We used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal ...gammopathy of undetermined significance MGUS, presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell–cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.
Limited data exists to show the correlation of (tumour protein 53)
mutation detected by Next generation sequencing (NGS) and the presence/absence of deletions of 17p13 detected by FISH. The study ...which is the largest series to date includes 2332 CLL patients referred for analysis of del(17p) by FISH and
mutations by NGS before treatment. Using a 10% variant allele frequency (VAF) threshold, cases were segregated into high burden mutations (≥10%) and low burden mutations (<10%).
aberrations (17p del(17p) and/or
mutation) were detected in 320/2332 patients (13.7%). Using NGS analysis, 429
mutations were identified in 303 patients (13%). Of these 238 (79%) and 65 (21%) were cases with high burden and low burden mutations respectively. In our cohort, 2012 cases did not demonstrate a
aberration (86.3%). A total of 159 cases showed
mutations in the absence of del(17p) (49/159 with low burden
mutations) and 144 cases had both
mutation and del(17p) (16/144 with low burden mutations). Only 17/2332 (0.7%) cases demonstrated del(17p) with no
mutation. Validated NGS protocols should be used in clinical decision making to avoid missing low-burden
mutations and can detect the vast majority of
aberrations.
In situ hybridisation in tissue sections Chiecchio, Laura
Diagnostic histopathology (Oxford, England : 2008),
November 2020, 2020-11-00, Letnik:
26, Številka:
11
Journal Article
Recenzirano
The identification of recurrent tumour-specific chromosomal abnormalities, including rearrangements and copy number changes, has shown increasingly important diagnostic, therapeutic and prognostic ...implications. In surgical pathology, fluorescence in situ hybridization (FISH) is a quick and reliable method for the detection of many genetics aberrations, and can be applied to a variety of specimens including formalin-fixed paraffin-embedded (FFPE) tissues; as such, it has been successfully incorporated into diagnostic practice for lymphomas, mesenchymal and other solid tumours. The aim of this review is to discuss the utility, strengths and limitations of FISH when applied to the study of tumour tissue sections, with particular focus on the challenges associated with interpretation.
Multiple myeloma, monoclonal gammopathy of undetermined significance and smoldering multiple myeloma harbor common chromosomal abnormalities but the prevalence and relative association of aberrations ...in these diagnostic groups remains controversial. We investigated these aspects in a large series of patients.
Chromosome 13 deletion (Delta13), deletion of TP53, ploidy status and immunoglobulin heavy chain (IgH) translocations were evaluated by fluorescence in situ hybridization in patients with monoclonal gammopathy of undetermined significance (n=189), smoldering multiple myeloma (n=127) and multiple myeloma (n=400).
Overall, Delta13 (25%, 34% and 47%), 16q23 deletions (6%, 8% and 21%) and 17p13 deletions (3%, 1% and 10%) were less frequent in patients with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in those with multiple myeloma. When distinct genetic groups were considered, no differences in the prevalence of Delta13 were found with t(4;14)(p16;q32) and t(14;16)(q32;q23) among the three diagnostic groups; in contrast Delta13 was rarer in t(11;14)(q13;q32) in patients with monoclonal gammopathy (1/28) and smoldering myeloma (2/13) than in those with multiple myeloma (40%). Similar results were seen for the few t(6;14)(p21;q32) cases: 0/3 patients with monoclonal gammopathy or smoldering myeloma had the Delta13, whereas 4/6 (67%) patients with multiple myeloma and this translocation also had the deletion. In multiple myeloma patients with both an IgH translocation and Delta13, the proportions of cells affected by the two abnormalities were similar, as was the case for t(4;14) and t(14;16) monoclonal gammopathy patients positive for Delta13. In contrast, in monoclonal gammopathy patients with t(14;20)(q32;q11), the translocation was present in almost all cells, while the Delta13 was present in only a sub-population.
These results indicate that the presence and time of occurrence of Delta13 depends on the presence of specific concurrent abnormalities. The observation that Delta13 was extremely rare in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma with translocations directly involving cyclin D genes (CCND1 and CCND3) suggest a possible role of Delta13 in the progression of the disease specifically in these genetic sub-groups. (clinicaltrials.gov identifier: ISRCTN 68454111; UKCRN ID 1176).
A large series of plasma cell dyscrasias (n=2207) was examined for translocations which deregulate the MAF genes, t(14;20)(q32;q12) and t(14;16)(q32;q23), and their disease behavior was compared to a ...group characterized by the t(4;14)(p16;q32) where CCND2 is also up-regulated. The t(14;20) showed low prevalence in myeloma (27/1830, 1.5%) and smoldering myeloma (1/148, <1%) with a higher incidence in MGUS (9/193, 5% P=0.005). Strong associations with del(13) (76%), non-hyperdiploidy (83%) and gain of 1q (58%) were seen but no association with an IgA M-protein or absence of bone disease was noted. All three translocations were associated with poor outcome in myeloma, but strikingly all t(14;20) MGUS/smoldering myeloma cases (n=10) had stable, low level disease. In contrast, the 10 t(14;16) and 25 t(4;14) MGUS/smoldering myeloma cases were associated with both evolving and non-evolving disease. None of the associated genetic abnormalities helped to predict for progression from MGUS or smoldering myeloma.
We performed fluorescent in situ hybridization (FISH) for 16q23 abnormalities in 861 patients with newly diagnosed multiple myeloma and identified deletion of 16q del(16q) in 19.5%. In 467 cases in ...which demographic and survival data were available, del(16q) was associated with a worse overall survival (OS). It was an independent prognostic marker and conferred additional adverse survival impact in cases with the known poor-risk cytogenetic factors t(4;14) and del(17p). Gene expression profiling and gene mapping using 500K single-nucleotide polymorphism (SNP) mapping arrays revealed loss of heterozygosity (LOH) involving 3 regions: the whole of 16q, a region centered on 16q12 (the location of CYLD), and a region centered on 16q23 (the location of the WW domain-containing oxidoreductase gene WWOX). CYLD is a negative regulator of the NF-κB pathway, and cases with low expression of CYLD were used to define a “low-CYLD signature.” Cases with 16q LOH or t(14;16) had significantly reduced WWOX expression. WWOX, the site of the translocation breakpoint in t(14;16) cases, is a known tumor suppressor gene involved in apoptosis, and we were able to generate a “low-WWOX signature” defined by WWOX expression. These 2 genes and their corresponding pathways provide an important insight into the potential mechanisms by which 16q LOH confers poor prognosis.
Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential ...target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material.
We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis.
By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor.
Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.
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