NOTCH1
 mutational status in chronic lymphocytic leukaemia: clinical relevance of subclonal mutations and mutation types

AbstractChronic lymphocytic leukaemia (CLL) exhibits variable clinical course and response to therapy, but the molecular basis of this variability remains incompletely understood. Data independent acquisition (DIA)-MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. Here, a CLL-specific spectral library (7736 proteins) is described alongside an analysis of sample replication and data handling requirements for quantitative SWATH-MS analysis of clinical samples. The analysis was performed on 6 CLL samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins across 54 SWATH-MS acquisitions: the sources of variation and different computational approaches for batch correction were assessed. Functional enrichment analysis of proteins associated with IGHV mutational status showed significant overlap with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was implemented. This study provides a valuable resource for researchers working on the proteomics of CLL. It also establishes a sound framework for the design of sufficiently powered clinical proteomics studies. Indeed, this study shows that it is possible to derive biologically plausible hypotheses from a relatively small dataset.

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Ighv Mutational Status By Deep Next Generation Sequencing Refines Ighv Sanger Sequencing Classification in Patients with Chronic Lymphocytic Leukaemia

Blood ◽

10.1182/blood-2019-129145 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3028-3028

Author(s):

Azahara Fuentes ◽

Alicia Serrano ◽

Blanca Ferrer Lores ◽

Veronica Lendinez ◽

Carolina Monzo ◽

...

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

High Throughput ◽

Research Funding ◽

Sanger Sequencing ◽

High Throughput Sequencing ◽

Magnetic Beads ◽

Lymphocytic Leukaemia ◽

Standard Technique ◽

Mutational Status ◽

Major Clone

Introduction: Determination of the mutational status of rearranged immunoglobulin heavy chain variable (IgHV) genes in patients with Chronic Lymphocytic Leukaemia (CLL), is considered one of the most important prognostic factors: patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; ≤98%). Mutational status, is currently determined by Sanger sequencing (Sseq) that allows the analysis of the major clone, however, international guidelines recommend caution in assigning mutational status in cases with "Borderline" IgHV identity (97-97.9%), and cases with double rearrangements with discordant mutational status. Objective: Analyze and determine the mutational status of the IgHV locus by High-throughput sequencing (HTS), in a cohort of CLL patients (n=51) with unclassifiable Sseq results: borderline status (n=22); double rearrangements (n=27) with discordant mutational status (n=2). Methods: We included 51 DNA samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2019 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and analysis of IgHV rearrangements were performed on DNA conforming to the updated ERIC recommendations. In all the cases we were able to determinate the IGVH identity. To switch high-throughput sequencing to the clinical practice, we assessed the reliability of different library preparation methods to sequence IGH locus in patients with CLL. Amplification was performed using the Sequencing Multiplex Kit based on IGH FR (forward primers) and consensus JH (reverse primer) multiplex. PCR products were purified using Magsi-NGS Prep magnetic beads (Magnamedics Diagnostics), normalized and pooled to create a library for sequencing using a MiSeq equipment. To simplify and make automatic the analysis of the same we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Results: This approach led to the identification of a dominant clone IgHV in all cases (n=51). Instead, the percentage of identity calculated by HTS analysis varies in: - 15/22 borderline cases whose mutational status could be recalculated into 10 MM and 5 UM. The rest 7 remaining in borderline group. - We could identify both clones in 29 double rearrangements cases, with concordant mutational status except 2/29 undetermined cases, included in UM group regarding HTS results. Our tool led to the identification of a dominant clonotypic IgHV in all cases, and when compared the HTS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, 15 out of 22 (68,18%), could be reclassified. This case showed a major clone with productive rearrangement mutated by Sseq but unmutated by HTS. Conclusions: Analyze and determine the mutational status of the IgHV locus by HTS, would potentially reveal multiple rearrangements and increase the prognostic precision of IgHV mutation analysis. IgHV-HTS classification is able to precisely classify patients with borderline status or/and multiple IgHV rearrangements for which Sseq is inconclusive. In this case, it has been possible to improved prognostication for 17 out of 24 patients. This is helping us to discover the advantages of the data obtained by HTS compared with current Sseq standard technique. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Terol: Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Research Funding; Roche: Consultancy; Astra Zeneca: Consultancy.

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Chronic lymphocytic leukaemia: An immunobiology approach

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh0806319k ◽

2008 ◽

Vol 136 (5-6) ◽

pp. 319-323 ◽

Cited By ~ 1

Author(s):

Efterpi Kostareli ◽

Tatjana Smilevska ◽

Kostas Stamatopoulos ◽

Anastasia Kouvatsi ◽

Achilles Anagnostopoulos

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

B Cell ◽

Lymphocytic Leukaemia ◽

Biological Significance ◽

Variable Region ◽

Cell Of Origin ◽

Region Gene ◽

Phenotypic Data ◽

Over Expression ◽

Mutational Status

B cell chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia that follows an extremely variable clinical course. Several important prognostic parameters defining pathogenic and clinical subgroups of CLL have been identified and validated recently. The biological significance of immunoglobulin (Ig) heavy chain variable region gene (IgHV) mutational status and associated ZAP-70 over-expression, CD38 and chromosomal aberrations have enabled to identify patients at high risk for early disease progression and inferior survival. Moreover, studies of the B cell antigen receptor (BCR) structure and receptor signaling have been most helpful in revealing some new aspects of the biology of this disease. In particular, the analysis of IG genes has revealed that the expressed IgHV/IgKV/IgLV gene repertoires of CLL cells differ from those of normal B cells. A further unique feature of the CLL IG repertoire is the existence of subsets of cases with "stereotyped" BCRs. Accumulating molecular and phenotypic data support the notion that CLL development and evolution is not a simple scholastic event and strongly indicates a role for antigen in driving the cell of origin for at least some subsets of CLL cases.

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HLA-G in B-chronic lymphocytic leukaemia: Clinical relevance and functional implications

Seminars in Cancer Biology ◽

10.1016/j.semcancer.2007.06.011 ◽

2007 ◽

Vol 17 (6) ◽

pp. 430-435 ◽

Cited By ~ 20

Author(s):

Vera Rebmann ◽

Holger Nückel ◽

Ulrich Dührsen ◽

Hans Grosse-Wilde

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Clinical Relevance ◽

Lymphocytic Leukaemia ◽

Functional Implications

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The combination of complex karyotype subtypes and IGHV mutational status identifies new prognostic and predictive groups in chronic lymphocytic leukaemia

British Journal of Cancer ◽

10.1038/s41416-019-0502-x ◽

2019 ◽

Vol 121 (2) ◽

pp. 150-156 ◽

Cited By ~ 9

Author(s):

Andrea Visentin ◽

Laura Bonaldi ◽

Gian Matteo Rigolin ◽

Francesca Romana Mauro ◽

Annalisa Martines ◽

...

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Lymphocytic Leukaemia ◽

Complex Karyotype ◽

Mutational Status

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Impaired Peripheral Blood Dendritic Cell Counts in B-Chronic Lymphocytic Leukaemia.

Blood ◽

10.1182/blood.v108.11.4966.4966 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4966-4966

Author(s):

Rudolf Horvath ◽

Vit Budinsky ◽

Robert Pytlik ◽

Pavel Klener ◽

Marek Trneny ◽

...

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Chronic Lymphocytic Leukaemia ◽

Peripheral Blood ◽

Lymphocytic Leukaemia ◽

Early Stage ◽

Primary Immune Response ◽

Cell Counts ◽

Mutational Status ◽

Plasmacytoid Dcs

Abstract B-cell chronic lymphocytic leukaemia (B-CLL) is the most common leukaemia of adults in Western worls accounting for about 40% of leukemias in adults over the age of 65 years. Although, B-CLL cells can, under certain conditions, act as antigen presenting cell (APCs) they fail to generate an efficient anti-leukemic immune response. Dendritic cells (DCs) represent the most potent APCs and they are the only cell type capable of initiating a primary immune response. To asses a potential role of DCs compartment in insufficient anti-leukemic and anti-infectious immunity in B-CLL, we have analysed peripheral blood DCs subsets in 17 patients with an early stage B-CLL and 8 age matched controls by multi colour flow cytometry. Dendritic cells were identified by lack of B, T, NK and monocyte markers, HLA-DR expression and expression of CD11c and CD123 for myeloid and plasmacytoid DCs, respectively. We found a significant reduction of absolute plasmacytoid DCs counts in patients with B-CLL (average count = 49,4×106/L) when compared to healthy donors (average count = 111,3 ×106/L, p<0,05). Myeloid DCs counts were not different from healthy controls (average count = 192,7×106/L vs. 196,7×106/L p=NS). Number of plasmacytoid DCs decreased with B-CLL cells number. DC counts did not depend on CD38 or ZAP70 expression or immunoglobulin mutational status. Phenotypic analysis of circulating plasmacytoid and myeloid DCs showed low co-stimulation profile comparing to isotype controls. Thus, the depletion of pDCs in B-CLL could represent a mechanism contributing to the poor anti-leukemic immune responses in B-CLL and/or to immune deficiency in CLL patients.

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Analysis of basal chromosome instability in patients with chronic lymphocytic leukaemia

Mutagenesis ◽

10.1093/mutage/gez009 ◽

2019 ◽

Vol 34 (3) ◽

pp. 245-252 ◽

Cited By ~ 1

Author(s):

Micaela Palmitelli ◽

Carmen Stanganelli ◽

Flavia Stella ◽

Andrea Krzywinski ◽

Raimundo Bezares ◽

...

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Lymphocytic Leukaemia ◽

Human Cancer ◽

Chromosome Instability ◽

In Situ Hybridisation ◽

Low Frequency ◽

Variable Region ◽

Western World ◽

Fluorescence In Situ Hybridisation ◽

Mutational Status

AbstractGenomic instability is a hallmark of cancer, contributing to tumour development and transformation, being chromosome instability (CIN) the most common form in human cancer. Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. In this study, we have evaluated basal CIN in untreated patients with CLL by measuring chromosome aberrations (CAs) and micronucleus (MN) frequency and their association with different prognostic factors. Seventy-two patients and 21 normal controls were analysed. Cytogenetic and fluorescence in situ hybridisation (FISH) studies were performed. IGHV (immunoglobulin heavy chain variable region) mutational status was evaluated by reverse transcription polymerase chain reaction and sequencing. An increased number of CA in patients compared with controls (P = 0.0001) was observed. Cases with abnormal karyotypes showed increased CA rate than those with normal karyotypes (P = 0.0026), with a particularly highest frequency in cases with complex karyotypes. Among FISH risk groups, a significant low frequency of CA was found in patients with no FISH alterations compared to those with del13q14 and ≥2 FISH alterations (P = 0.0074). When mean CA value (6.7%) was considered, significant differences in the distribution of low and high CA frequency between cases with normal and abnormal karyotypes (P = 0.002) were observed. By MN analysis, higher frequency in patients compared to controls (P = 0.0001) was also found, as well as between cases with ≥2 FISH abnormalities and those with no FISH alterations (P = 0.026). Similarly, significant differences were observed when patients were divided according to mean MN frequency (2.2%; P ≤ 0.04). Interestingly, patients with high MN frequency had shorter time to first treatment than those with low frequency (P = 0.024). Cases with mutated and unmutated IGHV status showed increased CA and MN frequencies compared to controls (P ≤ 0.0007), but no differences between both groups were found. Our results support the strong interaction between CIN and genomic complexity as well as their influence on poor outcome in this pathology.

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A biclonal case of chronic lymphocytic leukaemia with discordant mutational status of the immunoglobulin heavy chain variable region and bimodal CD49d expression

British Journal of Haematology ◽

10.1111/bjh.17257 ◽

2020 ◽

Author(s):

Anya K. Hotinski, ◽

Oliver G. Best, ◽

Lauren A. Thurgood, ◽

Karen M. Lower ◽

Bryone J. Kuss

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Heavy Chain ◽

Lymphocytic Leukaemia ◽

Variable Region ◽

Immunoglobulin Heavy Chain ◽

Heavy Chain Variable Region ◽

Mutational Status

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The diagnostic chronic lymphocytic leukaemia genome by nanopore sequencing

10.1101/750059 ◽

2019 ◽

Cited By ~ 1

Author(s):

Adam Burns ◽

Daniella DiSalvo-Williams ◽

David Bruce ◽

Pauline Robbe ◽

Adele Timbs ◽

...

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Large Scale ◽

Lymphocytic Leukaemia ◽

Low Cost ◽

Predictive Biomarkers ◽

Nanopore Sequencing ◽

Genomic Deletions ◽

Mutational Status ◽

Short Period ◽

Following Error

AbstractChronic lymphocytic leukaemia (CLL) is characterised by considerable clinical and biological heterogeneity, with specific recurrent genomic alterations, includingTP53mutations, deletions of chromosome 17p, and IgHV mutational status, impacting on response to chemo-immunotherapy and targeted agents. Consequently, diagnostic screening for these predictive biomarkers is recommended in both national and international clinical guidelines. Current conventional methods, including fluorescentin-situhybridisation and Sanger sequencing, exhibit shortcomings in terms of cost, speed and sensitivity, and even second-generation sequencing methods encounter technical limitations imposed by short-read lengths and bio-informatics analysis. The MinION platform from Oxford Nanopore Technologies generates exceptionally long (1-100kbp) read lengths in a short period of time and at low cost, making it a good candidate for diagnostic testing. In this paper, we present a nanopore-based CLL-specific screening assay, to simultaneously screen for bothTP53mutations and del17p13.1, as well as determining the IgHV mutation status for a single patient in one sequencing run. We sequenced 11 CLL patients and were able to generate a full diagnostic dataset for all. We identified somatic SNVs and indels in the coding region ofTP53, and demonstrate that, following error correction of the data, we could accurately define the somatically hypermutated IgHV region in all patients. We also demonstrated the ability of the MinION platform to detect large-scale genomic deletions through low-coverage whole-genome sequencing. We conclude that nanopore sequencing has the potential to provide accurate, low-cost and rapid diagnostic information, which could be applied to other cancer types.

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