Germline GATA1s-generating mutations predispose to leukemia with acquired trisomy 21 and Down syndrome-like phenotype.

Individuals with Down syndrome are at increased risk of myeloid leukemia in early childhood, which is associated with acquisition of GATA1 mutations that generate a short GATA1 isoform called GATA1s. Germline GATA1s-generating mutations result in congenital anemia in males. We report on 2 unrelated families that harbor germline GATA1s-generating mutations in which several members developed acute megakaryoblastic leukemia in early childhood. All evaluable leukemias had acquired trisomy 21 or tetrasomy 21. The leukemia characteristics overlapped with those of myeloid leukemia associated with Down syndrome, including age of onset at younger than 4 years, unique immunophenotype, complex karyotype, gene expression patterns, and drug sensitivity. These findings demonstrate that the combination of trisomy 21 and GATA1s-generating mutations results in a unique myeloid leukemia independent of whether the GATA1 mutation or trisomy 21 is the primary or secondary event and suggest that there is a unique functional cooperation between GATA1s and trisomy 21 in leukemogenesis. The family histories also indicate that germline GATA1s-generating mutations should be included among those associated with familial predisposition for myelodysplastic syndrome and leukemia.

Methylation microarray-based detection of clinical copy-number aberrations in CLL benchmarked to standard FISH analysis.

Copy-number aberrations (CNAs) are assessed using FISH analysis in diagnostics of chronic lymphocytic leukemia (CLL), but CNAs can also be extrapolated from Illumina BeadChips developed for genome-wide methylation microarray screening. Increasing numbers of microarray data-sets are available from diagnostic samples, making it useful to assess the potential in CNA diagnostics. We benchmarked the limitations of CNA testing from two Illumina BeadChips (EPIC and 450k) and using two common packages for analysis (conumee and ChAMP) to FISH-based assessment of 11q, 13q, and 17p deletions in 202 CLL samples. Overall, the two packages predicted CNAs with similar accuracy regardless of the microarray type, but lower than FISH-based assessment. We showed that the bioinformatics analysis needs to be adjusted to the specific CNA, as no general settings were identified. Altogether, we were able to predict CNAs using methylation microarray data, however, with limited accuracy, making FISH-based assessment of deletions the superior diagnostic choice.

IGHV-associated methylation signatures more accurately predict clinical outcomes of chronic lymphocytic leukemia patients than IGHV mutation load.

Currently, no molecular biomarker indices are used in standard care to make treatment decisions at diagnosis of chronic lymphocytic leukemia (CLL). We used Infinium MethylationEPIC array data from diagnostic blood samples of 114 CLL patients and developed a procedure to stratify patients based on methylation signatures associated with mutation load of the IGHV gene. This procedure allowed us to predict the time to treatment with a hazard ratio (HR) of 8.34 (95% confidence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: 2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases for which the two classification procedures gave discrepant results showed that these cases were incorrectly classified using IGHV status. Moreover, methylation-based classification stratified patients with different overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not possible using IGHV status. Furthermore, we assessed the performance of the developed classification procedure using published HumanMethylation450 array data for 159 patients for whom information on time to treatment, overall survival and relapse was available. Despite 450K array methylation data not containing all the biomarkers used in our classification procedure, methylation signatures again stratified patients with significantly better accuracy than did IGHV mutation load regarding all available clinical outcomes. Thus, stratification using IGHV-associated methylation signatures may provide better prognostic power than IGHV mutation status.

Comparative evaluation of the heterozygous variant standard deviation as a quality measure for next-generation sequencing.

Next-generation sequencing holds unprecedented throughput in terms of informational content to cost. The technology has entered the scene in laboratory diagnostics and offers flexible workflows in biomedical research. However, the rapid acquisition of genomic data also gives rise to a substantial fraction of sequencing artifacts, causing the detection of false-positive germline variants or erroneous somatic mutations. Consequently, there is a pressing need for efficient and practical quality assessment in sequencing projects. In this study, we investigate using heterozygous variant allele frequency (VAF) standard deviation (σ) for supplementary quality control. Whereas several proposed quality metrics are based on empirical assessments, the dispersion of the allele frequencies reflects a direct approximation of the inherent and discrete features of a diploid genome. Consequently, homologous chromosomes display heterozygous VAF of approximately 1/2. Based on the meta-analysis of 152 whole-exome sequencing data sets, we found that σ reflects both sequencing coverage and noise and can be effectively modeled. It is concluded that the relative comparison of heterozygous VAF σ provides a practical handle for quality assessment, even for samples afflicted with copy-number alterations. The approach can be implemented when performing whole-exome, whole-genome, or targeted panel sequencing and helps identify problematic samples, such as those retrieved from archived formalin-fixed paraffin-embedded tissue.

A decade with whole exome sequencing in haematology.

The first decade of capture-based targeted whole exome sequencing (WES) has now passed, while the sequencing modality continues to find more widespread usage in clinical research laboratories and still offers an unprecedented diagnostic assay in terms of throughput, informational content and running costs. Until quite recently, WES has been out of reach for many clinicians and molecular biologists, and it still poses issues or is met with some reluctance with regards to cost versus benefit in terms of effective assay costs, hands-on laboratory work and data analysis bottlenecks. Although WES is used more than ever, it may also be argued that the usage is peaking and that new implementations, or relevance in its current state, will likely be leveling off during the following decade as the price on whole genome sequencing continues to drop. In this review, we focus on the past decade of targeted whole exome sequencing in malignant hematology. We thematically revisit some of the significant discoveries and niches that use next-generation sequencing, and we outline what and how WES has contributed to the field - from clonal hematopoiesis of the aging bone marrow to profiling malignancies down to the single cell.

Measurable residual disease assessment by qPCR in peripheral blood is an informative tool for disease surveillance in childhood acute myeloid leukaemia.

Serial assessments of measurable (or minimal) residual disease (MRD) by qPCR may identify nascent relapse in children with acute myeloid leukaemia (AML) and enable pre-emptive therapy. We investigated the kinetics and prognostic impact of recurrent fusion transcripts (RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3 or KMT2A-ELL) in 774 post-induction samples from bone marrow (BM, 347) and peripheral blood (PB, 427) from 75 children with AML. BM MRD persistence during consolidation did not increase the risk of relapse, and MRD at therapy completion did not correlate to outcome (HR = 0·64/MRD log reduction (CI: 0·32-1·26), P = 0·19). In contrast, 8/8 patients with detectable MRD in PB after first consolidation relapsed. Persistence (n = 4) and shifting from negative to positive (n = 10) in PB during follow-up predicted relapse in 14/14 patients. All 253 PB samples collected during follow-up from 36 patients in continuous complete remission were MRD negative. In core-binding factor AML, persistent low-level MRD positivity in BM during follow-up was frequent but an increment to above 5 × 10-4 heralded subsequent haematological relapse in 12/12 patients. We demonstrate that MRD monitoring in PB after induction therapy is highly informative and propose an MRD increment above 5 × 10-4 in PB and BM as a definition of molecular relapse since it always leads to haematological relapse.

Measurable residual disease monitoring using Wilms tumor gene 1 expression in childhood acute myeloid leukemia based on child-specific reference values.

BACKGROUND: Measurable/minimal residual disease (MRD) monitoring can predict imminent hematological relapse in acute myeloid leukemia (AML). The majority of childhood AML patients do not harbor fusion genes or mutations applicable as MRD markers and overexpression of Wilms tumor gene 1 (WT1) may constitute a useful monitoring target. However, age-specific reference values in healthy hematopoiesis and standardization of WT1 assessment are prerequisites for clinical utility. PROCEDURE: We investigated WT1 expression across age in hematologically healthy controls (n = 109), during suspected infection (n = 90) and bone marrow (BM) regeneration (n = 13). WT1 expression in AML at diagnosis (n = 91) and during follow-up (n = 30) was compared with age-specific reference values. RESULTS: WT1 expression correlated with age and showed higher levels in both BM and peripheral blood (PB) in children compared with adults (P < 0.001 and P = 0.01). WT1 expression from healthy hematopoiesis was lower in PB compared with BM (WT1BM /WT1PB  = 8.6, 95% CI: 5.3-13.7) and not influenced by infection nor BM regeneration. At AML diagnosis, 66% had more than 20-fold WT1 overexpression in PB or BM (PB 74%; BM 45%). WT1 was quantified in 279 PB samples during follow-up. All 11 patients with PB sampling within 4 months of disease recurrence displayed WT1 overexpression by a median of 1.9 months (range, 0.7-9.7) before hematological relapse. CONCLUSIONS: This study defines child-specific reference values for WT1 expression in healthy hematopoiesis and demonstrates that WT1 expression in PB is a useful post-treatment monitoring tool in childhood AML. Based on these observations, we propose definitions for childhood AML molecular relapse using WT1 overexpression.

Treatment of Molecular Relapse by Cessation of Immunosuppression After Hematopoietic Stem Cell Transplantation in Pediatric FLT3-ITD AML Monitored by WT1 Expression in Peripheral Blood.

Relapse after hematopoietic stem cell transplantation in pediatric acute myeloid leukemia is a fatal event in the majority of cases. Immunotherapy may prevent an impending relapse if instituted at first molecular evidence of disease recurrence. Wilms tumor gene 1 (WT1) is overexpressed in the majority of children and may constitute a useful molecular marker of measurable residual disease applicable for disease monitoring in peripheral blood where the background amplification from healthy hematopoiesis is less prevalent compared with bone marrow. We report the measurable residual disease kinetics from a child with FLT3-internal tandem duplication acute myeloid leukemia where sequential WT1 monitoring in peripheral blood-guided withdrawal of immunosuppression.

Nature and nurture: a case of transcending haematological pre-malignancies in a pair of monozygotic twins adding possible clues on the pathogenesis of B-cell proliferations.

We describe a comprehensive molecular analysis of a pair of monozygotic twins, who came to our attention when one experienced amaurosis fugax and was diagnosed with JAK2+ polycythaemia vera. He (Twin A) was also found to have an asymptomatic B-cell chronic lymphocytic leukaemia (B-CLL). Although JAK2-, Twin B was subsequently shown to have a benign monoclonal B-cell lymphocytosis (MBL). Flow cytometric and molecular analyses of the B-cell compartments revealed different immunoglobulin light and heavy chain usage in each twin. We hypothesized that whole exome sequencing could help delineating the pattern of germline B-cell disorder susceptibility and reveal somatic mutations potentially contributing to the differential patterns of pre-malignancy. Comparing bone marrow cells and T cells and employing in-house engineered integrative analysis, we found aberrations in Twin A consistent with a myeloid neoplasm, i.e. in TET2, RUNX1, PLCB1 and ELF4. Employing the method for detecting high-ranking variants by extensive annotation and relevance scoring, we also identified shared germline variants in genes of proteins interacting with B-cell receptor signalling mediators and the WNT-pathway, including IRF8, PTPRO, BCL9L, SIT1 and SIRPB1, all with possible implications in B-cell proliferation. Similar patterns of IGHV-gene usage to those demonstrated here have been observed in inherited acute lymphoblastic leukaemia. Collectively, these findings may help in facilitating identification of putative master gene(s) involved in B-cell proliferations in general and MBL and B-CLL in particular.

Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission.

The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

hMICL and CD123 in combination with a CD45/CD34/CD117 backbone - a universal marker combination for the detection of minimal residual disease in acute myeloid leukaemia.

Real-time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so-called leukaemia-associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C-type Lectin (hMICL, also termed C-type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin-3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre-clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10(-4) in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post-induction time-point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123-based MFC assay is a promising MRD tool in AML.

SWIGH-SCORE: A translational light-weight approach in computational detection of rearranged immunoglobulin heavy chain to be used in monoclonal lymphoproliferative disorders.

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.

The potential of 3rd-generation nanopore sequencing for B-cell clonotyping in lymphoproliferative disorders.

Lymphoid malignancies are characterized by clonal cell expansion, often identifiable by unique immunoglobulin rearrangements. Heavy (IGH) and light-chain gene usage offers diagnostic insights and enables sensitive residual disease detection via next-generation sequencing. With its adaptable throughput and variable read lengths, Oxford Nanopore thirdgeneration sequencing now holds promise for clonotyping. This study analyzed CD138+ plasma-cell DNA from eight multiple myeloma patients, comparing clonotyping performance between Nanopore sequencing, Illumina MiSeq, and Ion Torrent S5. We demonstrated clonotype consistency across platforms through Smith-Waterman local alignment of nanopore reads. The mean clonal percentage of IGH V and J gene usage in the CD138+ cells was 69% for Nanopore, 67% for S5, and 76% for MiSeq. When aligned with known clonotypes, clonal cells averaged a 91% similarity, exceeding 85%. In summary, Nanopore sequencing, with its capacity for generating millions of high-quality reads, proves effective for detecting clonal IGH rearrangements. This versatile platform offers the potential for measuring residual disease down to a sensitivity level of 10-6 at a lower cost, marking a significant advancement in clonotyping techniques.

A novel RT-qPCR assay for quantification of the MLL-MLLT3 fusion transcript in acute myeloid leukaemia.

OBJECTIVES: Patients with acute myeloid leukaemia (AML) of the monocytic lineage often lack molecular markers for minimal residual disease (MRD) monitoring. The MLL-MLLT3 fusion transcript found in patients with AML harbouring t(9;11) is amenable to RT-qPCR quantification but because of the heterogeneity of translocation break points, the MLL-MLLT3 fusion gene is a challenging target. We hypothesised that MRD monitoring using MLL-MLLT3 as a RT-qPCR marker is feasible in the majority of patients with t(9;11)-positive AML. METHODS: Using a locked nucleic acid probe, we developed a sensitive RT-qPCR assay for quantification of the most common break point region of the MLL-MLLT3 fusion gene. Five paediatric patients with t(9;11)-positive AML were monitored using the MLL-MLLT3 assay. RESULTS: A total of 43 bone marrow (BM) and 52 Peripheral blood (PB) samples were collected from diagnosis until follow-up. Two patients relapsed, and both were MRD positive in BM after first induction course. A total of three relapses occurred, and they were detected by RT-qPCR 3 wks before haematological relapse was diagnosed. CONCLUSION: This MLL-MLLT3 RT-qPCR assay could be useful in MRD monitoring of a group of patients with AML who often lack reliable MRD markers.

Mitochondrial cytochrome c oxidase subunit II variations predict adverse prognosis in cytogenetically normal acute myeloid leukaemia.

Alterations in the two catalytic genes cytochrome c oxidase subunits I and II (COI and COII) have recently been suggested to have an adverse impact on prognosis in patients with acute myeloid leukaemia (AML). In order to explore this in further detail, we sequenced these two mitochondrial genes in diagnostic bone marrow or blood samples in 235 patients with AML. In 37 (16%) patients, a non-synonymous variation in either COI or COII could be demonstrated. No patients harboured both COI and COII non-synonymous variations. Twenty-four (10%) patients had non-synonymous variations in COI, whereas 13 (6%) patients had non-synonymous variations in COII. The COI and COII are essential subunits of cytochrome c oxidase that is the terminal enzyme in the oxidative phosphorylation complexes. In terms of disease course, we observed that in patients with a normal cytogenetic analysis at disease presentation (CN-AML) treated with curative intent, the presence of a non-synonymous variation in the COII was an adverse prognostic marker for both overall survival and disease-free survival (DFS) in both univariate (DFS; hazard ratio (HR) 4.4, P = 0.006) and multivariate analyses (DFS; HR 7.2, P = 0.001). This is the first demonstration of a mitochondrial aberration playing an adverse prognostic role in adult AML, and we argue that its role as a potentially novel adverse prognostic marker in the subset of CN-AML should be explored further.

Characterization and prognostic significance of mitochondrial DNA variations in acute myeloid leukemia.

Recent studies have suggested that mutations in the mitochondrial genome (mtDNA) may play a role in the development and response to treatment for human cancer. The aim of this study was to investigate whether mtDNA variations have any prognostic relevance, to clarify the spectra of mtDNA variation and to determine whether there was any correlation to known prognostic factors in acute myeloid leukemia (AML). To elucidate this, we sequenced the entire mtDNA in 56 AML patients and 14 control subjects. When analyzing the biologic impact of the non-synonymous variations in the mtDNA coding genes, we found an inferior disease-free survival for patients exhibiting variations in the two most important catalytic genes of the complex IV of the oxidative phosphorylation complexes (OXPHOS), that is, the cytochrome c oxidase subunit I and the cytochrome c oxidase subunit II (hazard ratio 2.6, P = 0.03; multivariate analysis). In addition, the most frequent variation was the T16311C in the control region, which was found in 11 (20%) of the 56 patients. This observation was confirmed in another cohort of 173 diagnostic AML samples. In this expanded group, the T16311C variation tended to be associated with chromosomal abnormalities.

Reproducibility of low-level residual myeloma immunoglobulin detection using ultra-deep sequencing.

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 longitudinal 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 normalization for MRD evaluation as a stable reference. These findings may guide MRD-level reporting and comparisons between laboratories.

Cell-free DNA for detection of clonal B cells in diffuse large B cell lymphoma by sequencing.

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.

Toward Cytogenomics: Technical Assessment of Long-Read Nanopore Whole-Genome Sequencing for Detecting Large Chromosomal Alterations in Mantle Cell Lymphoma.

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 with current next-generation sequencing modalities. 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. On the basis of 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.