BACKGROUND:Clonotyping of immunoglobulin heavy chain (IGH) gene rearrangements is critical for diagnosis, prognostication, and measurable residual disease monitoring in chronic lymphocytic leukemia (CLL). Although short-read next-generation sequencing (NGS) platforms, such as Illumina MiSeq, are widely used, they face challenges in spanning full VDJ rearrangements. Long-read sequencing via Oxford Nanopore Technologies (ONT) offers a potential alternative using the compact and cost-effective flow cells. METHODS:We evaluated IGH clonotyping in samples from 13 CLL patients using ONT MinION with Flongle flow cells and super-accuracy GPU-based base calling (Dorado), comparing results to MiSeq-based LymphoTrack assays. Amplicons were generated using the IGH FR1 assay and analyzed using Smith-Waterman alignment and IgBlast tools. RESULTS:All major and minor clonotypes identified by MiSeq were matched with 100% sequence identity using Nanopore sequencing. Clonal burden estimates were strongly correlated (Pearson ρ = 0.87, p < 10-4), although with considerable variability. Somatic hypermutation status was reliably assessed with super-accuracy base calling (Q30: 76%). Fast or CPU-only base calling was insufficient for accurate mutation analysis. CONCLUSION:Nanopore sequencing enables accurate and rapid IGH clonotyping in CLL, offering comparable performance to MiSeq with lower cost and laboratory footprint. This supports its potential utility in routine and decentralized hematopathology workflows.

Myeloma bone disease (MBD), a common complication in multiple myeloma (MM), causes increased risk of fractures leading to morbidity and impaired quality of life for patients. Proteasome inhibitors, a cancer-targeting therapy for MM, have been shown to have a beneficial off-target bone anabolic effect. However, side effects and toxicities of proteasome inhibitors limits their long-term use, especially in patients in disease remission. Ixazomib is an oral proteasome inhibitor with anti-myeloma effect, but a less severe toxicity profile compared to other approved proteasome inhibitors. To investigate the effect of ixazomib on MBD, we conducted a single-center clinical study where 30 patients with MM in remission received ixazomib, and evaluated bone-specific changes through serum markers, imaging, cell cultures, and bone histomorphometry. We have previously shown that short-term ixazomib treatment (3 months) induces increased trabecular bone volume and formation of enlarged bone structural units (BSU) without changing osteoblast number or activity. Here, we present evidence that long-term (24 months) ixazomib treatment inhibits the activation of new bone remodeling events through attenuation of both bone resorption and formation. The initial gains in percentage of superficial trabecular BSU bone volume remained stable and the proportion of large BSUs containing woven bone decreased, suggesting improved bone mineralization over time. Overall, our results indicate that long-term ixazomib treatment led to prolonged bone formation events during the initial treatment phase, followed by inhibition of new bone resorption and its coupled bone formation, preserving the gained bone and possibly preventing advancement of MBD in patients with MM in remission. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov ID NCT04028115.

Abstract: Positron emission tomography–computed tomography (PET-CT) is recommended for response evaluation in aggressive large B-cell lymphoma (LBCL) but cannot detect minimal residual disease (MRD). Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for real-time disease monitoring. This study evaluated longitudinal ctDNA monitoring as an MRD marker in LBCL. In this prospective, single-center study, 14 newly diagnosed patients with LBCL receiving first-line immunochemotherapy underwent frequent longitudinal blood sampling. A 53-gene targeted sequencing panel quantified ctDNA and evaluated its kinetics, correlating it with clinical parameters and PET-CT, including total metabolic tumor volume (TMTV) calculated using artificial intelligence–based analysis via RECOMIA. Baseline ctDNA was detected in 11 of 14 patients (79%) with a median variant allele frequency of 6.88% (interquartile range, 1.19%-10.20%). ctDNA levels correlated significantly with TMTV (ρ = 0.90; P < .0001) and lactate dehydrogenase. ctDNA kinetics, including after 1 treatment cycle, mirrored PET-CT metabolic changes and identified relapsing or refractory patients. This study demonstrates ctDNA-based MRD monitoring in LBCL using a fixed targeted assay with an analytical sensitivity of at least 10−3. The kinetics of ctDNA reflects the clinical course and PET-CT findings, underscoring its complementary potential to PET-CT.

Patients with chronic lymphocytic leukemia (CLL) undergoing ibrutinib treatment often experience incomplete response, yet the molecular level underlying clonal inertia remains to be explored. We investigated the molecular and clinical dynamics of CLL during 16 months of ibrutinib monotherapy by analyzing blood samples from two patients who continued having CLL cells in the peripheral blood during treatment. At diagnosis, the clonal burden within the B cell compartment was found to be 55% (pt1) and 86% (pt2) for the dominant clones. At 16 months following treatment these clones still constituted 66% and 89%, respectively. Utilizing multi-omic methodologies at the DNA and RNA levels, including single-cell transcriptomics, we aimed to establish a comprehensive framework for multi-omics analysis for longitudinal ibrutinib response evaluation. The presented study revealed genomically stable disease during ibrutinib treatment, but with intensified expression of genes involved in pathways related to apoptosis, cellular stress response, and canonical NF-κB signaling from diagnosis to 16 months of treatment.

INTRODUCTION:Formalin-fixed paraffin-embedded (FFPE) tumor biopsy is the current mainstay of genotyping, but is limited by its invasiveness and tumor heterogeneity. Plasma cell-free DNA (cfDNA) constitutes a minimally invasive alternative that may better capture tumor-derived profiles from circulating tumor DNA (ctDNA). This study compares the performance and genomic concordance of cfDNA and FFPE tumor DNA in aggressive non-Hodgkin large B-cell lymphoma. METHODS:Paired diagnostic FFPE tissue and plasma samples from 15 patients were sequenced with a custom 53-gene panel. RESULTS:Detection thresholds were empirically guided at 1% variant allele frequency (VAF) for cfDNA and 10% for unpaired FFPE DNA. The median number of cfDNA variants was 6 (interquartile range (IQR): 2-11) versus 63 (IQR: 15-250) in FFPE DNA at 1% VAF. Collectively, 102 somatic variants were shared between cfDNA and FFPE DNA with a median of 5 (range: 0-23). cfDNA showed a five-fold lower median VAF for shared variants than FFPE DNA (7% vs. 36%, p < 0.0001). Eighty percent of patients harbored at least one cfDNA variant. A maximum cfDNA recall rate of 83% was observed at FFPE DNA VAF > 50%. COSMIC database overlap was twice as high for cfDNA compared to FFPE DNA (22% vs. 11%) at 10% VAF. CONCLUSION:cfDNA has superior specificity for somatic mutation detection but lower sensitivity than FFPE DNA. Modest concordance was demonstrated between the two compartments. Our results support a complementary role of ctDNA in mutational profiling at a 1% VAF threshold in a pragmatic and clinically applicable setup.

IntroductionDiffuse large B-cell lymphoma (DLBCL) exhibits striking clinical and biological heterogeneity. Recent studies have identified new subgroups within germinal center B-cell like (GCB) DLBCL, associated with inferior prognosis, irrespective of MYC and BCL2 translocations. We explored the existence of such a DLBCL high-risk subgroup, based on multilevel aberrations, especially focusing on MYC and BCL2.MethodsTissue samples from 111 DLBCL patients were sequenced with a 90-gene lymphoma panel, followed by integrative analyses combining sequencing data, immunohistochemistry, fluorescent in situ hybridization, and clinical data.ResultsWe identified a high-risk subgroup in DLBCL defined by: dual immunohistochemical MYC and BCL2 expression (DEL), concurrent MYC and BCL2 translocations (DHL-BCL2), mutations in MYC, CXCR4, or both, and/or BCL2 amplification. The high-risk subgroup constituted 41% of the cohort and included DHL-BCL2, DEL, a GCB subgroup likely representing the recently described GCB subgroups, and a subset of non-GCB patients. In multivariate analysis, high-risk features provided independent predictive value from age and IPI. The 5-year overall survival was 36% in high-risk patients, compared to 76% in non-high-risk patients.ConclusionWe identified a distinct high-risk DLBCL subgroup, characterized by MYC and BCL2 aberrations, beyond conventional DHL-BCL2 and DEL, and irrespective of cell-of-origin, thereby expanding the poor-prognosis group.

The approach to managing chronic lymphocytic leukemia (CLL) has changed considerably. Treatment strategies vary, ranging from watchful waiting in the initial stages to Bruton tyrosine kinase (BTK) inhibitor monotherapy, chemo-immunotherapy, or stem cell transplantation in advanced stages of the disease. A notable aspect of BTK inhibitors, such as ibrutinib, is the phenomenon of prolonged lymphocytosis, a condition shown not to impact survival rates. Despite a high response rate, complete molecular remission is infrequent with ibrutinib monotherapy (Strati & al. 2020), unlike cases treated with venetoclax, rituximab, or combinations thereof (Roberts & al. 2015). This persistent residual lymphocytosis during ibrutinib treatment does not indicate a lack of response or disease progression (Herman & al. 2014; Barrientos & al. 2019). Because the extent of this clonal inertia remains to be thoroughly investigated at the molecular level, this methodological study investigated a multi-omics approach to profile the clonal, molecular CLL architecture from treatment initiation and response follow-up. Methods In this proof-of-concept study, we characterized purified B cells at diagnosis and follow-up (8 and 16 months) from two patients (pt.) with CLL, unmutated IGHV, receiving ibrutinib as first-line treatment. Blood samples showed a lymphocyte count of 127 and 20.9·109/L. Clonotyping using rearranged IGH locus was done at all three time points, averaging 0.6 million (M) reads. Whole-exome (90 M reads), full-transcript mRNA (63 M reads), and single-cell sequencing (~56000 cells) were performed at the time of diagnosis and last follow-up. Results The mono-therapeutic intervention reduced peripheral lymphocytes to 3.67-6.86·109/L at 8 months and 3.66-8.87·109/L at 16 months follow-up after ibrutinib initiation. Only minor changes of the clonal CLL burden within the B-cell compartment were found from 54.80% clonotypic rearrangements (pt. 1, IGHV3-23*04 J4*02) at diagnosis and 60.12 to 66.81% at follow-up. For pt. 2 (IGHV1-69*13 J6*02), this was 86.41% at diagnosis, followed by 85.96 to 88.62% at the time of follow-up. Genomic lesions were confirmed transcriptionally, showing archetypical NOTCH1, SF3B1 point mutations, and ASXL1frameshift in C-terminal exon 12/13. Also, allelic and chromosomal imbalances from 13q14 deletions were observed in both samples at the DNA level, in agreement with cytogenetics. Additionally, mutated TP53, loss of 10q23, monoallelic deletion of ATM (11q21-q24), and the IGH locus from 14q32 were found for pt. 2. At the molecular level, pt. 2, several factors contribute to a high-risk profile. However, both patients were molecularly stable nearly 1.5 years after initiating ibrutinib treatment. In summary, a direct correlation between putative somatic variants was found between diagnosis and relapse (ρPearson=0.91, Pt-statistics=0.0001) with a variant overlap of 82% (132/162, >0.05 variant allele frequency, VAF). No significant change in VAF from diagnosis to follow-up was observed for DNA or RNA (PU-test=0.92 and PU-test=0.79). As no clonal progression was indicated by at the DNA level, we turned to single-cell resolution of the B cells compartment, showing an expressional shift from baseline to follow-up in biological pathways related to apoptosis, cellular response to stress, and canonical NFKB signaling transduction specific with significantly increased expression profiles at follow-up. Bulk RNA sequencing of the coding transcriptome quantitatively confirmed this increased expression of apoptotic markers. Conclusion We show that the CLL burden in the B-cell compartments remains high in either case, with no indications of clonal evolution, progression, or clearance of lymphocytosis at the molecular level. Collectively, striking clonal concordance and stability are evident, genomically and transcriptionally. Thus, we extend the early findings of Byrd & al., Woyach & al., and others, showing that lymphocytosis is a feature of ibrutinib. However, the results also show a molecular persistency of this lymphocytosis with precision for follow-up evaluation, with relevance for high-risk profiles. We are currently applying this methodology to profile a cohort to 3.5 years of follow-up.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells due to constitutive B-cell receptor (BCR) signaling, leading to apoptosis resistance and increased proliferation. This study evaluates the effects of the Bruton Tyrosine Kinase (BTK) inhibitor ibrutinib on the molecular composition, clonality, and kinetics of B cells during treatment in CLL patients. Employing a multi-omics approach of up to 3.2 years of follow-up, we analyzed data from 24 CLL patients, specifically focusing on nine patients treated with ibrutinib monotherapy. In this study, clonal stability was observed within the ibrutinib-treated group following an effective initial clinical response, where clonotype frequencies of residual CLL cells remained high and stable, ranging from 74.9% at 1.5 years to 87.7% at approximately 3 years. In contrast, patients treated with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax exhibited substantial reductions in clonal frequencies, approaching molecular eradication. Deep whole-exome sequencing revealed minimal genomic progression in the ibrutinib group, maintaining somatic drivers and variant allele frequencies (VAF) above 0.2 throughout treatment. At the single-cell level, the NF-κB pathway inhibition and apoptotic signals were detected or even augmented during treatment in ibrutinib-treated patients. These findings may corroborate the role of ibrutinib in stabilizing the genomic landscape of CLL cells, preventing significant genomic evolution despite maintaining a high clonal burden within the residual B-cell compartment.

We present a lightweight tool for clonotyping and measurable residual disease (MRD) assessment in monoclonal lymphoproliferative disorders. It is a translational method that enables computational detection of rearranged immunoglobulin heavy chain gene sequences.•The swigh-score clonotyping tool emphasizes parallelization and applicability across sequencing platforms.•The algorithm is based on an adaptation of the Smith-Waterman algorithm for local alignment of reads generated by 2nd and 3rd generation of sequencers.For method validation, we demonstrate the targeted sequences of immunoglobulin heavy chain genes from diagnostic bone marrow using serial dilutions of CD138+ plasma cells from a patient with multiple myeloma. Sequencing libraries from diagnostic samples were prepared for the three sequencing platforms, Ion S5 (Thermo Fisher Scientific), MiSeq (Illumina), and MinION (Oxford Nanopore), using the LymphoTrack assay. Basic quality filtering was performed, and a Smith-Waterman-based swigh-score algorithm was developed in shell and C for clonotyping and MRD assessment using FASTQ data files. Performance is demonstrated across the three different sequencing platforms.

Background: Diffuse large B-cell lymphoma (DLBCL) harbors striking clinical and molecular heterogeneity, which continues to encumber risk stratification with a consequently high rate of refractory/relapsed patients (pts). Though the genomic landscape of DLBCL has been comprehensively uncovered, the search for eligible molecular biomarkers continues. Exploratively, we set out to retrospectively evaluate differential mutational distributions in a DLBCL cohort using targeted sequencing. Patients and method: A cohort of 105 de novo DLBCL pts, uniformly treated with first-line R-CHOP, were studied. Thirty pts relapsed, of which 70% had early relapses (<2 years after completing therapy), while 30% had late relapses (≥2 years). Targeted next-generation sequencing (90 gene panel) was applied on archival paraffin-embedded diagnostic samples and, if available, first relapse samples. Results: In total, 118 samples were sequenced, each displaying a median of 12 (range 1–46) putative somatic protein-coding mutations. Germinal center B-cell (GCB) DLBCL (n = 60) displayed a differential somatic signature with enrichment of EZH2, TNFRSF14, BCL2, ACTB, SOCS1, and FAS mutations (Fisher's Exact, p < 10-4), whereas mutations in CDKN2A, PRDM1, MYD88, and CD79B were significantly enriched in non-GCB pts (p < 10-4, n = 44). Survival analysis confirmed inferior overall survival (OS) in non-GCB DLBCL (5-year OS, log-rank (Mantel-Cox), p = 0.01, hazard ratio (HR) 1.7). Pts with double-hit biology (DHB), defined as either MYC and BCL2 and/or BCL6 fluorescent in situ hybridized positive and/or with MYC and BCL2 immunohistochemical double expression (n = 32), did not display a significant mutational signature or differential OS. However, a subgroup of DHB pts characterized by mutations in MYC, PRDM1, or IRF4 (n = 15) had markedly inferior OS (5-year OS, log-rank, p = 0.0003, HR 3.34) compared to negative non-DHB pts (n = 55) or negative non-DHB/DHB pts (p = 0.0017, HR 2.7, n = 72). Noticeably, ACTB mutations were only present in non-DHB pts. The mutational distribution at diagnosis did not differ between pts with relapse and in sustained remission. However, in the relapse group, GNA13 mutation at diagnosis was more frequent in early relapses than late relapses (43% vs. 0%, Fisher’s Exact without correction, p = 0.03). The presence of GNA13 in the entire cohort showed a trend towards inferior survival. Conclusion: Differential distribution with significant mutational signatures were expectedly found in GCB versus non-GCB, but not in subgroups with DHB or relapse. Interestingly, mutations in MYC, PRDM1 or IRF4 identified a distinct subset of DHB pts with inferior survival. Furthermore, GNA13 mutations were significantly enriched in early relapse pts. These preliminary findings call for validation of GNA13 as a potential marker of early relapse as well as the prognostic value of MYC, PRDM1 and IRF4 mutations in DHB DLBCL. The research was funded by: The Danish Cancer Society and The Vissing Foundation Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, tumor biology and heterogeneity Conflicts of interests pertinent to the abstract T. S. Larsen Consultant or advisory role Roche, Gilead, Novartis, Celgene/BMS Research funding: Genentech

Multiple myeloma, a mature B-cell neoplasm, is the second most common hematologic malignancy. Despite advancements in treatment, the disease remains incurable, with more than 100,000 annual deaths worldwide. As recommended by the International Myeloma Working Group, measurable residual disease (MRD) should be addressed at a 10-5 sensitivity level or beyond for practical purposes. Next-generation sequencing (NGS) has provided new opportunities with deep sequencing of clonal rearrangements of the immunoglobulin heavy chain (IGH) locus in B-cell malignancies. Although the ability to resolve one cancerous cell in a million other B cells is becoming attractive as a prognostic indicator in sustained patients who are MRD-negative, reaching consistent sensitivity levels is challenging because of sample stochasticity and the substantial amount of deoxyribonucleic acid (DNA) required for library preparation. Thus, in the presented study, we implemented ultra-deep sequencing of rearranged IGH to investigate the reproducibility and consistency aimed at the 10-5 sensitivity level. In this controlled setup, our data provided stable MRD detection of 1.2 clonal cells per 100,000 analyzed cells and lon-gitudinal reproducibility. We also demonstrated a low false-negative rate using 4-5 replicates and 700-800 ng DNA per sequencing replicate. In conclusion, adding an internal control to the replicates enabled clonal cell nor-malization for MRD evaluation as a stable reference. These findings may guide MRD-level reporting and com-parisons between laboratories.(c) 2023 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Detection of t(11;14)(q13;q32), a hallmark of mantle cell lymphoma (MCL), remains challenging when relying on next-generation sequencing (NGS) in contrast to fluorescence in-situ hybridization. First, the breakpoint locations vary from patient to patient. Secondly, the translocation does not result in a chimeric fusion protein and can, thus, not be detected by RNA sequencing or PCR of cDNA. Furthermore, the breakpoint does not alter the gene or coding region of CCND1 but deregulates transcriptional regulation kilobases upstream.

Osteolytic bone disease is present in about 80% of patients with multiple myeloma at the time of diagnosis. Managing bone disease in patients with multiple myeloma is a challenge and requires a multi-faceted treatment approach with medication, surgery, and radiation. The established treatments with intravenous or subcutaneous antiresorptives can cause debilitating adverse events for patients, mainly osteonecrosis of the jaw, which, traditionally, has been difficult to manage. Now, oral surgery is recommended and proven successful in 60–85% of patients. Patients with spinal involvement may benefit from surgery in the form of vertebroplasty and kyphoplasty for pain relief, improved mobility, and reestablished sagittal balance, as well as the restoration of vertebral height. These procedures are considered safe, but the full therapeutic impact needs to be investigated further. Ixazomib, the first oral proteasome inhibitor, increases osteoblast differentiation, and recently published preliminary results in patients treated with Ixazomib maintenance have promisingly shown increased trabecular volume caused by prolonged bone formation activity. Other novel potential treatment strategies are discussed as well.

The current advances and success of next-generation sequencing hold the potential for the transition of cancer cytogenetics toward comprehensive cytogenomics. However, the conventional use of short reads impedes the resolution of chromosomal aberrations. Thus, this study evaluated the detection and reproducibility of extensive copy number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies whole-genome sequencing compared with short-read Illumina sequencing. Using the mantle cell lymphoma cell line Granta-519, almost 99% copy-number reproducibility at the 100-kilobase resolution between replicates was demonstrated, with 98% concordance to Illumina. Collectively, the performance of copy number calling from 1.5 million to 7.5 million long reads was comparable to 1 billion Illumina-based reads (50x coverage). Expectedly, the long-read resolution of canonical translocation t(11;14)(q13;q32) was superior, with a sequence similarity of 89% to the already published CCND1/IGH junction (9x coverage), spanning up to 69 kilobases. The cytogenetic profile of Granta-519 was in general agreement with the literature and karyotype, although several differences remained unresolved. In conclusion, contemporary long-read sequencing is primed for future cytogenomics or sequencing-guided cytogenetics. The combined strength of long-and short-read sequencing is apparent, where the high-precision junctional mapping complements and splits paired-end reads. The potential is emphasized by the flexible single-sample genomic data acquisition of Oxford Nanopore Technologies with the high resolution of allelic imbalances using Illumina short-read sequencing.

The current advances and success of next-generation sequencing hold the potential for the transition of cancer cytogenetics toward comprehensive cytogenomics. However, the conventional use of short reads impedes the resolution of chromosomal aberrations. Thus, this study evaluated the detection and reproducibility of extensive copy number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies whole-genome sequencing compared with short-read Illumina sequencing. Using the mantle cell lymphoma cell line Granta-519, almost 99% copy-number reproducibility at the 100-kilobase resolution between replicates was demonstrated, with 98% concordance to Illumina. Collectively, the performance of copy number calling from 1.5 million to 7.5 million long reads was comparable to 1 billion Illumina-based reads (50× coverage). Expectedly, the long-read resolution of canonical translocation t(11;14)(q13;q32) was superior, with a sequence similarity of 89% to the already published CCND1/IGH junction (9× coverage), spanning up to 69 kilobases. The cytogenetic profile of Granta-519 was in general agreement with the literature and karyotype, although several differences remained unresolved. In conclusion, contemporary long-read sequencing is primed for future cytogenomics or sequencing-guided cytogenetics. The combined strength of long- and short-read sequencing is apparent, where the high-precision junctional mapping complements and splits paired-end reads. The potential is emphasized by the flexible single-sample genomic data acquisition of Oxford Nanopore Technologies with the high resolution of allelic imbalances using Illumina short-read sequencing. The current advances and success of next-generation sequencing hold the potential for the transition of cancer cytogenetics toward comprehensive cytogenomics. However, the conventional use of short reads impedes the resolution of chromosomal aberrations. Thus, this study evaluated the detection and reproducibility of extensive copy number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies whole-genome sequencing compared with short-read Illumina sequencing. Using the mantle cell lymphoma cell line Granta-519, almost 99% copy-number reproducibility at the 100-kilobase resolution between replicates was demonstrated, with 98% concordance to Illumina. Collectively, the performance of copy number calling from 1.5 million to 7.5 million long reads was comparable to 1 billion Illumina-based reads (50× coverage). Expectedly, the long-read resolution of canonical translocation t(11;14)(q13;q32) was superior, with a sequence similarity of 89% to the already published CCND1/IGH junction (9× coverage), spanning up to 69 kilobases. The cytogenetic profile of Granta-519 was in general agreement with the literature and karyotype, although several differences remained unresolved. In conclusion, contemporary long-read sequencing is primed for future cytogenomics or sequencing-guided cytogenetics. The combined strength of long- and short-read sequencing is apparent, where the high-precision junctional mapping complements and splits paired-end reads. The potential is emphasized by the flexible single-sample genomic data acquisition of Oxford Nanopore Technologies with the high resolution of allelic imbalances using Illumina short-read sequencing. Cytogenetics has been a mainstay of laboratory hematology for half a century; its success is partly owing to highly recurrent aberrations, such as the Philadelphia chromosome, t(9;22)(q34;q11), in chronic myeloid leukemia, t(14;18)(q32;q21) detected in follicular lymphoma, and t(11;14)(q13;q32) detected in mantle cell lymphoma (MCL). The large structural variants provide diagnostic handles and hold prognostic information, with TP53 loss, del(17p) being one of the most prominent examples of acquired copy number alterations (CNAs). The importance of cytogenetic analyses is emphasized by most hematologic malignant cancers carrying structural aberrations in one or more chromosomes. For example, this includes approximately half of all chronic lymphocytic leukemia cases based on chromosome 13q alone. As many as four of five patients with chronic lymphocytic leukemia may carry large structural variants1Dohner H. Stilgenbauer S. Benner A. Leupolt E. Krober A. Bullinger L. Dohner K. Bentz M. Lichter P. Genomic aberrations and survival in chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2814) Google Scholar in one or more chromosomes, and structural variants account for virtually all MCL cases. A complex karyotype is the strongest predictor of refractoriness and inferior outcome in both entities.2Thompson P.A. O'Brien S.M. Wierda W.G. Ferrajoli A. Stingo F. Smith S.C. Burger J.A. Estrov Z. Jain N. Kantarjian H.M. Keating M.J. Complex karyotype is a stronger predictor than del(17p) for an inferior outcome in relapsed or refractory chronic lymphocytic leukemia patients treated with ibrutinib-based regimens.Cancer. 2015; 121: 3612-3621Crossref PubMed Scopus (199) Google Scholar,3Sarkozy C. Terre C. Jardin F. Radford I. Roche-Lestienne C. Penther D. Bastard C. Rigaudeau S. Pilorge S. Morschhauser F. Bouscary D. Delarue R. Farhat H. Rousselot P. Hermine O. Tilly H. Chevret S. Castaigne S. Complex karyotype in mantle cell lymphoma is a strong prognostic factor for the time to treatment and overall survival, independent of the MCL international prognostic index.Genes Chromosomes Cancer. 2014; 53: 106-116Crossref PubMed Scopus (56) Google Scholar However, many cytogenetic laboratory analyses are based on specifically targeted regions or use microarrays that do not reveal translocations. In contrast, next-generation and third-generation whole-genome sequencing approaches offer consolidation by eliminating investigations of targeted regions, thereby potentially leveraging the field of cytogenetics toward cytogenomics. Whole-exome and whole-genome short-read sequencing can already be implemented to detect copy number variation and other chromosomal lesions. However, long-read third-generation sequencing, such as that provided by Oxford Nanopore Technologies (ONT; Oxford, UK), provides a clear potential to decrease false-positive large structural variant detection. Several computational strategies exist, such as identifying anomalies in insert sizes or interchromosomal mapping of paired-end short reads. However, employing long reads enables a possible severalfold increase in detectable structural variants4English A.C. Salerno W.J. Hampton O.A. Gonzaga-Jauregui C. Ambreth S. Ritter D.I. Beck C.R. Davis C.F. Dahdouli M. Ma S. Carroll A. Veeraraghavan N. Bruestle J. Drees B. Hastie A. Lam E.T. White S. Mishra P. Wang M. Han Y. Zhang F. Stankiewicz P. Wheeler D.A. Reid J.G. Muzny D.M. Rogers J. Sabo A. Worley K.C. Lupski J.R. Boerwinkle E. Gibbs R.A. Assessing structural variation in a personal genome-towards a human reference diploid genome.BMC Genomics. 2015; 16: 286Crossref PubMed Scopus (109) Google Scholar or offers complementary confirmation of germline susceptibility variants.5Thibodeau M.L. O'Neill K. Dixon K. Reisle C. Mungall K.L. Krzywinski M. Shen Y. Lim H.J. Cheng D. Tse K. Wong T. Chuah E. Fok A. Sun S. Renouf D. Schaeffer D.F. Cremin C. Chia S. Young S. Pandoh P. Pleasance S. Pleasance E. Mungall A.J. Moore R. Yip S. Karsan A. Laskin J. Marra M.A. Schrader K.A. Jones S.J.M. Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing.Genet Med. 2020; 22: 1892-1897Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Unfortunately, higher error rates6Mahmoud M. Gobet N. Cruz-Davalos D.I. Mounier N. Dessimoz C. Sedlazeck F.J. Structural variant calling: the long and the short of it.Genome Biol. 2019; 20: 246Crossref PubMed Scopus (276) Google Scholar and inaccessibility of the method have been an issue for long-read sequencing. Thus, sequencing in cancer research is yet slightly biased toward detecting short nucleotide variants, such as somatic point mutations and insertions/deletions. For more information on the development and technology behind single-molecule, real-time, long-read sequencing and structural variant detection, see reviews by Rovigatti7Rovigatti U. Cancer modelling in the NGS era - part I: emerging technology and initial modelling.Crit Rev Oncol Hematol. 2015; 96: 274-307Crossref PubMed Scopus (11) Google Scholar and Sakamoto et al.8Sakamoto Y. Zaha S. Suzuki Y. Seki M. Suzuki A. Application of long-read sequencing to the detection of structural variants in human cancer genomes.Comput Struct Biotechnol J. 2021; 19: 4207-4216Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Mantle cell lymphoma is a hematologic malignant cancer marked by a wide span of genomic aberrations of somatic mutations and acquired copy number alterations and translocations. Detection of t(11;14)(q13;q32), a diagnostic hallmark of MCL, remains challenging when relying on next-generation sequencing in contrast to fluorescence in situ hybridization. First, the breakpoint locations vary from patient to patient. Second, the translocation does not result in a chimeric fusion protein, like with the Philadelphia chromosomal BCR-ABL1, and is thus not detectable by RNA sequencing or RT-PCR. Furthermore, the breakpoint on chromosome 11 does not alter the gene or coding region of CCND1 but abrogates the transcriptional regulation kilobases (Kb) upstream of this proto-oncogene. Likewise, the chromosome 14 breakage involves not a discrete gene but the Ig heavy chain (IGH) locus and gene cluster. These challenges are not specific to MCL but are encountered in most B-cell malignant cancers, often involving 14q32 as the translocation partner. The fact that clonal rearrangement of the Ig heavy chains occurs in both normal and derailed development of B cells adds a layer of complexity. With its complex karyotype investigated in-depth in several studies,9Jadayel D.M. Lukas J. Nacheva E. Bartkova J. Stranks G. De Schouwer P.J. Lens D. Bartek J. Dyer M.J. Kruger A.R. Catovsky D. Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas: functional studies in a cell line (Granta 519).Leukemia. 1997; 11: 64-72Crossref PubMed Scopus (100) Google Scholar, 10Rudolph C. Steinemann D. Von Neuhoff N. Gadzicki D. Ripperger T. Drexler H.G. Mrasek K. Liehr T. Claussen U. Emura M. Schrock E. Schlegelberger B. Molecular cytogenetic characterization of the mantle cell lymphoma cell line GRANTA-519.Cancer Genet Cytogenet. 2004; 153: 144-150Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 11de Leeuw R.J. Davies J.J. Rosenwald A. Bebb G. Gascoyne R.D. Dyer M.J. Staudt L.M. Martinez-Climent J.A. Lam W.L. Comprehensive whole genome array CGH profiling of mantle cell lymphoma model genomes.Hum Mol Genet. 2004; 13: 1827-1837Crossref PubMed Scopus (108) Google Scholar Granta-519 is a suitable cellular model of high-grade MCL to evaluate the potential role of ONT long-read sequencing in future cytogenetics. Hence, Granta-519–derived DNA was used with DNA from a healthy control donor to perform a contemporary technical assessment of ONT long-read sequencing in detecting extensive somatic structural alterations at a genomic scale. Blood from the control donor was collected in an EDTA tube. Signed consent was provided by the donor, and blood collection for research use was approved by the Regional Ethics Committee for the Region of Southern Denmark (approval number S-20160069). Mononuclear cells were lysed with RNA/DNA-compatible MagNA Pure LC mRNA isolation kit I lysis buffer (Roche, Basel, Switzerland) to preserve nucleic acids after Ficoll gradient centrifugation. Lysate was subsequently stored at −80°C. According to the manufacturer's protocol, DNA from GRANTA-519 (ACC 342; Leibniz-Institut DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and the donor was extracted with the AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany). Concentration was measured using Qubit 3.0 Fluorometer with either the Qubit dsDNA BR Assay or the Qubit dsDNA HS Assay Kits [Life Technologies, Thermo Fisher Scientific (TFS), Waltham, MA]. The Granta-519 cell line and control donor samples underwent long- and short-read sequencing for direct comparison. For long-read whole-genome sequencing library preparation, 1 μg DNA was diluted with nuclease-free water to reach a total volume of 49 μL. Library was prepared using the Ligation Sequencing Kit (SQK-LSK110; ONT). Adapter ligation mix was prepared according to protocol enrichment of long fragments (>3 Kb; Genomic DNA by Ligation; SQK-LSK110; ONT; November 10, 2020). Fragments were recovered with a 10-minute incubation at 37°C in the elution buffer. Following end repair, adapter ligation, and bead purification, the libraries were loaded onto primed R9.4. flow cells (FLO-MIN106D; ONT) and sequenced on MinION Mk1C (ONT). Data were gathered until output plateaued 60 to 70 hours after the sequencing started. Base calling was performed with Guppy integrated into the Mk1C MinKNOW software version 22.03 (ONT). Raw sequencing reads (QPhred > 7) were concatenated (cat command) and aligned to GRCh38 using Minimap2 version 2.24.12Li H. Minimap2: pairwise alignment for nucleotide sequences.Bioinformatics. 2018; 34: 3094-3100Crossref PubMed Scopus (4674) Google Scholar Mapped Granta-519 Nanopore reads (GRCh38) are available for download at dx.doi.org/10.6084/m9.figshare.23282954. Whole-genome sequencing on NovaSeq 6000 System (Illumina, San Diego, CA) was performed using 500 ng of the previously purified DNA for long-read sequencing. The Illumina DNA PCR-Free Prep kit and protocol were used for library generation to obtain uniform coverage. Following end repair, fragmented DNA was size selected (550 bp insert-size protocol) using bead purification with subsequent adapter ligation. The pooled libraries were sequenced using an S4 flow cell. Alignment of reads was performed with BWA (Burrows-Wheeler Aligner version 0.7.17).13Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (30551) Google Scholar Detection of copy number alterations was based on two methods for cross-checking of results: the analyzed coverage profile obtained through BEDTools multicov version 2.26,14Quinlan A.R. Hall I.M. BEDTools: a flexible suite of utilities for comparing genomic features.Bioinformatics. 2010; 26: 841-842Crossref PubMed Scopus (13878) Google Scholar with fixed resolution 100-Kb genomic intervals for further computational analyses, and by implementing QDNAseq version 1.22,15Scheinin I. Sie D. Bengtsson H. van de Wiel M.A. Olshen A.B. van Thuijl H.F. van Essen H.F. Eijk P.P. Rustenburg F. Meijer G.A. Reijneveld J.C. Wesseling P. Pinkel D. Albertson D.G. Ylstra B. DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly.Genome Res. 2014; 24: 2022-2032Crossref PubMed Scopus (264) Google Scholar with a 100-Kb segmentation for unpaired confirmation. Coverage profiles for each sample were normalized to the median number of reads and smoothened for noise reduction (gaussian and median filter; segment neighborhood range of 9 × 100 Kb). Sorting, indexing, and read summarizing were performed with SAMtools version 1.7.16Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe sequence alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (36218) Google Scholar Split read alignments spanning across different chromosomes were derived from mapping output (Minimap2 and BWA13Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (30551) Google Scholar and SAMtools view) based on the secondary alignment flag of the SAM specification (grep SA:Z:) together with inter-chromosomal paired-end reads (BWA; short reads only) for confirmation. Junctional long reads were identified using the following criteria: i) junctions observed more than once within a 100-bp window, ii) junctions confirmed in replicates with a read length of at least 5 Kb, and iii) junctions confirmed by short-read sequencing. Finally, breakpoint sequences were characterized using BLAT (University of California, Santa Cruz, CA),17Kent W.J. BLAT--the BLAST-like alignment tool.Genome Res. 2002; 12: 656-664Crossref PubMed Scopus (6479) Google Scholar BLAST (National Center for Biotechnology Information and NIH, Bethesda, MD),18Boratyn G.M. Camacho C. Cooper P.S. Coulouris G. Fong A. Ma N. Madden T.L. Matten W.T. McGinnis S.D. Merezhuk Y. Raytselis Y. Sayers E.W. Tao T. Ye J. Zaretskaya I. BLAST: a more efficient report with usability improvements.Nucleic Acids Res. 2013; 41: W29-W33Crossref PubMed Scopus (788) Google Scholar and the local realignment Smith-Waterman algorithm (EMBOSS version 6.6; default settings: gapopen 10.0, gapextend 0.05, EDNAFULL matrix).19Rice P. Longden I. Bleasby A. EMBOSS: the European molecular biology open software suite.Trends Genet. 2000; 16: 276-277Abstract Full Text Full Text PDF PubMed Scopus (6815) Google Scholar The sequencing bioinformatics workflow was performed on Ubuntu 18.04 and 20.04 LTS, with downstream analyses using Mathematica version 11 (Wolfram Research, Champaign, IL) and R version 3.6.1 (https://www.anaconda.com/download). Plots were prepared for publication in Prism version 9 (GraphPad Software, Boston, MA). Selected target regions, CDKN2A, CDKN2B, BCL6, TP53 (dual assay), and CCNL1 (Supplemental Table S1) were investigated by real-time quantitative PCR using TaqMan Copy Number Assays (Applied Biosystems, TFS) together with the selected reference gene (TERT) using the QuantStudio 12K Flex System and CopyCaller Software version 2 (Applied Biosystems, TFS). A pathologist (B.P.) evaluated the G-banded karyotype of Granta-519 independently to compare sequencing results. The karyotype was generated by incubation of 20 × 106 cells with 500 mg of Colcemid (KaryoMax Colcemid; Gibco, TFS) in Dulbecco's modified Eagle's medium (Gibco, TFS) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, TFS), 2 mmol/L l-glutamine, and 2% penicillin/streptomycin (concentration of 2 × 106 cells/mL) for 16 hours at 37°C. The effective yield from ONT sequencing was 14.9 to 19.2 × 109 mapped bases (Table 1 and Supplemental Table S2), theoretically corresponding to 5× to 6× average coverage per run. A markedly lower average read length was observed for the cell line sample compared with the donor sample (2416 to 3537 versus 6144 to 9861 bp) but inversely reflected the number of reads (4.4 million to 7.5 million versus 1.5 million to 3.1 million mapped reads; 96% to 98% mapping efficiency). For ONT long-read sequencing, the median reads per 100-Kb bin was 131 (interquartile range, 68 to 221) (Supplemental Table S3).Table 1Sequencing MetricsVariableOxford Nanopore Technologies long-read sequencingIllumina short-read sequencingGranta-519 number 1Granta-519 number 2Donor number 1Donor number 2Granta-519DonorSequences4,557,8667,783,6893,162,3941,530,214986,847,985977,781,924Reads mapped4,371,1357,463,5883,085,3411,467,777986,847,985977,781,924Reads mq075,971140,85135,03112,58043,380,85343,675,192Non-primary alignments1,274,5062,133,408869,118423,8629,263,1848,554,566Total length (bases)16,123,126,56618,810,444,05719,430,803,36915,090,344,333149,014,045,735147,645,070,524Bases mapped15,708,418,80118,269,393,22519,231,520,83114,898,483,187149,014,045,735147,645,070,524Bases mapped (cigar)15,401,733,82517,881,776,35819,105,863,42514,856,040,594146,559,361,028145,144,822,496Mismatches (bases)1,318,976,1111,537,107,2491,749,345,7871,520,410,9371,203,839,5261,133,097,727Error rate0.090.090.090.10.010.01Average length (bases)3537241661449861151151Maximum length (bases)157,885212,351281,245157,591151151Average quality21.421.520.920.735.135.1Insert size average (bases)0000557.6555.9Insert size SD (bases)0000228.9229.7Pairs on different chromosomes00005,908,1164,672,729Median reads per bin (100 Kb)1662791135533,08832,387Inter-chromosomal chimeric reads526,621813,654249,87489,59012,253,96211,426,480The estimated error rate is based on the ratio between mismatches/bases mapped (Samtools stats), whereas the average quality estimates the likelihood of correct base calling as a Phred score. Inter-chromosomal read pair mapping and insert size estimations were not applicable for Nanopore sequencing (underlined). The average read lengths were longer for the donor samples (6.1 to 9.9 Kb versus 2.4 to 3.5 Kb), whereas the total numbers of sequenced and mapped bases for Granta-519 and donor replicates were comparable (33 million versus 34 million reads).Kb, kilobases; mq0, mapping quality zero; SD, standard deviation. Open table in a new tab The estimated error rate is based on the ratio between mismatches/bases mapped (Samtools stats), whereas the average quality estimates the likelihood of correct base calling as a Phred score. Inter-chromosomal read pair mapping and insert size estimations were not applicable for Nanopore sequencing (underlined). The average read lengths were longer for the donor samples (6.1 to 9.9 Kb versus 2.4 to 3.5 Kb), whereas the total numbers of sequenced and mapped bases for Granta-519 and donor replicates were comparable (33 million versus 34 million reads). Kb, kilobases; mq0, mapping quality zero; SD, standard deviation. Illumina sequencing yielded 147 to 149 × 109 mapped bases, corresponding to 49× to 50× coverage with 977 million and 986 million mapped reads. The median reads per 100-Kb bin were 32,586 (interquartile range, 31,231 to 33,659) (Table 1 and Supplemental Table S2). The copy number assessment included nearly three billion covered bases (chromosomes 1 to 22 and X; 100-Kb bins) (Supplemental Table S3A). A comparison of the control donor sample replicates identified 10.7% [323 megabases (Mb)] deviating from the expected relative coverage (>50% deviation). These regions were categorized as low confident (Figure 1). Overall, 98.8% of the chromosomal copy numbers outside these regions were shared between the ONT-sequenced Granta-519 replicates (2676 Mb), and 98.3% were identical to Illumina sequencing (2623 Mb). In total, 319-Mb copy number alterations were shared between the subsets, corresponding to 10.5% of the sequenced genome, with gains in 48 Mb and 271-Mb chromosomal losses. Direct concordance and reproducibility were found between CNAs of at least 1 Mb (Figure 1 and Table 2) and QDNAseq copy number calls (325 Mb in total) (Supplemental Table S3B and Supplemental Figure S1). A minor subset of CNAs unique to QDNAseq was identified in low-confidence regions.Table 2Position, Size, and Concordance of Large Granta-519 Chromosomal Copy Number Alterations Comprising ≥1 MbChromosomePositionMbCytobandCNAReproducibility, %11,500,000–2,500,000∗May involve the start of the chromosome.1p36.33-p36.32Gain99.2 (98.3)3,500,000–9,500,0006p36.32-p36.22Loss11,000,000–34,000,000†Interrupted by region deviating in coverage.23p36.22-p35.1Loss48,000,000–52,000,0004p33-p32.3Loss65,000,000–118,000,00053p31.3-p12Loss159,000,000–164,500,0005.5q23.1-q23.3Loss165,000,000–174,500,0009.5q23.3-q25.1Gain181,500,000–191,500,00010q25.3-q31.2Loss3500,000–84,500,000∗May involve the start of the chromosome.84p26.3-p12.1Loss98.8 (99.1)920,000,000–30,000,00010p21.3-p21.1Loss99.2 (97.2)1231,500,000–34,000,0002.5p11.21-p11.1Loss99.9 (99.8)1349,500,000–51,000,0001.5q14.2-q14.3Loss99.8 (99.4)1423,000,000–25,000,0002q11.2-q12Loss99.4 (98.8)103,000,000–106,000,0003q32.32-q32.33Gain17500,000–18,000,00017.5p13.3-p11.2Loss97.6 (88.1)29,500,000–30,500,0001q11.2Loss61,500,000–66,000,0004.5q23.2-q24.1Loss75,000,000–77,000,0002q25.1Loss181,000,000–27,500,000∗May involve the start of the chromosome.†Interrupted by region deviating in coverage.26.5p11.32-q11.2Loss99.6 (98.2)29,000,000–30,500,0001.5q12.1Loss38,500,000–74,000,00035.5q12.2-q22.3Gain74,000,000–80,000,0006q22.3-q23Loss200–7,500,0007.5p13-p12.3Loss99.7 (99.1)11,000,000–23,500,00012.5p12.2-p11.21Loss32,000,000–33,500,0001.5q11.21-q11.22LossThe reproducibility and concordance between Oxford Nanopore Sequencing replicate and Illumina sequencing (in parentheses) was high. The percentage was calculated by excluding problematic regions identified from the donor control replicates. Size estimations were confirmed and rounded by inspection.CNA, copy number alteration; Mb, megabases.∗ May involve the start of the chromosome.† Interrupted by region deviating in coverage. Open table in a new tab The reproducibility and concordance between Oxford Nanopore Sequencing replicate and Illumina sequencing (in parentheses) was high. The percentage was calculated by excluding problematic regions identified from the donor control replicates. Size estimations were confirmed and rounded by inspection. CNA, copy number alteration; Mb, megabases. A total of 25 large CNAs were detected, spanning from a single to several megabases, counting 21 losses and 4 copy gains (Table 2). Most notably, the findings included loss of TP53, whereas frequently observed BCL6 copy gain and ATM loss in MCL20Le Bris Y. Magrangeas F. Moreau A. Chiron D. Guerin-Charbonnel C. Theisen O. Pichon O. Canioni D. Burroni B. Maisonneuve H. Thieblemont C. Oberic L. Gyan E. Pellat-Deceunynck C. Hermine O. Delfau-Larue M.H. Tessoulin B. Bene M.C. Minvielle S. Le Gouill S. Whole genome copy number analysis in search of new prognostic biomarkers in first line treatment of mantle cell lymphoma: a study by the LYSA group.Hematol Oncol. 2020; 38: 446-455Crossref PubMed Scopus (4) Google Scholar were absent. The afflicted regions ranged from bi-allelic deletions of chromosome 9 CDKN2A/B and up to eight regional copies of chromosome 18, in line with the findings by Jadayel et al9Jadayel D.M. Lukas J. Nacheva E. Bartkova J. Stranks G. De Schouwer P.J. Lens D. Bartek J. Dyer M.J. Kruger A.R. Catovsky D. Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas: functional studies in a cell line (Granta 519).Leukemia. 1997; 11: 64-72Crossref PubMed Scopus (100) Google Scholar and Rudolph et al.10Rudolph C. Steinemann D. Von Neuhoff N. Gadzicki D. Ripperger T. Drexler H.G. Mrasek K. Liehr T. Claussen U. Emura M. Schrock E. Schlegelberger B. Molecular cytogenetic characterization of the mantle cell lymphoma cell line GRANTA-519.Cancer Genet Cytogenet. 2004; 153: 144-150Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar The relative copy numbers for TP53, BCL6, and CDKN2A/B were confirmed by real-time quantitative PCR (Figure 2 and Supplemental Table S3C). The most comprehensive allelic imbalances arising from CNAs were directly identifiable from the chromosomal variant allele frequencies (Supplemental Figure S2). Although evaluation of G-banded karyotype independently confirmed copy number alterations involving chromosomes 1, 3, 12 to 14, 17, 18, and 20, the extent was not directly comparable (Supplemental Figure S3). Loss on chromosome 9p was not detectable by G banding. Because of a large number of ambiguously mapped reads, with collectively 1.7 million marked as chimeric from the long-read alignment (Supplemental Table S4), the following basic principles were implemented to identify translocations: i) most false-positive observations are irreproducible events; ii) true biological features are reproducible in replicates; and iii) coaligned junctions display identical or closely spaced breakpoints with fixed 5′ or 3′ ends. No chromosomal translocations were shared between donor replicates. In contrast, three translocations were resolved for Granta-519. The CCND1/IGH fusion t(11;14)(q13.3;q32.33) (69,576,560; 105,864,262) was supported by nine junctional long reads (ONT) (Supplemental Table S5), confirmed by five short reads (Illumina) (Supplemental Table S6) and 39 split read pairs mapped to each chromosome (Illumina; data not shown). The identified breakpoint was positioned approximately 44 Kb downstream from the major translocation cluster sequence published by Rimokh et al,21Rimokh R. Berger F. Delsol G. Digonnet I. Rouault J.P. Tigaud J.D. Gadoux M. Coiffier B. Bryon P.A. Magaud J.P. Detection of the chromosomal translocation t(11;14) by polymerase chain reaction in mantle cell lymphomas.Blood. 1994; 83: 1871-1875Crossref PubMed Google Scholar in keeping with previous findings.22Willis T.G. Jadayel D.M. Coignet L.J. Abdul-Rauf M. Treleaven J.G. Catovsky D. Dyer M.J. Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction.Blood. 1997; 90: 2456-2464Crossref PubMed Google Scholar The breakpoint sequence, flanked by mapped region upstream CCND1 and IGH sequences of 676 to 68,899 bp (94.7% to 98% identical to GRCh38 reference; BLAT17Kent W.J. BLAT--the BLAST-like alignment tool.Genome Res. 2002; 12: 656-664Crossref PubMed Scopus (6479) Google Scholar), displayed a mean sequence similarity of 89% (range, 83.7% to 94.7%) (Supplemental Table S7, A and B) to the 493-bp CCND1/IGH JH4 translocation breakpoint sequence submitted by Willis et al22Willis T.G. Jadayel D.M. Coignet L.J. Abdul-Rauf M. Treleaven J.G. Catovsky D. Dyer M.J. Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction.Blood. 1997; 90: 2456-2464Crossref PubMed Google Scholar (GenBank; http://ncbi.nlm.nih.gov/nuccore/

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in the western world. It is highly heterogeneous with a variable clinical course, but curable with chemo-immunotherapy in up to 70% of all cases. The lymphoma presents in lymph nodes and/or extranodal lymphoid tissue, and the diagnosis is based on invasive procedures for histopathologic evaluation. Methods: In this technical study, we evaluated cell-free DNA (cfDNA) from blood plasma to detect clonal B cells in patients with DLBCL using rearranged immunoglobulin heavy chain gene as targets by next-generation sequencing. Clonal B cell sequences and frequencies were determined from blood plasma cfDNA and cellular DNA from matched excised lymphoma tissues and mononuclear cells isolated from diagnostic bone marrow and blood samples from 15 patients. Results: We showed that identical clonal rearrangements could be detected in blood plasma and excised lymphoma tissue and that plasma cfDNA was superior in detecting clonal rearrangements compared to blood or bone marrow-derived cellular DNA. Conclusion: These findings consolidate the role of blood plasma as a reliable and easily accessible source for detecting neoplastic cells in DLBCL.