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

Blood ◽  
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.

Next-Generation Deep Sequencing Reveals Multiple Ighv Clones in One Third of CLL Patients Defining New Prognostic Subgroups and Improving Previous Classification

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4127-4127
Author(s):  
Basile Stamatopoulos ◽  
Adele Timbs ◽  
Hélène Dreau ◽  
Ruth Clifford ◽  
Pauline Robbe ◽  
...  
Keyword(s):  
B Cell ◽  
Sanger Sequencing ◽  
Magnetic Beads ◽  
Lymphocytic Leukemia ◽  
Median Frequency ◽  
Next Generation ◽  
Mutational Status ◽  
Median Error ◽  
Major Clone ◽  
The Impact

Abstract Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by a heterogeneous clinical course. Currently, the mutational status of the immunoglobulin heavy chain variable (IgHV) region defines 2 risk groups: patients with ≥ 2% difference from the germline are considered as unmutated (UM) and have a poor prognosis while the opposite is observed for mutated (M) patients (Hamblin et al, 1999). Until now, clonality has been determined using PCR/gel electrophoresis and the percentage of IgHV mutations by Sanger sequencing using the Biomed-2 method (van Dongen, Leukemia 2003) which only allows for analysis of the major clone and assumes that only one clone per patient should be considered. Methods: in the present study, we sequenced the IgHV gene for 200 CLL patients with a median follow-up of 70 months (range, 1-309) by next-generation (NGS) and Sanger sequencing (SS) and investigated the impact on prognosis for the different subgroups. Briefly, 100ng of cDNA generated from RNA extracted from highly purified CD19+ cells obtained at diagnosis was amplified using IGH LEADER master mixes. For SS, clonality of PCR product was determined and monoclonal samples were directly sequenced. For NGS, PCR products were purified using magnetic beads, normalized and pooled to create a library for sequencing using the MiSeq v3 Reagent kit (600 cycles). Sequencing data was analyzed using LymphoTrack™ bioinformatics software and percentage of clone was based on the first 200 most abundant clones detected. Clones with an abundancy of < 2.5% were not considered. The IMGT database was used to calculate the percentage of mutation compared to germline. Results: Using SS, 49% (98/200) of patients were IgHV M, 38% (76/200) UM, while 13% (26/200) were polyclonal and further Sanger sequencing was inconclusive. Concordance between SS and NGS was 98.3%: for 90.2%, the major clone detected by NGS was the same by SS, for 8.5% the clone was the same but the percentage mutated compared to germ-line differed slightly with a median error of 0.6%, while for 1.7%, SS detected another clone which was less abundant by NGS. Interestingly, among patients considered as monoclonal by SS, 25.3% (44/174) were found to be polyclonal by NGS. NGS revealed that 35% (70/200) of patients display different IgHV re-arrangements in the same patient: the median frequency of the first two most abundant clones were 78.2% (range, 28.5-94.9) and 16.4% (range, 2.5-49.0) respectively. This implies that at least in these patients, leukemia-initiating events must occur prior to IgHV re-arrangement in a pro-B cell. Further in-depth studies of the HSC and early B cell progenitor compartments in these patients will be required to confirm these observations. In terms of prognosis, M, UM and polyclonal patients determined by SS had a median treatment-free survival (TFS) of 178, 29 and 129 months respectively (P<0.0001) and a median overall survival (OS) of >309, 183 and >309 months (P<0.0001). Since NGS was able to highlight different clones, we were able to create 5 different categories: patients with (a) multiple M clones, (b) 1 M clone, (c) a mix of M-UM clones, (d) 1 UM clone, (e) multiple UM clones. Using this new NGS classification, we found patients with different prognosis among patients already classified by SS. For example, SS-M patients were classified in 3 subgroups with a median TFS of >251 (a), 178 (b), 56 (c) months (P=0.0014). Similar results were observed for SS-UM patients who were divided in 3 subgroups with a median TFS of 94 (c), 24 (d), and 19 (e) month (P=0.0296). When all patients where considered, IgHV-NGS classification stratified patients in 5 different subgroups with median TFS of >251 (a), 178 (b), 57 (c), 24 (d), 19 (e) months (P<0.0001) and a median OS of 270, >309, >309, 183, 88 months (P<0.0001). Conclusions: determination of IgHV mutational status by NGS has excellent concordance with classical SS and in addition enables the detection of small clones which has a significant impact on prognosis. This allows the analysis of all clones without labor intensive manipulations especially for polyclonal patients. Here we showed for the first time that one third of CLL patients present with multiple IgHV subclones that impact on prognosis and refine the previous SS-IgHV classification. Figure 1. Figure 1. Disclosures Schuh: Acerta Pharma BV: Research Funding.

scholarly journals Detection of Immunoglobulin Heavy Chain Gene Clonality By High-Throughput Sequencing for Minimal Residual Disease Monitoring in Chronic Lymphocytic Leukaemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1747-1747
Author(s):  
Alicia Serrano ◽  
Azahara Fuentes ◽  
Blanca Ferrer Lores ◽  
Veronica Lendinez ◽  
Carolina Monzo ◽  
...  
Keyword(s):  
Research Funding ◽  
High Throughput Sequencing ◽  
Lymphocytic Leukaemia ◽  
Residual Disease ◽  
Data Summarization ◽  
Bioinformatic Pipeline ◽  
Mutational Status ◽  
Final Data ◽  
Minimal Residual

Introduction: The negative minimal residual disease (MRD) after treatment has been recently accepted as endpoint for Chronic Lymphocytic Leukaemia (CLL) clinical trials. Conventionally, MRD can be detected by using multi-color Flow Cytometry (FC) with high sensitivity. Determination of the clonal immunoglobulin gene rearrangement can be a useful monitoring marker in a broad range of B-cell lymphoproliferative neoplasms. Moreover, the mutational status of immunoglobulin heavy chain variable (IgHV) rearrangement is considered one of the most important prognostic factors in CLL. Therefore, the identification of the IgHV rearrangement can be a useful marker both at diagnostic and as monitoring marker for MRD. Nowadays, high-throughput sequencing (HTS) technologies has enabled highly sensitive cancer genomic testing in clinical laboratories. There are same initiatives based on HTS to use IgHV rearrangement as marker for MRD monitoring in Acute lymphoblastic leukemia or multiple myeloma, but it remains unharmonized for application on CLL in the clinical laboratory. Objective: We evaluated the performance and clinical applicability of HTS assay for IgHV rearrangement in CLL MRD monitoring in 69 samples from 19 CLL patients treated. Methods: The libraries including IGH locus were performed using the Sequencing Multiplex Kit on IGH consensus primers. To simplify and make automatic the analysis of the data obtained, 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. Assessment of IgHV mutational status by Sseq, genomic DNA (gDNA; 50-100 ng), were used for IgHV analysis. gDNA was amplified using locus-specific primer sets for IgHV designed to allow for the amplification of all known alleles of the germline IgH sequence, as described previously. Inmunophenotypic studies were performed on erythrocyte-lysed whole PB samples according to Euroflow procedures. PB white blood cells (WBC) was systematically stained with the eight color combination panel recently proposed by the ERIC group for MRD detection (Rawstron AC et al. 2016). Data acquisition was performed on a FACSCanto II flow cytometer Becton-Dickinson Biociences using the FACSDiva software (V8.0; BD). For data analysis, the Infinicyt softwareTM (Cytognos SL, Salamanca, Spain) was used. The MRD levels were reported as fraction of CLL cells of all nucleated cells. MRD negativity was define as a fraction <10 -4 However, for treatments that preferentially clear the blood (for example monoclonal antibodies) the MRD was confirm in the bone marrow as has been recommended. Results: Patient demographics and the results of IGH clonality tests are summarized in Table 1. Interpretable results were obtained with higher sensitivity compared with Sseq at diagnosis stage (19/19 samples), and we are able to determine the same clone at subsequent samples. Among 50 follow-up samples, 44 MRD were positive in both techniques (HTS and FC); 3 follow-up samples, were negative in both determinations (HTS and FC); in contrast, 3 follow-up samples, were negative by FC but positive by HTS (8.3, 17.2, 18.2 samples). This is the case of Patient 8, in which the last follow-up sample, the detection of the primary clone is positive by HTS, while by FC is undetected (Figure 1). In addition, it was possible to detect IgHV clone in all Patient 7 samples. It was under long-term monitoring by HTS and FC, and 17 months after initial diagnosis and first line of treatment (7.5 sample), it was detected a loss of response (Figure 2.). Conclusions: We evaluated an HTS IgHV assay using initial and follow-up samples of 19 CLL patients. Using our pre-designed primer set in library preparation, and developed our specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation, we were able to demonstrate that our method was more sensitive than FC in detecting positive follow-up samples and could be able to propose it for MRD monitoring in CLL. However, the prognostic impact of these low-level MRD detected by HTS should be validated with further investigations. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Ortiz: GILEAD SCIENCES: Research Funding. Terol:Abbvie: Consultancy; Gilead: Research Funding; Janssen: Consultancy, Research Funding; Astra Zeneca: Consultancy; Roche: Consultancy.

Emergence of Fc-Gamma-Riib-Dominance Contributes to Resistance to Therapeutic Antibodies in Patients with Chronic Lymphocytic Leukaemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 447-447
Author(s):  
Melinda Burgess ◽  
Sally Mapp ◽  
Roberta Mazzieri ◽  
Jonathan Ellis ◽  
Catherine Cheung ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukaemia ◽  
Primary Cultures ◽  
Differential Sensitivity ◽  
Moderate Activity ◽  
Therapeutic Antibodies ◽  
Advisory Committees ◽  
Antibody Resistance

Abstract Aim: Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia. Whilst therapeutic antibodies show clinical activity in CLL patients, resistance develops. Thus, identifying mechanisms of antibody resistance and methods to reduce resistance would be valuable in managing CLL. Results: In this study we show that a therapeutic antibody against CD62L is able to induce antibody-dependent cell mediated cytotoxicity (ADCC) and phagocytosis (ADP) in primary cultures of CLL cells. Significantly, we observed that patients with stable disease retained sensitivity to CD62L-Ab whilst untreated patients, whose disease progressed, became progressively resistant to CD62L-Ab. Using strategies to enrich for monocytes we were able to show that the CD62L-Ab dependent killing was attributable to an FcγR-dependent mechanism within the monocyte derived cell (MDC) fraction of PBMCs. Transcriptomic profiling and marker analysis indicated that the MDCs acquired a macrophage phenotype. Both MDCs from antibody-sensitive or antibody-resistant patients were able to bind Ab-bound CLL cells equally. Moreover, resistance could not be attributed to reduced numbers of monocytes or macrophages or to distinct subtypes of monocytes or macrophages. Using pharmacological inhibitors of the activating pathway of FcγR signaling and the inhibitory FcγRIIB pathway we were able to show that the antibody resistance in MDCs, derived from patients with CLL, was due to the emerging dominance of the FcγRIIb pathway relative to the activating FcγR pathways. We examined whether the differential sensitivity to CD62L-Ab was also evident for anti-CD20 antibodies used clinically for CLL. Rituximab showed only moderate activity in vitro and no clear difference in cytotoxicity was observed between patients who were previously identified as being resistant or sensitive to the CD62L antibody. Obinutuzumab invoked similar differential cell killing in PBMCs from patients sensitive to, or resistant to, CD62L-Ab. Further comparison indicated that CD62L-Ab and obinutuzumab induced similar malignant B cell binding to MDCs and ADP in contrast to rituximab. Finally, similar to anti-CD62L, ADCC/ADP response to obinutuzumab was reduced following treatment of sensitive cultures with a syk or BTK inhibitor and increased in MDCs derived from resistant patients treated with a Ship1 inhibitor. Conclusions: These data establish, for the first time, that MDCs derived from CLL patients may switch from an antibody sensitive phenotype to an antibody-resistant phenotype as disease progresses. Significantly, we show that the resistance to MDC-mediated ADCC/ADP may be reversed by the inhibition of FcγRIIB with pharmacological modifiers. Disclosures Mollee: Onyx: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Gill:Sanofi Aventis: Research Funding; Roche: Honoraria; AbbVie: Honoraria; Roche: Research Funding.

Novel Targeted Therapies and Their Impact on Survival from Multiple Myeloma (MM) and Chronic Lymphocytic Leukaemia (CLL) in the Real World

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3546-3546
Author(s):  
Alexandra G Smith ◽  
Timothy Bagguley ◽  
Eve Roman ◽  
Andy C Rawstron ◽  
James R Bailey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Chronic Lymphocytic Leukaemia ◽  
Combination Chemotherapy ◽  
Real World ◽  
Research Funding ◽  
Lymphocytic Leukaemia ◽  
Alkylating Agents ◽  
First Line ◽  
Time Periods ◽  
Line Chemotherapy

Abstract Introduction The treatment landscape for many mature B-cell malignancies is evolving rapidly, with patients and clinicians facing increasingly complex choices about therapeutic options that differ in efficacy, toxicity and cost. Accounting for around a quarter of all haematological cancer diagnoses, multiple myeloma (MM) and chronic lymphocytic leukaemia (CLL) are two conditions where increases in the number and combinations of potentially life-prolonging therapies has been particularly marked; ranging from the use of single alkylating agents to immunomodulatory drugs and proteasome inhibitors for MM, and combination chemotherapy, immuno-chemotherapy, novel monoclonal antibodies and tyrosine-kinase inhibitors (TKIs) for CLL. Contemporary data enabling the success of such therapeutic changes to be evaluated in the general patient population is, however, lacking. With centralized diagnostics and a unified clinical network covering a catchment population of 4 million, the UK's Haematological Malignancy Research Network (www.hmrn.org) was specifically established to provide timely real-world data to answer such questions; and findings from this unique population-based cohort are reported here. Methods Patients newly diagnosed 2004-13 with MM (n=2084) or CLL (n=1866) were followed-up until January 2016. Demographic, prognostic, first-line treatment and outcome data for the time-periods 2004-07, 2008-10 and 2011-15 were examined using standard statistical methods; relative survival (RS) was estimated using national life tables. Results The median age at diagnosis of MM was 73 years (17% <60 years); 39% of patients presented with an ISS score of III and 25% were asymptomatic (CRAB score 0). In total, 1514 (73%) patients received first-line chemotherapy either at diagnosis or as a consequence of disease progression. Regimens were classified by their main agent, and the therapy changes over the 11-year period are shown in Figure 1a; in 2004-07, 44% of treated patients received single-agent alkylating therapy, in 2008-10 76% were treated with combination immunomodulatory therapy and by 2011-15 this had increased to 92%. The 3-year overall survival (OS) and RS estimates for all patients combined were 45.9% (95% Confidence Interval 43.4-48.4) and 52.0% (49.1-54.8) respectively. Differences in outcome by treatment year are clearly evident (Figure 1b): 3-year RS 2004-07, 46.5% (41.8-51.2); 2008-10, 48.4% (43.5-53.2); and 2011-15, 62.1% (56.8-66.9). The improvement in survival for patients treated in 2011-15 compared to 2004-07 was confirmed by multivariate Cox regression (Hazard Ratio 0.65, 0.56-0.76). With a median diagnostic age of 71 years (18% <60 years); the majority of CLL patients had early-stage disease (BinetStage A, 78%). In total 547 patients were treated with first-line chemotherapy, with the regimen again changing over time (Figure 1c). Patients treated 2004-07 generally received single alkylating agents (56%) or combination chemotherapy (42%), by 2008-10 32% of patients had a monoclonal antibody added to chemotherapy (chemo-immunotherapy), increasing to 72% among those treated 2011-15. The 3-year OS and RS for all treated patients combined were 69.5% (65.3-73.3) and 80.3% (75.5-84.3) respectively. However, there was no incremental statistically significant change in 3-year RS (Figure 1d); 2004-07, 76.4% (65.2-84.4); 2008-10, 78.3% (69.8-84.6); and 2011-15 84% (76.3-89.4); and taking 2004-07 as the reference, the corresponding hazard ratios for the 3 time-periods were 1 (reference), 1.00 (0.78-1.37) and 0.79 (0.58-1.09). The cost implications of the changing treatment landscape are currently being examined, and by December 2016 the findings presented above will include more recently diagnosed patients (2014-15), which is particularly pertinent for CLL, where a step-change may have occurred due to the introduction of TKIs. Conclusions Our analyses confirm that first-line chemotherapy for MM and CLL is changing markedly; highlighting the importance of monitoring the impact of therapeutic change in a real-world setting. The improvement in MM survival currently contrasts with CLL, suggesting that encouraging results from clinical trials may not always translate directly into similar improvements at a population level. Clearly, additional analysis of data from patients diagnosed >2014 are required. Figure 1 Figure 1. Disclosures Smith: Novartis: Research Funding; Janssen-Cilag: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Cook:Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda Oncology: Consultancy, Research Funding, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Glycomimetics: Consultancy. Patmore:Roche: Honoraria; Janssen Cilag: Honoraria.

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

2017 ◽  
Vol 182 (4) ◽  
pp. 597-602 ◽  
Author(s):  
Tiziana D'Agaro ◽  
Tamara Bittolo ◽  
Vanessa Bravin ◽  
Michele Dal Bo ◽  
Federico Pozzo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Clinical Relevance ◽  
Lymphocytic Leukaemia ◽  
Mutational Status

scholarly journals A TP53-mutáció-analízis jelentősége krónikus lymphocytás leukaemiában

2017 ◽  
Vol 158 (6) ◽  
pp. 220-228
Author(s):  
Viktória Fésüs ◽  
Dóra Marosvári ◽  
Béla Kajtár ◽  
Péter Attila Király ◽  
Judit Demeter ◽  
...  
Keyword(s):  
High Risk ◽  
Chronic Lymphocytic Leukaemia ◽  
Mutation Analysis ◽  
Sanger Sequencing ◽  
Tp53 Mutation ◽  
Lymphocytic Leukaemia ◽  
Tp53 Mutations ◽  
Risk Patients ◽  
Innovative Medicine ◽  
Made In

Abstract: Introduction: In recent years much progress has been made in the therapy of chronic lymphocytic leukaemia, as the new innovative medicine proved to be effective in managing patients carrying TP53 abnormalities. To identify all these patients, it is essential to screen for both forms of TP53 defects, including both 17p deletions and TP53 mutations. Aim: The aim of this study was to determine the frequency of TP53 mutations and their association with 17p deletions in a large Hungarian cohort of 196 patients suffering from chronic lymphocytic leukaemia. Method: We performed mutation analysis of TP53 (exons 3–10) using Sanger sequencing. Results: TP53 mutations were present in 15.8% of patients, half of which were associated with 17p deletion. By analysing both forms, TP53 defect was identified in 25.4% of the patients. Conclusions: Our study demonstrates that by performing a TP53 mutation analysis, an additional 10% of high-risk patients can be detected. Orv. Hetil., 2017, 158(6), 220–228.

scholarly journals Single Cell Approaches - Drilling Down Deeper Using Technology and Integrated Data Analyses

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-28-SCI-28
Author(s):  
Dan A. Landau
Keyword(s):  
High Resolution ◽  
Chronic Lymphocytic Leukemia ◽  
Chronic Lymphocytic Leukaemia ◽  
Single Cell ◽  
Research Funding ◽  
Somatic Mutations ◽  
Lymphocytic Leukaemia ◽  
Lymphocytic Leukemia ◽  
Cancer Evolution ◽  
Cell Identity

Cancer progression, relapse and resistance are the result of an evolutionary optimization process. Vast intra-tumoral diversity provides the critical substrate for cancer to evolve and adapt to the selective pressures provided by effective therapy. Our previous work has shown that genetically distinct subpopulations compete and mold the genetic makeup of the malignancy (1, 2). Additionally, we have shown that epigenetic changes in cancer may be similar to the process of genetic diversification, in which stochastic trial and error leads to rare fitness enhancing events (3). These studies demonstrate the need to integrate genetic, epigenetic and transcriptional information in the study of cancer evolution, specifically at the single-cell resolution - the atomic unit of somatic evolution. To enable this work, we have developed a single-cell multi-omics toolkit, and apply it to chart the evolutionary history and developmental topographies of normal and malignant blood cells. First, we have applied single-cell multi-omics to chronic lymphocytic leukaemia (CLL), a highly informative model for cancer evolution (4). We applied multiplexed single-cell reduced-representation bisulfite sequencing to healthy B and CLL cells, and demonstrated that epimutations serve as a molecular clock. Heritable epimutation information therefore allows to infer high-resolution lineages with single-cell data, directly in patient samples. CLL tree topography showed earlier branching and longer branch lengths than normal B cell trees. These features reflect rapid drift after malignant transformation and CLL's greater proliferative history. Multi-omic single-cell Integration of methylome sequencing with whole transcriptome and genotyping capture validated tree topology inferred solely on the basis of epimutation information. To examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles involving TLR pathway activation. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy. Second, charting the transcriptomes of clonally mutated cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To tackle this challenge, we developed Genotyping of Transcriptomes (GoT), a technology to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing(5). With GoT we profiled thousands of CD34+ cells from patients myeloproliferative neoplasms to study how somatic mutations corrupt the process of human hematopoiesis. These data allow to superimpose the two differentiation trees; the native wildtype tree and the one corrupted by mutation. High-resolution mapping of malignant versus normal progenitors showed increased fitness with myeloid differentiation with CALR mutation. We identified the unfolded protein response as a predominant outcome of CALR mutations, with dependency on cell identity. Notably, stem cells and more differentiated progenitors show distinct transcriptional programs as a result of somatic mutation, suggesting differential sensitivity to therapeutic targeting. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in blood neoplasms is dependent on the native cell identity. Landau, D. A., Carter, S. L., Stojanov, P. et al., Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell152, 714-726 (2013).Landau, D. A., Tausch, E., Taylor-Weiner, A. N. et al., Mutations driving CLL and their evolution in progression and relapse. Nature526, 525-530 (2015).Landau, D. A., Clement, K., Ziller, M. J. et al., Locally disordered methylation forms the basis of intratumor methylome variation in chronic lymphocytic leukemia. Cancer Cell26, 813-825 (2014).Gaiti, F., Chaligne, R., Gu, H. et al., Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia. Nature569, 576-580 (2019).Nam, A. S., Kim, K. T., Chaligne, R. et al., Somatic mutations and cell identity linked by Genotyping of Transcriptomes. Nature571, 355-360 (2019). Disclosures Landau: Pharmacyclics: Research Funding; Celgene: Research Funding; Illumina Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Sanger sequencing of mitochondrial hypervariable region of ancient samples for DNA authentication and screening before high-throughput sequencing

2021 ◽  
Vol 40 ◽  
pp. 103216
Author(s):  
Anna Šenovská ◽  
Eva Drozdová ◽  
Kristýna Brzobohatá ◽  
Eva Chocholová ◽  
Dana Fialová ◽  
...  
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Hypervariable Region
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