A decade with whole exome sequencing in haematology Hansen, Marcus C.; Haferlach, Torsten; Nyvold, Charlotte G.
British journal of haematology,
February 2020, 2020-Feb, 2020-02-00, 20200201, Letnik:
188, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Summary
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.
•Ultradeep sequencing of immunoglobulin genes can identify residual disease.•Low-level detection of clonally rearranged immunoglobulins is a stochastic process.•A sufficient amount of DNA must be ...analyzed to detect one cell in a million.•Standardization of measurable disease detection by sequencing is found imperative.
Malignant lymphoproliferative disorders collectively constitute a large fraction of the hematological cancers, ranging from indolent to highly aggressive neoplasms. Being a diagnostically important hallmark, clonal gene rearrangements of the immunoglobulins enable the detection of residual disease in the clinical course of patients down to a minute fraction of malignant cells. The introduction of next-generation sequencing (NGS) has provided unprecedented assay specificity, with a sensitivity matching that of polymerase chain reaction-based measurable residual disease (MRD) detection down to the 10–6 level. Although reaching 10–6 to 10–7 is theoretically feasible, employing a sufficient amount of DNA and sequencing coverage is placed in the perspective of the practical challenges when relying on clinical samples in contrast to controlled serial dilutions. As we discuss, the randomness of subsampling must be taken into account to accommodate the sensitivity threshold—in terms of both the required number of cells and sequencing coverage. As a substantial part of the reviewed studies do not state the depth of coverage or even amount of DNA in some cases, we call for increased transparency to enable critical assessment of the MRD assays for clinical implementation and feasibility.
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.
Summary
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.
T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and ...curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging.
We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models.
We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19
, CD19
, and mixed CD19
/CD19
B-cell lymphoma.
Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.
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.
Overexpressed genes may be useful for monitoring of measurable residual disease (MRD) in patients with childhood acute myeloid leukemia (AML) without a leukemia-specific target. The normal expression ...of five leukemia-associated genes (SPAG6, ST18, MSLN, PRAME, XAGE1A) was defined in children without hematologic disease (n = 53) and children with suspected infection (n = 90). Gene expression at AML diagnosis (n=50) and during follow-up (n = 21) was compared with child-specific reference values. At diagnosis, 34/50 children (68%) had high expression of at least one of the five genes, and so did 16/31 children (52%) without a leukemia-specific target. Gene expression was quantified in 110 peripheral blood (PB) samples (median, five samples/patient; range, 1 to 10) during follow-up in 21 patients with high expression at diagnosis. All nine patients with PB sampling performed within 100 days of disease recurrence displayed overexpression of SPAG6, ST18, PRAME, or XAGE1A at a median of 2 months (range, 0.6 to 9.6 months) before hematologic relapse, whereas MSLN did not reach expression above normal prior to hematologic relapse. Only 1 of 130 (0.8%) follow-up analyses performed in 10 patients in continuous complete remission had transient expression above normal. SPAG6, ST18, PRAME, and XAGE1A expression in PB may predict relapse in childhood AML patients and facilitate MRD monitoring in most patients without a leukemia-specific target.
•Changes in the clonal composition of acute myeloid leukemia occur during therapy.•Rare cases of outgrowth of clones related to nonmalignant, but clonal hematopoiesis unrelated to the leukemic clone ...can be seen on regeneration after chemotherapy.•In cases in which changes occur in the proportions of leukemic subclones, the largest subclone is not necessarily the one with relapse-initiating potential.•Caution is warranted when correlating residual disease levels to prognosis in patients whose leukemic subclones exhibit differential responses to chemotherapy.•It remains to be seen whether these findings can be extrapolated to the minimal residual disease setting when overall reduction of leukemic subclones is much greater.
Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and surveying several subclones simultaneously. There is little experience with interpretation of differential clonal responses to therapy. We hypothesized that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level >0.02. Five patients had a rising clone at the time of evaluation. In a representative case, the rising clone was present only in the putative healthy stem cells (CD45lowCD34+CD38–CD123–CD7–) and not in the putative leukemic stem cells (CD34+CD38–CD123+CD7+) cells, thus indicating nonmalignant clonal hematopoiesis. In contrast, 17 of 43 evaluable patients exhibited a differential response in genes related to the leukemic clone. Twenty-six of 43 patients exhibited a clonal response that followed the overall treatment response. Patients with a differential response had better event-free survival (EFS) and overall survival (OS) than those in whom the clonal response followed the overall response (log-rank test, EFS: p = 0.045, OS: p = 0.050). This indicates that when following multiple leukemia-related clones, the less chemotherapy-responsive clone could, in some cases, have lower relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies on the precise interpretations of multiclone NGS–MRD assays.
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