49 methyl sites downregulated. Notably, methylated PAPB1 C-terminus peptide containing R493 was most significantly depleted upon PRMT9-KD. PABP1 potentiates translation initiation by binding to the poly(A) mRNA tail and interacting with factors like eIF4G. We thus generated an anti-R493 methylation antibody. Indeed, PRMT9 was confirmed to catalyze R493 methylation through in-vitro and cellular methylation assays (Fig. 1F, G). To define the function of R493 methylation, we ectopically expressed either WT PABP1 or the R493K mutant in Molm13 cells and then engineered those cells to express shPABP1 to KD endogenous PABP1. Notably, unlike WT PABP1, expression of R493K suppressed protein synthesis and increased apoptosis after endogenous PABP1-KD, suggesting that PRMT9 mediated PABP1-R493 methylation promotes AML viability (Fig. 1H, I).To discover PRMT9 inhibitors, we conducted a structure-based virtual screen of 960,000 compounds from NCI and Zinc compound libraries (Fig. 1J). We requested the top 300 candidates to assess their anti-leukemia activity in Molm13 cells (Fig. 1K). The top 20 most effective compounds were further analyzed via PRMT9 methylation assay (R493 antibody detection). NSC641396 exhibited the most potent PRMT9 inhibitory effects, as evidenced by decreased PABP1 R493 methylation levels at a dose <1 µΜ (Fig. 1L). NSC641396 treatment on AML CD34 + cells altered polysome profiling, decreased protein biosynthesis and reduced levels of short-lived proteins, suggesting translation inhibition (Fig. 1M).Taken together, our results suggest that PRMT9 plays an oncogenic role in AML or LSC maintenance, by promoting PABP1 methylation mediated translation activity. Further studies are needed to explore if targeting PRMT9 with newly identified lead compound ablates LSCs activity. [Display omitted] DisclosuresMarcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

Recurrent Chromosomal Abnormalities in Tissues Involved by Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma.

Prognostically relevant chromosomal abnormalities in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) are routinely identified by fluorescence in situ hybridization (FISH) on peripheral blood or bone marrow specimens. We studied the prevalence of chromosomal abnormalities on extramedullary tissues involved by CLL/SLL and evaluated their association with prominent proliferation centers (PPCs). FISH for recurrent abnormalities in CLL/SLL was performed on formalin-fixed, paraffin-embedded biopsy sections. PPCs were identified on H&E-stained sections. Available FISH results on peripheral blood or bone marrow specimens were also reviewed. Recurrent FISH abnormalities were detected in 69% of 320 CLL/SLL biopsy specimens studied, including +12 (35%), 13q- (24%), 11q- (15%), 17p- (6%), 6q- (2%), and IGH/BCL2 (0.9%). Forty-three patients had abnormal blood or bone marrow FISH analyses, of whom 7 (16%) had discordant +12 and/or 13q-, and 3 (7%) had discordant 17p- or 11q-. Morphology was positive (17%), negative (78%), or equivocal (6%) for PPCs on 247 evaluable biopsy specimens, a finding not significantly associated with FISH results (P = .7). Trisomy 12 is overrepresented in tumoral CLL/SLL involvement, compared with the known predominance of 13q- in blood. Discrepancies between leukemic and tissue FISH findings are occasionally encountered. FISH results do not correlate with the presence of PPCs.

Whole-Genome Mate Pair Sequencing Transforms Cytogenetic Analysis of Hematologic Malignancies: Development of a Targeted Panel to Detect Diagnostic/Prognostic Chromosomal Rearrangements and Copy Number Changes in AML

Acute myeloid leukemia (AML) is the most common form of acute leukemia affecting both children and adults. Once recognized as a single disease, the World Health Organization now groups AML into ten subtypes based on genetic abnormality. This is based on differences among recurrent genetic abnormalities with respect to response to therapy, relapse risk and overall survival. Currently most testing of AML patients occurs by karyotype analysis, FISH or RT-PCR. Here we describe a next generation sequencing approach to better classify and accurately detect abnormalities associated with AML.For the validation of whole genome mate pair sequencing (MPseq) targeted panel to test patients with AML, twenty-five abnormal and ten normal samples extracted from each sample type (peripheral blood (PB) and bone marrow (BM)) were run with the MPseq assay. Samples were chosen based on reported abnormalities previously tested by conventional karyotyping and/or FISH. The mate pair panel is designed to detect rearrangements involving genes ABL1, BCR, CBFB, CREBBP, DEK, KAT6A(MYST3), MECOM(EVI1), MLF1, MLL(KMT2A), MYH11, NPM1, NUP214(CAN), NUP98, PML, RARA, RPN1, RUNX1(AML1), RUNX1T1(ETO) and copy number changes on chromosomes 5, 7, 8, 13, 17, and 20. DNA was processed using the Illumina Nextera Mate Pair library preparation kit, multiplexed at 2 samples/lane and sequenced on the Illumina HiSeq. Data was aligned to the reference genome (GRCh38) using BIMA and abnormalities were identified using SVAtools, both in-house developed tools. A targeted analysis approach was utilized using a well-defined list of recurrent chromosomal abnormalities associated with known diagnostic and prognostic significance as well as targeted therapies in AML. The karyotype, FISH and MPseq results were compared to determine concordance.To test the limit of detection based on percent tumor, samples previously tested with FISH were diluted. Tumor percentage was determined by the abnormal FISH result and was diluted with a normal sample to mimic various percentages (100%, 50%, 25%, 10%, 5%, 2%). For both PB and BM specimens, the rearrangements were detected down to the 10% dilution and the copy number changes were detected down to 25%. Based on these cutoffs, all 50 abnormal samples (25 PB, 25 BM) used for the validation were 100% concordant with abnormalities targeted by the panel that were previously detected by FISH and/or karyotype. In addition, MPseq identified additional abnormalities not detected by karyotype or FISH and further characterized several complex abnormalities. One example includes a case with MLLT10/MLL(KMT2A) fusion detected by MPseq which was missed by the AML FISH panel because the rearrangement involved an insertional translocation. Another example is the identification of BCR/FGFR1 fusion in a case when AML FISH identified three copies of BCR.In conclusion, we validated a whole genome mate pair sequencing targeted panel to detect diagnostic/prognostic chromosomal rearrangements and copy number changes in patients with AML. Due to the limitations in detecting very low level abnormalities, MPseq would not be recommended for follow-up post therapy or minimal residual disease testing unless the coverage is increased with deeper sequencing. However, our validation study demonstrate the clinical utility of MPseq as a potential replacement assay to conventional FISH on diagnostic AML samples and highlight the increased diagnostic yield. Thus, MPseq represents a next generation sequencing technology that has the potential to revolutionize the diagnosis of hematologic malignancies and provide an opportunity to advance precision medicine. DisclosuresNo relevant conflicts of interest to declare.