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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
12.
Toward Cytogenomics Hansen, Marcus H.; Cédile, Oriane; Kjeldsen, Marie L.G. ...
The Journal of molecular diagnostics : JMD,
November 2023, 2023-11-00, Volume:
25, Issue:
11
Journal Article
Peer reviewed
Open access
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.
During a 10‐year period (1992–2001) in the region of Southern Denmark, 337 patients aged 15 years or older (range 16–93 years, median 67 years) were diagnosed with acute myeloid leukaemia (AML). ...Cytogenetic analysis was carried out in 90%, of whom 53% had clonal chromosome aberrations. Some 24% and 31% had only numerical or structural abnormalities respectively. The remaining patients showed both types of abnormalities. Ploidy levels in decreasing order were: pseudodiploidy, 41%; hyperdiploidy, 32%; and hypodiploidy, 27%. Pseudodiploidy characterizes type M3 (70%) and hypodiploidy M6 (56%). Recurrent cytogenetic abnormalities – t(8;21), t(15;17) and inv(16) – were found in 3·3%, 3·3% and 2·0% of all patients respectively. Prognostically intermediate and adverse aberrations were found in 39% and 44%, respectively, of those with an abnormal karyotype. Rare recurrent aberrations were found in two patients in this material. A previously described non‐recurrent abnormality was found to be recurrent in one patient der(20)t(11;20)(q13.2;p13). New, previously undescribed abnormalities were found in 41 patients. Statistically significant correlations were found between t(15;17) and young age (P < 0·001), inv(16) and young age (P < 0·006), −17 and M6 (P = 0·007), and M6 and complex karyotype with five or more unrelated aberrations (P = 0·004). We conclude that this truely population‐based cytogenetic study of adult AML showed distributions of chromosome abnormalities that differ from those described so far.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Minimal residual disease (MRD) monitoring has a strong prognostic value in childhood lymphoblastic leukemia (ALL) and is currently utilized in all major pediatric ALL protocols. MRD monitoring is ...done by multiparameter flow cytometry, IG/TCR quantitative PCR or reverse transcriptase quantitative PCR of leukemic fusion transcripts providing a reliable measurement of treatment response. However, occasionally bone marrow (BM) aspirates may not yield representative material or be misinterpreted due to treatment‐induced changes in MRD marker profile, undetected subclones at diagnosis, contamination with peripheral blood or cell adhesion and stroma cell interactions posing a risk for underestimating MRD levels and misclassifying resistant disease that may be detected by traditional BM morphology methods, immunohistochemistry, karyotyping and FISH. We present four cases with high MRD levels where MRD monitoring failed to provide the correct stratification information. Through these cases, we discuss the continued need to consider all available information including BM smears, touch imprints and trephine biopsy preparations not only at diagnosis but throughout remission monitoring in pediatric ALL.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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.
Daratumumab is an integral part of the treatment of multiple myeloma (MM) but its real-world efficacy has only been described in small cohorts. MAMMOTH is the only large multi-center study that ...reported the outcomes of CD38-refractory MM. The present study includes a complete, Danish, nation-wide cohort of 635 MM patients who initiated treatment a daratumumab-containing index regimen (IR) prior to 1.1.2019, and describes the outcomes of 472 patients who discontinued their IR until 1.1.2021. Patients received a median of 3 lines of therapy (LOT) prior to the IR. The median time from diagnosis to discontinuation of the IR (T 0) was 4 years. At T 0, 73% of patients were quadruple drug class exposed (CE). The median overall survival (mOS) after T 0 was 12.2 months in the entire cohort, 15.3 months in double CE, 22.5 months in triple CE, 12.6 months in quadruple CE and 8.3 months in alkylator-bortezomib-carfilzomib-daratumumab-lenalidomide-pomalidomide-exposed patients. After T 0, 79%, 48%, 29%, 17%, 10% and 6% of patients received 1, 2, 3, 4, 5, 6 additional LOT, respectively, achieving overall response rates ranging from 44% to 11% and median time to next treatment (TNT) from 138 to 54 days. In the first subsequent LOT after T 0, 51% of patients were retreated with daratumumab. Despite the lack of benefit in terms response and TNT, daratumumab retreatment was associated with superior OS on multivariate analysis adjusting for age, previous transplantation, IR, drug class exposure at T 0, treating site, time from diagnosis to T 0 and presence of cytogenetic high-risk markers. Median OS was 24.6 months in patients retreated with and 11.3 months in patients treated without daratumumab (p<0.0001). In conclusion, patients with MM who discontinue their first daratumumab-containing regimen have a life expectancy of approximately one year. Our results support a rationale behind daratumumab retreatment.
A
Overall survival after T 0; B: Overall survival after T 0 by cytogenetic risk; C: Overall survival after T 0 by IR; D: Overall survival after T 0 by prior exposure.
T 0=time of discontinuation of the first daratumumab-containing line of therapy; IR=index regimen; high-risk=t(4;14), t(14;16) or del17p by FISH; D-mono=daratumumab monotherapy; D-bor=daratumumab-bortezomib-dexamethasone; D-len=daratumumab-lenalidomide-dexamethasone; D-other=daratumumab in other combinations; Double_CE=exposed to daratumumab and another class of drugs; Triple_CE=exposed to daratumumab and two other classes of drugs; Quadruple_CE=exposed to daratumumab and three other classes of drugs; ABCDLP-exposed=exposed to daratumumab, bortezomib, carfilzomib, lenalidomide and pomalidomide
Display omitted
Szabo: Takeda: Consultancy; Sanofi: Consultancy; Janssen: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation ...of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4
T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8
T cells able to contain chronic virus infections.
We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4
T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8
lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pT
, p = 0.01), reduced frequencies of peripheral CD4
Bcl-6
T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pT
formation, pT
and CD4
IL-21
frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman's rho: - 0.86, p < 0.001.
To the best of our knowledge, this is the first report of elevated CD4
IL-21
T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aim:In multiple myeloma (MM), high intensity of FDG uptake in focal lesions measured as the standard uptake value (SUV) at diagnosis is associated to aggressive disease and reduced overall survival. ...However, the reason for high intensity FDG uptake in some focal lesions is unknown, but hypothetically such "hot" lesions could represent evolving myeloma sub-clones with particular characteristics, e.g. higher proliferative activity. We aimed to explore and characterize focal lesions with high FDG uptake and to compare the findings with random diagnostic bone marrow biopsies with lower FDG intensity or a biopsy from another lesion with lower FDG uptake. Thus, we have created a paired biopsy biobank that will be explored for molecular, biological, physiological and myeloma prognostic markers. Here we present our first data including results on morphology, immunohistochemistry, mutational, proliferative and cytogenetic status in the biopsies
Material and methods: CT-guided biopsies from FDG-PET positive CT-visible focal lesions in sternum, sacrum, humerus, femoral, pubic and iliac bones were taken without complications depending on accessibility and safety in 14 newly diagnosed, untreated MM patients, 2 females and 12 males, aged 53-77 years. Patients that had received steroids or bisphosphonates were excluded. Bone marrow biopsies were taken as part of the normal diagnostic work-up, but included an extra biopsy and aspirate for research. FDG uptake in the regions of interest (ROI) at focal and random bone marrow biopsy was quantified by dedicated software (ROVER, ABX, Radeberg, Germany) to obtain the following variables: Lesion volumes, SUVmax, SUVpeak, cSUVmean (SUVmean corrected for partial volume effect). The paired biopsies were analyzed for: Myeloma plasma cells percentage (PC%), myeloma cell proliferation (Ki67 positive fraction of CD138 positive cells (Ki67/CD138%), myeloma cell MYC protein expression (MYC/CD138%), FISH aberrations in % of myeloma cells (del17p, del13q, del1p, amp1q, t(11;14), t(4;14), BRAF mutation and specific p16, p27, and p53 protein expressions by IHC in % of MM cells.
Results: 13 patients were evaluable for analysis with paired datasets. One patient was excluded due to a normal bone marrow examination (multifocal myeloma). ROI SUVmax values ranged from 2.6 to 22.16 and differences in SUVmax between paired biopsies ranged from 0.4 to 17.1. First of all, we found myeloma malignancy in all PET-positive CT-guided biopsies. Comparing the findings in "hot" versus "random" biopsy groups we found significantly higher PC%s in the "hot" biopsy group (p=0.01) but this was not a consistent finding in all patients. PC proliferation rate (Ki67/CD138) was higher in some of the "hot" biopsies but this was neither a uniform observation. No difference in "primary event" chromosome 14q translocations was observed, whereas we identified subclones with typical cytogenetic secondary or late events in several "hot" biopsies that were not present in the random bone marrow biopsy: amp1q in 3 patients, del1p in 1 patient, del13q and del17p in 1 patient. MYC protein expression was higher in "hot" biopsies in 4 patients, and downregulation of p27 was evident in 2 patients. We found no unbalanced expression of p53 or p16, and in only one patient we identified a BRAF mutated subclone that was equally present in the "hot" and "random" biopsy (20% vs 30%). Few patients presented more adverse findings in the "hot" biopsies. Overall, MM adverse findings were present unbalancedly in 8 of 13 patients.
Conclusion: We did not identify a mutual factor that explained the more intense FDG uptake in CT guided biopsies than in random bone marrow, e.g. a higher PC Ki67 expression. However, in 8 of 13 patients we identified 1 or more prognostic adverse, unbalanced findings in the PET positive focal lesions indicating presence of more aggressive subclones. These findings are concordant with the adverse prognostic importance of finding high-intense PET-positive focal lesions on FDG-PET/CT in newly diagnosed MM patients.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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