Follicular lymphoma is an incurable malignancy, with transformation to an aggressive subtype representing a critical event during disease progression. Here we performed whole-genome or whole-exome ...sequencing on 10 follicular lymphoma-transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 ...mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.
Key Points
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and ...phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra‐deep next‐generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single‐agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow‐up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance‐associated BTK mutations in individual patients treated with ibrutinib.
What's new?
Although the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia, 20% of patients still show disease progression. Comprehensive characterisation of mechanisms underlying ibrutinib resistance and the related changes in the subclonal architecture induced by the selective pressure of the treatment may usher in new clinical advances. This time‐resolved ultra‐deep genomic scrutiny of mutation target genes reveals unique patterns of highly dynamic clonal variegation associated with BTK inhibition and identifies novel resistance‐associated BTK mutations in individual patients. Furthermore, evidence suggests that sensitive molecular monitoring of treatment response can facilitate the early detection of impending relapse.
Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease ...progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPA
). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPA
-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPA
provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.
While most myelodysplastic syndrome/acute myeloid leukemia cases are sporadic, rare familial cases occur and provide some insight into leukemogenesis. The most clearly defined familial cases result ...from inherited mutations in RUNX1 or CEBPA. Recently, novel germline mutations in GATA2 have been reported. We, therefore, investigated individuals from families with one or more first-degree relatives with myelodysplastic syndrome/acute myeloid leukemia with wild-type RUNX1 and CEBPA, for GATA2 mutations. Screening for other recurrent mutations was also performed. A GATA2 p.Thr354Met mutation was observed in a pedigree in which 2 first-degree cousins developed high-risk myelodys-plastic syndrome with monosomy 7. They were also observed to have acquired identical somatic ASXL1 mutations and both died despite stem cell transplantation. These findings confirm that germline GATA2 mutations predispose to familial myelodysplastic syndrome/acute myeloid leukemia, and that monosomy 7 and ASXL1 mutations may be recurrent secondary genetic abnormalities triggering overt malignancy in these families.
Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. ...Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based
mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in
mutant follicular lymphoma.
variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of
mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated
variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different
mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based
mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of
mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.
Summary
MicroRNAs (miRNA, miR) are negative regulators of gene expression that play an important role in diverse biological processes such as development, cell growth, apoptosis and haematopoiesis, ...suggesting their association with cancer. Here we analysed the expression signatures of 157 miRNAs in 58 diffuse large B‐cell lymphoma (DLBCL), 46 follicular lymphoma (FL) and seven non‐neoplastic lymph nodes (LN). Comparison of the possible combinations of DLBCL‐, FL‐ and LN resulted in specific DLBCL‐ and FL‐signatures, which include miRNAs with previously published function in haematopoiesis (MIRN150 and MIRN155) or tumour development (MIRN210, MIRN10A, MIRN17‐5P and MIRN145). As compared to LN, some miRNAs are differentially regulated in both lymphoma types (MIRN155, MIRN210, MIRN106A, MIRN149 and MIRN139). Conversely, some miRNAs show lymphoma‐specific aberrant expression, such as MIRN9/9*, MIRN301, MIRN338 and MIRN213 in FL and MIRN150, MIRN17‐5P, MIRN145, MIRN328 and others in DLBCL. A classification tree was computed using four miRNAs (MIRN330, MIRN17‐5P, MIRN106a and MIRN210) to correctly identify 98% of all 111 cases that were analysed in this study. Finally, eight miRNAs were found to correlate with event‐free and overall survival in DLBCL including known tumour suppressors (MIRN21, MIRN127 and MIRN34a) and oncogenes (MIRN195 and MIRNLET7G).
Follicular lymphoma (FL) is an indolent, B-cell, non-Hodgkin’s lymphoma with varying cytological appearance and clinical behavior. The genetic hallmark of FL is the t(14;18) translocation, and as a ...germinal center derived entity it is also characterized by somatic hypermutation of the immunoglobulin heavy chain (IgH) gene. In an attempt to correlate this molecular signature with the cytological grading of FL, we have analyzed the IgH variable (IgV
H
), regions in all cytological grades of FL. Four FL cases showing t(14;18) translocation were classified into grade I-III categories according to the current WHO guidelines. The IgV
H
gene segments were PCR-amplified, sequenced, and compared to their respective germline IgV
H
sequences. The neoplastic cells of grade I and II FLs revealed clonally related, but highly divergent IgV
H
gene sequences indicating the ongoing nature of somatic hypermutation. Grade III FL also showed extensive presence of somatic hypermutation, but these mutations were not associated with intraclonal divergence. Thus, these results suggest that grade I-II and grade III FL may represent different biological entities. The presence of ongoing somatic hypermutation of IgV
H
sequences in grade I and II FLs is compatible with direct follicular origin of these tumor cells, contrasting the homogenous, stable clones of grade III FL resembling a post-follicular stage of B-cell development. Our findings demonstrate that contrary to the three tiered cytological grading, molecular features of IgH genes classify FL into two distinct subcategories. These studies also suggest that with progression FL gains post-follicular–like molecular features and becomes independent of the germinal center microenvironment.
Background
Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain‐of‐function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of ...patients. However, these analyses relied on single‐site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations.
Objectives
We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies TB) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study.
Methods
Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in‐house‐designed, highly sensitive multiplex droplet digital PCR assay.
Results
EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild‐type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones.
Conclusion
The in‐depth spatio‐temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.
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