Copy-Neutral Loss-of-Heterozygosity Adds Diagnostic and Prognostic Information to the Molecular Profiling of Hematological Malignancies

Abstract Background: Copy-neutral loss-of-heterozygosity (CN-LOH) - not detectable by chromosome banding analysis - is gaining importance as a prognostic factor and can either cause the duplication of an activating mutation in an oncogene, the deletion of a tumor suppressor gene or the gain/loss of specific methylated regions. However, examination for possible CN-LOH in hematological diagnostics is at present not routinely performed and, hence, data regarding the occurrence of CN-LOH across different entities as well as the association of relevant genes is limited. Aim: (1) Frequency assessment of CN-LOH by target enrichment sequencing (TES) in a diagnostic setting, (2) evaluation of whole genome sequencing (WGS) data to estimate the prevalence of CN-LOH in a larger cohort, to pinpoint relevant genes for CN-LOHs with so far unknown associations, and to determine cross-entity variability. Patients and Methods: 1196 patients (507 female, 689 male, median age: 66 years), sent between 04/2021-07/2021 for diagnostic work-up, were analyzed by TES with a median coverage of 1765x for the gene panel and 52x for the CNV spike-in panel (IDT, Coralville, IA). Amplification-free WGS libraries of 3851 different patients were sequenced with a median coverage of 102x. Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner). Cnvkit (v 0.9.9) was used to call copy number variations (CNVs) and CN-LOH for TES and HadoopCNV (Yang et al. 2017) was used to call CN-LOH for WGS. Results: 1196 patients were analyzed by TES. For 10% of the patients at least one CN-LOH event was detected without any association to age or gender but a slightly higher incidence in myeloid compared to lymphoid neoplasms (10% vs 6%). In 14 patients, CN-LOH affected more than one chromosome arm. CN-LOH occurred most frequently in 4q (n = 15), 7q (n = 16), 9p (n = 25) and 11q (n = 10). As expected, 4q CN-LOH co-occurred with high variant allele frequencies (VAF) of TET2. Based on WGS data, 4q CN-LOH occurred predominately in AML (35%), CMML (22%), and MDS (20%). In rare cases, 4q CN-LOH was associated with FBXW7 variants in T-ALL. 7q CN-LOH occurred nearly exclusively in myeloid neoplasms (95%) and was associated with high VAFs in EZH2 in 69% of TES and 82% of WGS cases. CUX1 variants with high VAFs were detected in 80% (TES) and 45% (WGS) of the remaining cases, respectively. The well-known 9p CN-LOH led to JAK2V617F homozygosity in all myeloid neoplasms and occurred most often in MPNs. In T-ALL, regions of 9p CN-LOH harbored CDKN2A/B deletions. 11q CN-LOH occurred more often in myeloid than lymphoid neoplasms (79% vs 21%) and was associated with CBL variants in 61% and KMT2A-PTD in 19% of the cases. In contrast, ATM was the relevant gene in all lymphoid cases with 11q CN-LOH. CN-LOH in 11p was detected less frequently and only in 25% of cases an association with WT1 variants could be identified. Our WGS data confirmed the known associations between 1p CN-LOH and high allele burden in MPL, CSF3R and NRAS, 2p CN-LOH and DNMT3A variants, 13q CN-LOH and FLT3-ITD, the near exclusive occurrence of 16p CN-LOH in follicular lymphoma (FL, 98%) with high CREBBP-mutant allele burden , 17p CN-LOH and TP53 homozygosity, and the exclusive occurrence of 21q CN-LOH in AML and its association with RUNX1 mutations. Besides, 12q CN-LOH was associated with KMT2D in FL, with SH2B3 in MDS/MPN overlaps and in rare cases with KDM2B. For 17q CN-LOH the relevant gene was not unequivocally identifiable with high mutant allele variants in SRSF2, STAT5B, and NF1. 18q CN-LOH was a very rare event but consistently associated with a high VAF of MBD2, which presumably influences cell proliferation (Cheng et al. 2018). 19q CN-LOH was mostly (63%) associated with a high VAF of CEBPA variants, except for patients with hairy cell leukemia: in these cases nonsense mutations in CIC (VAF > 90%) were detected. CN-LOH in 22q was more common in myeloid malignancies (65% vs 35%) and associated with PRR14L mutations in the majority of myeloid cases (62%). Of note, this association occurred neither in AML samples nor in lymphoid neoplasms. No recurrent mutations were found for 6p and 14q CN-LOHs. For all other chromosomes, CN-LOH events were very rare. Conclusions: By using a CNV spike-in panel, TES adds additional diagnostic and prognostic information by enabling simultaneous detection of selected gene mutations and genome-wide CNVs, as well as CN-LOH, without increase in sequencing costs and turn-around times. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

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Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms

Blood ◽

10.1182/blood-2013-02-486050 ◽

2013 ◽

Vol 121 (21) ◽

pp. 4388-4395 ◽

Cited By ~ 65

Author(s):

Elisa Rumi ◽

Daniela Pietra ◽

Paola Guglielmelli ◽

Roberta Bordoni ◽

Ilaria Casetti ◽

...

Keyword(s):

Loss Of Heterozygosity ◽

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Neutral Loss ◽

Molecular Event ◽

Allele Burden ◽

Chromosome 1P ◽

Key Points ◽

Exon 10 ◽

Marrow Fibrosis

Key Points In MPL exon 10–mutated myeloproliferative neoplasms, the MPL-mutant allele burden varies considerably from about 1% to almost 100%. High mutation burdens originate from acquired copy-neutral loss of heterozygosity of chromosome 1p and are associated with marrow fibrosis.

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Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases

Oncohematology ◽

10.17650/1818-8346-2020-15-2-85-91 ◽

2020 ◽

Vol 15 (2) ◽

pp. 85-91

Author(s):

T. N. Subbotina ◽

I. E. Maslyukova ◽

A. A. Faleeva ◽

P. A. Nikolaeva ◽

A. S. Khazieva ◽

...

Keyword(s):

Mutant Allele ◽

Sanger Sequencing ◽

Quantitative Assessment ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Mutant Gene ◽

Positive Sample ◽

Allele Burden ◽

Minor Variant ◽

Allelic Burden

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

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Antibody Response to Vaccination with BNT162b2, mRNA-1273, and ChADOx1 in Patients with Myeloid and Lymphoid Neoplasms

Blood ◽

10.1182/blood-2021-146832 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 218-218

Author(s):

Jil Rotterdam ◽

Margot Thiaucourt ◽

Juliana Schwaab ◽

Andreas Reiter ◽

Sebastian Kreil ◽

...

Keyword(s):

Antibody Response ◽

Board Of Directors ◽

Research Funding ◽

Low Grade ◽

Hematological Disease ◽

Advisory Committees ◽

Hematological Diseases ◽

Myeloid Neoplasms ◽

Lymphoid Neoplasms ◽

Treatment Naïve

Abstract Background: In general, patients with hematological diseases are predisposed to develop infections. Severe COVID-19 infection associated with high mortality is more likely in these patient cohorts compared to the general population. Due to immune defects related to the primary disease and/or to immunosuppressive treatment regimes, vaccination efficacy may be reduced in patients with hematological diseases. So far, data on this area are limited. Aim: To evaluate vaccination-related antibody response to BNT162b2, mRNA-1273, and ChADOx1 in patients with hematological disorders. Patients and methods: In this interim analysis of a prospective, observational single-center study, we report antibody levels at least 2 weeks after COVID-19 vaccination. A FDA/CE approved electrochemiluminescent assay (ECLIA) (Elecsys®, Roche, Mannheim, Germany) was used to quantify antibodies, pan Ig (including IgG) against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The assay has a measurement range of 0.4 to 250 U/mL, with a concentration ≥0.8 U/ml considered as positive. Data were analyzed for patients without detection of anti-N (nucleocapsid) SARS-CoV-2 antibody (i.e., without having passed SARS-CoV-2 infection). All tests were performed according to the manufacturer's instructions in an accredited laboratory at the University Hospital Mannheim. Results: Between February 2021 and July 2021, a total of 175 patients with hematological diseases were included in this study. The median age was 66 years (range 21-90 years), and 81 (46.3%) were female. The antibody levels were measured at least 14 days (median, 58 days) after the 2 nd vaccination. The patients were vaccinated with BNT162b2 (BioNTech, n=134), mRNA-1273 (Moderna, n=19), ChADOx1 (AstraZeneca, n=12), or got the first vaccination with BNT162b2 and the second with ChADOx1 (n=10). Overall, 145/175 (82.9%) were diagnosed with a malignant hematological disease (myeloid neoplasms, n=108; lymphoid neoplasms, n=37) and 30/175 with a non-malignant hematological disease (autoimmune disease, n=24; benign, n=6). 124 patients (70.1%) were on active therapy, and 51 patients (29.1%) were previously treated or treatment naïve. Correlation to specific therapies is ongoing and will be presented. In general, vaccination-related antibody response was positive (≥0.8 U/mL) in 148/175 (84.6%) patients with a median level of 208.6 U/mL (range 0.8-250.00) and negative (<0.8 U/mL) in 27/175 (15.4%) patients. The distribution of the negative cohort regarding the disease subgroups were as followed: myeloid neoplasms 7/27 (25.9%), lymphoid neoplasms 16/27 (59.3%), non-malignant hematological disease 4/27 (14.8%). Within the negative cohort, 21/27 (77.8%) were treated on active therapy, 6/27 (22.2%) were previously treated or treatment naïve. In myeloid neoplasms, patients with classical myeloproliferative neoplasm (MPN) had the highest negative result for antibodies with 4/7 (57.1%) followed by myelodysplastic syndrome (MDS) 2/7 (28.6%). Interestingly, all patients with chronic myeloid leukemia (CML) had a measurable immune response. In lymphoid neoplasms, patients with low-grade non-hodgkin lymphoma (NHL) (predominately chronic lymphocytic leukemia, CLL) had the highest negative antibody result 13/16 (81.3%) followed by high-grade NHL 4/8 (50%; predominately diffuse large b-cell lymphoma, DLBCL). In non-malignant hematological diseases, only patients with autoimmune diseases had a negative result. Conclusion: A remarkable group of patients with hematological disease were measured with no or low immune response after 2 nd COVID-vaccination, especially those with low-grade NHL, MDS and autoimmune disease. It seems that the percentage of patients with MPN and low response is less critical. No problems appeared in CML patients. Further explorations are needed with focus on potential risk of COVID infections despite full vaccination: The potential of 3 rd booster vaccination should be explored within clinical trials. Disclosures Reiter: AOP Orphan Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Abbvie: Membership on an entity's Board of Directors or advisory committees; Deciphera: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses. Kreil: Novartis: Research Funding. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Jawhar: Takeda: Honoraria, Other: Travel support; Blueprint Medicines: Honoraria; Stemline: Consultancy, Honoraria; Celgene: Other: Travel support; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Saussele: Roche: Honoraria; Pfizer: Honoraria; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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Recurrent Mutations within the Nfkbie gene: A Novel Mechanism for NF-κB Deregulation in Aggressive Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.297.297 ◽

2014 ◽

Vol 124 (21) ◽

pp. 297-297

Author(s):

Larry Mansouri ◽

Lesley-Ann Sutton ◽

Viktor Ljungstrom ◽

Sina Bondza ◽

Linda Arngarden ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Mutant Allele ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

B Cell Lymphomas ◽

Tlr Signaling ◽

B Cell Malignancies ◽

Recurrent Mutations ◽

Genomic Aberrations

Abstract Dysregulated NF-κB signaling appears to be particularly important in B-cell malignancies, with recurrent mutations identified within both the canonical and non-canonical NF-κB pathways, as well as in components of the B-cell receptor (BcR) and Toll-like receptor (TLR) signaling pathways. In chronic lymphocytic leukemia (CLL), although recurrent mutations have been identified in MYD88 (TLR signaling) and BIRC3 (non-canonical NF-κB pathway), their frequency is low (<3%) and hence the extent to which genetic aberrations may contribute to constitutional NF-κB activation remains largely unknown. To gain further insight into this issue, we designed a HaloPlex gene panel (Agilent Technologies) and performed targeted next-generation sequencing (NGS) (HiSeq 2000/Illumina) of 18 NF-κB genes in a discovery cohort of 124 CLL patients, intentionally biased towards poor-prognostic patients with either unmutated IGHV genes or high-risk genomic aberrations. Using a conservative cutoff of >10% for the mutant allele, we identified mutations (n=35) within 30/124 (24%) patients in 14/18 NF-κB genes analyzed. IκB genes, which encode for cytoplasmic inhibitor proteins, accounted for 20/35 (57%) mutations, with IκBε (encoded by NFKBIE) mutated in 8 patients; notably, 3/8 cases carried an identical 4bp deletion within exon 1 of NFKBIE. Prompted by these findings, we proceeded to validate our findings in an independent CLL cohort (n=168) using the same methodology as above and primarily focusing on cases with poor-prognostic features. We identified 30 mutations within 28 CLL patients in 11/18 NF-κB genes analyzed. Strikingly, 13/30 mutations were found within IκBε, with 10/13 patients carrying the same 4bp NFKBIE deletion. Notably, investigations into whether additional cases (within both the discovery and validation cohort) may harbor mutations of low clonal abundance (<10% mutant allele), led to the detection of the NFKBIE deletion in another 18 cases. Owing to the prevalence of this 4bp deletion within the NFKBIE gene, we developed a GeneScan assay and screened an additional 312 CLL cases. Collectively, 40/604 (6.6%) CLL patients were found to carry this frame-shift deletion within the NFKBIE gene, which is in line with a recent publication reporting that 10% of Binet stage B/C patients carried this mutation (Damm et al. Cancer Discovery 2014). Remarkably, the majority of these NFKBIE mutations (16/40) were found in a subgroup of patients that expressed highly similar or stereotyped BcRs and are known to have a particularly poor outcome, denoted as subset #1. This finding thus alludes to a subset-biased acquisition and/or selection of genomic aberrations, similar to what has been reported for subset #2 and SF3B1, perhaps as a result of particular modes of BcR/antigen interaction. We utilized proximity-ligation assays to test the functional impact of the NFKBIE deletion by investigating protein-protein interactions. This analysis revealed reduced interaction between the inhibitor IκBε and the transcription factor p65 in NFKBIE-deleted CLL cells; IκBε-knock-down shRNA experiments confirmed dysregulated apoptosis/NF-κB signaling. Finally, to assess whether the NFKBIE deletion could also be present in other B-cell malignancies, we screened 372 mature B-cell lymphoma cases using NGS or the GeneScan assay and found the deletion in 7/136 (5.1%) mantle cell lymphomas, 3/66 (4.5%) diffuse large B-cell lymphomas and 3/170 (1.8%) splenic marginal zone lymphomas. Taken together, our analysis revealed that inactivating mutations within the NFKBIE gene lead to NF-κB activation in CLL and potentially several other B-cell-derived malignancies. Considering the central role of BcR stimulation in the natural history of CLL, the functional loss of IκBε may significantly contribute to sustained CLL cell survival and shape the disease evolution. This novel data strongly indicates that components of the NF-κB signaling pathway may be prime targets for future targeted therapies not only in CLL but also other mature B-cell lymphomas. Disclosures No relevant conflicts of interest to declare.

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The consequences of uniparental disomy and copy number neutral loss-of-heterozygosity during human development and cancer

Biology of the Cell ◽

10.1042/bc20110013 ◽

2011 ◽

Vol 103 (7) ◽

pp. 303-317 ◽

Cited By ~ 55

Author(s):

Pablo Lapunzina ◽

David Monk

Keyword(s):

Human Development ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Neutral Loss ◽

Uniparental Disomy

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Classifying and Reporting Acquired Copy Number Variants (CNVs) and Copy Neutral Loss of Heterozygosity (CN-LOH) in Neoplastic Disorders: Joint Recommendations From the American College of Medical Genetics and Genomics (ACMG), and the Cancer Genomics Consortium (CGC)

Cancer Genetics ◽

10.1016/j.cancergen.2017.04.016 ◽

2017 ◽

Vol 214-215 ◽

pp. 36-37 ◽

Cited By ~ 1

Author(s):

Gordana Raca ◽

Jaclyn Biegel ◽

Linda Cooley ◽

Adrian Dubuc ◽

Betsy Hirsch ◽

...

Keyword(s):

Loss Of Heterozygosity ◽

Copy Number ◽

Cancer Genomics ◽

Neutral Loss ◽

Copy Number Variants ◽

Medical Genetics ◽

Genetics And Genomics ◽

Neoplastic Disorders

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A Novel Case of Extreme Thrombocytosis in Acute Myeloid Leukemia Associated With Isochromosome 17q and Copy Neutral Loss of Heterozygosity

Annals of Laboratory Medicine ◽

10.3343/alm.2015.35.3.366 ◽

2015 ◽

Vol 35 (3) ◽

pp. 366-369 ◽

Cited By ~ 1

Author(s):

Eunkyoung You ◽

Sun Young Cho ◽

John Jeongseok Yang ◽

Hee Joo Lee ◽

Woo-In Lee ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Loss Of Heterozygosity ◽

Myeloid Leukemia ◽

Neutral Loss ◽

Acute Myeloid

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Analysis of the clinical impact of NPM1 mutant allele burden in a large cohort of younger adult patients with acute myeloid leukaemia

British Journal of Haematology ◽

10.1111/bjh.16239 ◽

2019 ◽

Vol 188 (6) ◽

pp. 852-859 ◽

Cited By ~ 3

Author(s):

David C. Linch ◽

Robert K. Hills ◽

Alan K. Burnett ◽

Nigel Russell ◽

Rosemary E. Gale

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Mutant Allele ◽

Clinical Impact ◽

Large Cohort ◽

Adult Patients ◽

Allele Burden ◽

Acute Myeloid

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Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms

Blood ◽

10.1182/blood-2009-02-205690 ◽

2009 ◽

Vol 113 (25) ◽

pp. 6403-6410 ◽

Cited By ~ 252

Author(s):

Anna M. Jankowska ◽

Hadrian Szpurka ◽

Ramon V. Tiu ◽

Hideki Makishima ◽

Manuel Afable ◽

...

Keyword(s):

Loss Of Heterozygosity ◽

Chromosomal Abnormalities ◽

Myeloproliferative Neoplasms ◽

Neutral Loss ◽

Regulatory Gene ◽

Clinical Analysis ◽

Gene Marker ◽

Compound Heterozygous ◽

Single Nucleotide ◽

Myeloid Malignancies

Abstract Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number–neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders.

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