| 1. |
- Marincevic, Millaray, 1983-
(författare)
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Array-based Characterization of Chronic Lymphocytic Leukemia : - with Focus on Subsets Carrying Stereotyped B-cell Receptors
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In chronic lymphocytic leukemia (CLL), the presence of multiple subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has implicated antigen(s) in leukemogenesis. These stereotyped subsets display similar immunoglobulin (IG) gene usage, almost identical complementarity determining region 3’s and may share clinical features. For instance, subsets #1 (IGHV1/5/7/IGKV1-39) and #2 (IGHV3-21/IGLV3-21) have inferior outcome compared to non-subset patients, whereas subset #4 (IGHV4-34/IGKV2-30) display a favourable prognosis. The aim of this thesis was to investigate genomic aberrations, gene expression patterns and methylation profiles in stereotyped subsets and compare epigenetic profiles in CLL and mantle cell lymphoma (MCL). In paper I, we investigated genomic aberrations in subsets #2, #4 and #16 and in non-stereotyped samples (n=101) using high-density 250K SNP arrays. Subset #2 and non-subset #2 IGHV3-21 cases displayed a higher frequency of aberrations than subset #4 cases. The high incidence of del(11q) in both subset #2/non-subset #2 may reflect the adverse survival reported for IGHV3-21 patients. In contrast, the lower frequency of genetic events and lack of poor-prognostic aberrations in subset #4 may partially explain their indolent disease. In paper II, we analysed the global RNA expression in subset #4, #16 and non-subset IGHV4-34 CLL patients (n=25). Subsets #4 and 16 showed distinct gene expression profiles, where genes involved in cell regulatory pathways were significantly lower expressed in subset #4, in line with their low-proliferative disease. In paper III, a genome-wide methylation array was applied to investigate methylation profiles in subsets #1, #2 and #4 (n=39). We identified differential methylation patterns for all subsets and found affected genes to be involved in e.g. apoptosis and therapy resistance. When performing functional annotation, a clear enrichment of genes involved in adaptive immunity was observed. These genes were preferentially methylated in subset #1 when compared to either subset #2 or #4, possibly due to different antigen responses. In paper IV, the genome-wide methylation profiles for 30 CLL and 20 MCL patients were investigated. Distinct methylation profiles were observed, where MCL displayed a more homogeneous profile. Homeobox transcription factor genes showed a higher degree of methylation in MCL, while apoptosis-related genes and proliferation-associated genes were methylated in CLL. In summary, this thesis demonstrates that stereotyped CLL subsets display differences in gene expression profiles, genetic aberrations and methylation patterns, underscoring the functional relevance of subgrouping according to BCR stereotypy. The distinct methylation profiles of CLL and MCL suggests that different epigenetic mechanisms are involved in the pathogenesis of these B-cell malignancies.
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| 2. |
- Marincevic, Millaray, 1983-
(författare)
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Chronic lymphocytic leukemia subsets with stereotyped B cell receptors are distinguished by unique methylation patterns
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Epigenetic modifications, such as DNA methylation, are crucial for both normal and tumor cell development. In chronic lymphocytic leukemia (CLL), multiple subsets have been reported that carry closely homologous, “stereotyped” B cell receptors (BCRs) with varying clinical outcome. To compare the methylation profiles of stereotyped CLL subsets, we here applied high-resolution methylation arrays (27,578 CpG sites) to 3 major subsets, i.e. poor-prognostic subsets #1 (IGHV1/5/7/IGKV1-39/1D-39, n=15) and #2 (IGHV3-21/IGLV3-21, n=9) and favorable-prognostic subset #4 (IGHV4-34/IGKV2-30, n=15). Overall, our results demonstrated significantly different and unique methylation profiles between these stereotyped subsets. Of particular interest was the observation that genes involved in apoptosis (e.g. RIPK3, AATK and TCF3) were methylated in subset #1 compared to subset #4, whilst genes involved in therapy resistance (e.g. ABCB11 and ITGAM) were unmethylated in subset #2 when compared to subset #4. Functional annotation of the differentially methylated genes revealed marked differences in genes involved in the adaptive immune response (e.g. CD80 and CD86), which generally remained unmethylated in subsets #2 and #4 in contrast to subset #1, indicating potential differences in antigenic response between subsets. Taken together, this data convincingly illustrates the distinct methylation profiles amongst stereotyped CLL cases and highlights a key role for epigenetics in disease pathogenesis.
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| 3. |
- Marincevic, Millaray, 1983-, et al.
(författare)
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Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B cell receptors
- 2010
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 95:12, s. 2072-2079
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Tidskriftsartikel (refereegranskat)abstract
- Background Numerous subsets of patients with chronic lymphocytic leukemia display similar immunoglobulin gene usage with almost identical complementarity determining region 3 sequences. Among IGHV4-34 cases, two such subsets with "stereotyped" B-cell receptors were recently identified, i.e. subset #4 (IGHV4-34/IGKV2-30) and subset #16 (IGHV4-34/IGKV3-20). Subset #4 patients appear to share biological and clinical features, e.g. young age at diagnosis and indolent disease, whereas little is known about subset #16 at a clinical level. DESIGN AND METHODS: We investigated the global gene expression pattern in sorted chronic lymphocytic leukemia cells from 25 subset/non-subset IGHV4-34 patients using Affymetrix gene expression arrays. RESULTS: Although generally few differences were found when comparing subset to non-subset 4/16 IGHV4-34 cases, distinct gene expression profiles were revealed for subset #4 versus subset #16. The differentially expressed genes, predominantly with lower expression in subset #4 patients, are involved in important cell regulatory pathways including cell-cycle control, proliferation and immune response, which may partly explain the low-proliferative disease observed in subset #4 patients. Conclusions Our novel data demonstrate distinct gene expression profiles among patients with stereotyped IGHV4-34 B-cell receptors, providing further evidence for biological differences in the pathogenesis of these subsets and underscoring the functional relevance of subset assignment based on B-cell receptor sequence features.
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| 4. |
- Marincevic, Millaray, 1983-
(författare)
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Genome-wide array-based methylation profiling reveals preferential methylation of homeobox transcription factor genes in mantle cell lymphoma and pro-apoptotic genes in chronic lymphocytic leukemia
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies that differ with regards to biological and clinical characteristics. Aberrant DNA methylation has been described for each disorder, but comparison between the two entities is lacking. We employed high-resolution methylation microarrays (27,578 CpG sites) to compare methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 IGHV mutated, stereotyped subset #4 and 15 IGHV unmutated, stereotyped subset #1) samples. Distinct differences in methylation patterns were revealed between MCL and CLL, where MCL displayed a more homogenous profile. When comparing MCL and CLL/subsets, 51 genes were consistently identified as differentially methylated in all comparisons. Among 19 genes methylated in MCL, six were homeobox transcription factors (e.g. HLXB9, HOXA13), whereas unmethylated loci in MCL included genes enhancing proliferation and tumor progression (e.g. MERTK and CAMP). In contrast, apoptosis-related genes were prominent among genes methylated in CLL (e.g. DYRK2 and CYFIP2). Correlation between promoter methylation and gene expression was confirmed for selected genes using RQ-PCR. Notably, homeobox genes were not expressed in either entity, implying different gene silencing mechanisms in MCL and CLL. In summary, the differences in global methylation profiles between MCL and CLL have unfolded distinct epigenetic mechanisms operating in each disease.
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| 5. |
- Marincevic, Millaray, 1983-, et al.
(författare)
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High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors
- 2010
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 95:9, s. 1519-1525
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Tidskriftsartikel (refereegranskat)abstract
- Background The existence of multiple subsets of chronic lymphocytic leukemia expressing 'stereotyped' B-cell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that 'stereotypy' may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets. Design and Methods We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 ICHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients. Results Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and non-subset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy. Conclusions Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.
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| 6. |
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| 7. |
- Modvig, S, et al.
(författare)
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Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia
- 2019
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Ingår i: Leukemia. - : Nature Publishing Group. - 0887-6924 .- 1476-5551. ; 33:6, s. 1324-1336
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Tidskriftsartikel (refereegranskat)abstract
- Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10-3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10-3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10-3 compared with MRD < 10-3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10-4-<10-3 had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10-4 or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10-4 had a cumulative incidence of relapse of 2.3% (95% CI 0-6.8, n = 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.
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| 8. |
- Modvig, S, et al.
(författare)
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Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.
- 2021
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Ingår i: Leukemia. - : Springer Science and Business Media LLC. - 1476-5551 .- 0887-6924. ; 35, s. 1894-1906
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Tidskriftsartikel (refereegranskat)abstract
- PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p<0.0001), 29 (HzR 2.7, p<0.0001), and 79 (HzR 3.5, p<0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4×10-2 versus 5.2×10-3, p<0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y=3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD>10-4 associated with a CIR5y=22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.
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| 9. |
- Paulsen, Karin, et al.
(författare)
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LymphoTrack Is Equally Sensitive as PCR GeneScan and Sanger Sequencing for Detection of Clonal Rearrangements in ALL Patients
- 2022
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Ingår i: Diagnostics. - : MDPI AG. - 2075-4418. ; 12:6
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal rearrangements of immunoglobulin (Ig) genes and T-cell receptor (TCR) genes are used for minimal measurable disease in acute lymphoblastic leukemia (ALL). The golden standard for screening of gene rearrangements in ALL has been PCR GeneScan and Sanger sequencing, which are laborsome and time-consuming methods. More rapid next-generation sequencing methods, such as LymphoTrack could possibly replace PCR GeneScan and Sanger sequencing for clonality assessment. Our aim was to evaluate to what extent LymphoTrack can replace PCR GeneScan and Sanger sequencing concerning sensitivity and quantifiability in clonality assessment in 78 ALL samples. With LymphoTrack, clonality assessment was based on the %Total reads, where >= 10% was used as cut off for clonal rearrangements. The patients displayed 0 to 4 clonal rearrangements per assay. The detection rate (rearrangements detected with PCR GeneScan and/or Sanger sequencing, also detected with LymphoTrack) was 85/85 (100%) for IGH, 64/67 (96%) for IGK, 91/93 (98%) for TCRG and 34/35 (97%) for TCRB. Our findings demonstrate that LymphoTrack was equally sensitive in detecting clonal rearrangements as PCR GeneScan and Sanger Sequencing. The LymphoTrack assay is reliable and therefore applicable for clonal assessment in ALL patients in clinical laboratories.
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