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.

Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2337-2337
Author(s):  
Lesley-Ann Sutton ◽  
Efterpi Kostareli ◽  
Anastasia Hadzidimitriou ◽  
Nikos Darzentas ◽  
Athanasios Tsaftaris ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
B Cell Receptors ◽  
Valuable Insight ◽  
Cell Receptors ◽  
Mutational Status ◽  
The Impact

Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.

Serum BAFF (B-CELL Activating Factor Of The TNF Family) predicts time to First Treatment in Early B-CELL Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4158-4158
Author(s):  
Stefano Molica ◽  
Gaetano Vitelli ◽  
Giovanna Cutrona ◽  
Giovanna Digiesi ◽  
Rosanna Mirabelli ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Serum Concentration ◽  
B Cell ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
B Cell Activating Factor ◽  
Mutational Status ◽  
Time To First Treatment ◽  
The Impact ◽  
Cell Chronic Lymphocytic Leukemia

Abstract We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70− and CD38− expression) and serum levels of BAFF (B-cell activating factor of the TNF family) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 125 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA (R & D Systems, USA) we found that higher levels of BAFF characterized more frequently females (P=0.01), patients with Rai stage 0 (P=0.03), mutated IgVH disease (P=0.008) and low ZAP-70 expression (P=0.04). In contrast, age (P=0.35), peripheral blood lymphocytosis (PBL)(P=0.09), hemoglobin (Hb) level (P=0.64), platelet (PLT) count (P=0.12), serum β2-m (P=0.49), LDH (P=0.85) and percentage of CD38-positive B-CLL cells (P=0.63) did not reflect circulating levels of BAFF. We used an optimal cut-point search to determine how to best split soluble BAFF data. Maximally selected log-rank statistics plots identified a BAFF serum concentration of 311 ng/mL as the best cut-off (P&lt;0.0001). Accordingly, patients who had BAFF levels higher than 311 ng/mL experienced a longer TFT (median 108 months) in comparison to patients whose BAFF levels were lower than 311 ng/mL (median 30 months; P&lt;0.0001). Along with serum concentration of BAFF, the univariate Cox proportional hazard model identified Rai substage I–II (P=0.003), lower PLT count (P=0.04), higher PBL count (P=0.01), increased LDH (P=0.01), ZAP-70 expression &gt; 20% (P=0.02) and absence of mutation of IgVH (P&lt;0.0001) as predictor of shorter TFT. In multivariate analysis only soluble BAFF (Hazard ratio [HR], 6.13; CI 95%, 2.31–16.25) and mutational status of IgVH, (HR= 2.99; CI 95% 1.33–6.76, P=0.008) maintained their discriminating power. The effects of BAFF on TFT were masked by mutational status of IgVH in patients with unmutated IgVH. However, serum levels of BAFF and mutational status of IgVH had a joint effect on TFT in patients with mutated IgVH which translates into a segregation of patients with mutated IgVH in two groups with different TFT according to BAFF levels (HR= 8.9; P&lt;0.0001). Our results indicate that in early B-cell CLL biological profile including among other parameters soluble BAFF may provide a useful insight into the complex interrelationship of prognostic variables. Furthermore, BAFF along with mutational status of IgVH can be adequately used to predict clinical behaviour of patients with low biological risk.

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.

scholarly journals Relation of Gene Expression Phenotype to Immunoglobulin Mutation Genotype in B Cell Chronic Lymphocytic Leukemia

2001 ◽  
Vol 194 (11) ◽  
pp. 1639-1648 ◽  
Author(s):  
Andreas Rosenwald ◽  
Ash A. Alizadeh ◽  
George Widhopf ◽  
Richard Simon ◽  
R. Eric Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Germ Line ◽  
Gene Expression Signature ◽  
Lymphocytic Leukemia ◽  
Common Mechanism ◽  
Human Leukemia ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression “signature,” irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

U.S. budget impact analysis of biosimilar versus originator intravenous rituximab for the treatment of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), rheumatoid arthritis (RA), granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e18820-e18820
Author(s):  
Elizabeth James ◽  
Holly Trautman ◽  
Ali McBride ◽  
Azhar Choudhry ◽  
Stephen Thompson
Keyword(s):  
B Cell ◽  
Impact Analysis ◽  
Budget Impact ◽  
Drug Acquisition ◽  
B Cell Lymphoma ◽  
Economic Benefits ◽  
Lymphocytic Leukemia ◽  
Sensitivity Analyses ◽  
Cost Share ◽  
The Impact

e18820 Background: Rituximab-abbs is an anti-CD20 monoclonal antibody and an important immuno-oncology agent for the treatment of B-cell malignancies NHL (diffuse large B-cell lymphoma [DLBCL] and follicular lymphoma [FL]) and CLL. It is also indicated for patients with RA, GPA, and MPA. Rituximab-abbs was the first rituximab biosimilar approved in the US and is expected to reduce drug acquisition costs. This budget impact model (BIM) estimated the impact of replacing a share of originator rituximab (IV-R-REF) use with rituximab-abbs (IV-R-BIOSIM) for NHL (DLBCL and FL), CLL, RA, GPA, and MPA. The objective was to project incremental annual cost differences between IV-R-BIOSIM and IV-R-REF for a hypothetical 1-million-member US healthcare insured (Medicare) population. Methods: An illustrative BIM estimated changes in 1-year drug and administration costs for an increased IV-R-BIOSIM uptake from 17.5% to 22.0%. Values for epidemiology, market share distribution, drug dosing, administration, and costs were derived from scientific literature, product labels, and publicly available cost resources. Dosing was based on a mean patient body surface area of 1.8 m2. Annual dose counts per patient were: 10 untreated FL with maintenance; 8 untreated FL (without maintenance), relapsed/refractory FL, or untreated DLBCL; 6 CLL, and 4 for RA, GPA, or MPA. All treatments were assumed to infuse over 3 hours. Drug acquisition and administration costs were from 2020 Average Sales Price pricing file and Centers for Medicare and Medicaid Services Physician Fee Schedule. Patient cost share was based on 2020 Medicare Part B 20% cost-share for office visits and drug products. Univariate sensitivity analyses were conducted. A scenario analysis was performed to project 2-year costs for extended FL maintenance treatment. Results: Estimated total annual plan incremental savings for a 1-million-member payer after the utilization shift were $312,379, equating to $0.31 per enrolled member per year (PMPY). Per-patient incremental drug cost savings with IV-R-BIOSIM for 1-year were $5,474–$12,924 (Table). The model was most sensitive to IV-R-REF cost and proportion of patients with RA. Conclusions: This analysis estimated annual savings of over $310,000 ($0.31 PMPY) for a 1-million-member US payer following a 4.5% utilization shift from IV-R-REF to IV-R-BIOSIM, demonstrating that IV-R-BIOSIM may confer considerable economic benefits vs originator rituximab.[Table: see text]

Association between the proliferative rate of neoplastic B cells, their maturation stage, and underlying cytogenetic abnormalities in B-cell chronic lymphoproliferative disorders: analysis of a series of 432 patients

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5130-5141 ◽  
Author(s):  
Sandra Quijano ◽  
Antonio López ◽  
Ana Rasillo ◽  
Susana Barrena ◽  
Maria Luz Sánchez ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Maturation Stage ◽  
Cytogenetic Abnormalities ◽  
M Phase ◽  
The Impact

Abstract Limited knowledge exists about the impact of specific genetic abnormalities on the proliferation of neoplastic B cells from chronic lymphoproliferative disorders (B-CLPDs). Here we analyze the impact of cytogenetic abnormalities on the proliferation of neoplastic B cells in 432 B-CLPD patients, grouped according to diagnosis and site of sampling, versus their normal counterparts. Overall, proliferation of neoplastic B cells highly varied among the different B-CLPD subtypes, the greatest numbers of proliferating cells being identified in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Compared with normal B cells, neoplastic B-CLPD cells showed significantly increased S + G2/M-phase values in mantle cell lymphoma (MCL), B-chronic lymphocytic leukemia (B-CLL), BL, and some DLBCL cases. Conversely, decreased proliferation was observed in follicular lymphoma, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM), and some DLBCL patients; hairy cell leukemia, splenic marginal zone, and MALT-lymphoma patients showed S + G2/M phase values similar to normal mature B lymphocytes from LN. Interestingly, in B-CLL and MCL significantly higher percentages of S + G2/M cells were detected in BM versus PB and in LN versus BM and PB samples, respectively. In turn, presence of 14q32.3 gene rearrangements and DNA aneuploidy, was associated with a higher percentage of S + G2/M-phase cells among LPL/WM and B-CLL cases, respectively.

FMNL-1 (Formin-Like 1) Gene in Chronic Lymphocytic Leukemia (CLL) Is Overexpressed in Young Patients with Adverse Prognostic Factors and Poor Outcome.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2775-2775 ◽  
Author(s):  
Eva Calpe ◽  
Patricia M.B. Favaro ◽  
Marta Crespo ◽  
Maria Joao Baptista ◽  
Ana Muntañola ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Survival ◽  
Lymphocyte Count ◽  
Biological Diversity ◽  
Young Patients ◽  
Lymphocytic Leukemia ◽  
Time To Progression ◽  
Mutational Status ◽  
Wide Range

Abstract Prognosis of patients with CLL has been traditionally assessed by using clinical parameters. Although useful, such parameters are a mere reflection of the biological diversity of CLL. In this regard, the mutational status of VH genes or ZAP-70 expression separates CLL into two clinical forms with different presenting features and outcome. Formins are multidomain proteins characterized by the presence of two conserved prolin-rich regions, namely formin homology 1 and 2. These proteins are implicated in a wide range of processes, including regulation of the cytoskeleton and in the regulation of the signal for cell survival. Formin is normally expressed in spleen, lymph node, and bone marrow cells, and it has been recently found to be overexpressed in T-cell lymphomas. The aim of this study was to analyze the expression of FMNL-1 in normal B-cell subsets and in a series of 73 patients (median age, 59 years; male/female 40/33; Binet A: 90.2%) with CLL. FMNL-1 expression was analyzed by Western Blot in separate subpopulations and by quantitative RT-PCR using expression in Jurkat as baseline. Among normal lymphocytes, FMNL-1 was only expressed in memory (CD19+CD27+) B-cells and in T-cells. In CLL cases with a low percentage of T-cells, mean of FMNL-1 expression was 2.18 AU (SD, 1.01 AU). Using an arbitrary cut-off of 3.2 AU, cases with increased expression of FMNL-1 were associated with a younger age at diagnosis (< 50 yrs), elevated lymphocyte count, high serum β2-microglobulin (β2-m) levels, increased ZAP-70 and CD38 expression, shorter time to progression, and shorter survival as compared to cases with low FMNL-1 expression. No relationship was observed with genetic abnormalities (table). In summary, among B-cell lymphoproliferative disorders, FMNL-1, a gene that regulates cell survival, is found only in CLL and its overexpression correlates with adverse clinical and biological parameters, particularly in young patients. Variable FMNL-1 normal FMNL-1 increased p value Age < 50 yrs 15% 86% 0.0001 Lymphocyte count > 50.000/ μL 14% 57% 0.018 Increasedβ2-microglobulin 17% 66% 0.038 CD38 > 30% 28% 100% 0.001 ZAP-70≥20% 50% 100% 0.014 Time to progression (median) 5.1 yrs 0.5 yrs 0.003 Survival (median) 16.4 yrs 8.5 yrs 0.009

Serum BAFF (B-Cell Activating Factor of the TNF Family) Compared with Immunoglobulin Heavy-Chain Mutation Status, ZAP-70 and CD38 as a Predictor of Time to First Treatment in Early B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3093-3093
Author(s):  
Stefano Molica ◽  
Gaetano Vitelli ◽  
Giovanna Cutrona ◽  
Giovanna Digiesi ◽  
Rosanna Mirabelli ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
B Cell Activating Factor ◽  
Mutational Status ◽  
Time To First Treatment ◽  
Cell Chronic Lymphocytic Leukemia

Abstract We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70- and CD38-expression) and serum levels of BAFF (B-cell activating factor of the TNF family) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 69 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA (R & D Systems, USA) we found that higher levels of BAFF characterized more frequently patients with Rai stage 0 (P=0.008) and mutated IgVH (P=0.03). In contrast, peripheral blood lymphocytosis (P=0.06), serum β2-m (P=0.159), LDH (P=0.333) and percentage of ZAP-70-positive (P=0.242) or CD38-positive B-CLL cells (P=0.142) did not reflect circulating levels of BAFF. The relationship among various bio-pathological parameters, analyzed by the multiple correspondence analysis (MCA), showed two different clinico-biological profiles. The first, characterized by higher BAFF serum levels (i.e., > 336 ng/mL), presence of mutation in the IgVH, low percentage of CD38-positive B-CLL cells (< 30%) and low LDH was associated with a stable pattern of disease generally not requiring therapy. The second, defined by lower BAFF levels, absence of mutation in the IgVH, high percentage of CD38- positive B-CLL cells and high LDH was associated with a more progressive pattern of disease and a shorter TFT. After a median follow-up time of 35 months (range, 2–120 months) 26 (37.6%) out of 69 patients experienced a need for chemotherapy. Kaplan-Meier estimates of patientsTFT, plotted after searching the best cut-off for BAFF (i.e., 336 ng/mL), demonstrated that low BAFF concentration was associated with a shorter TFT (median TFT 36 months) while median was not reached by patients with BAFF levels higher than 336 ng/mL (P<0.0001). Along with lower serum levels of BAFF (Hazard Ratio [HR], 0.19; P<0.0001), the univariate Cox proportional hazard model identified absence of mutation in IgVH (HR, 0.17; P<0.0001), CD38-positivity (HR, 3.32; P=0.01) and lower platelet count (HR, 0.19; P=0.03) as predictor of shorter TFT. Finally, in multivariate analysis only mutational status of IgVH (HR, 0.25; P=0.007) and serum concentration of BAFF (HR, 034; P=0.04) affected significantly TFT. Our results indicate that in early B-cell CLL clinico-biological profile including among other parameters BAFF may provide a useful insight into the complex interrelationship of prognostic variables and semplify their interpretation. The possible presence of BAFF isoform in B-CLL could peraphs account for the unexpected correlation between low soluble BAFF levels and poor clinical outcome in patients with early disease.

Clinical Impact of Clonal and Subclonal TP53, SF3B1, BIRC3, and ATM Mutations in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4138-4138
Author(s):  
Ferran Nadeu ◽  
Julio Delgado ◽  
Cristina Royo ◽  
Tycho Bauman ◽  
Tatjana Stankovic ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Allele Frequency ◽  
Lymphocytic Leukemia ◽  
Molecular Characteristics ◽  
Prognostic Impact ◽  
Driver Genes ◽  
Germline Variants ◽  
Tp53 Mutations ◽  
Mutational Status ◽  
The Impact

Abstract Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

Immunophenotypic Profile and Recurrent Somatic Mutations in 131 Chronic Lymphocytic Leukemia Patients: Low Expression of CD25 in NOTCH1-Mutated Cases

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3201-3201
Author(s):  
Miriam Castillo ◽  
Ana María Hurtado ◽  
Tzu Hua Chen-Liang ◽  
Julia Muñoz-Ballester ◽  
Bartlomiej P Przychodzen ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Somatic Mutations ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Limited Information ◽  
Male Predominance ◽  
Mutational Status ◽  
The Impact ◽  
Notch1 Mutations ◽  
Low Expression

Abstract Background: We and others have reported on the impact of recurrent somatic mutations not only in the multistep pathogenetic process, but also in the clinical heterogeneity of chronic lymphocytic leukemia (CLL) patients. Immunophenotyping, as part of the diagnostic workout, is used for assessing clonality, as a differential diagnosis tool, and to examine the expression of molecules associated with a worse prognosis. Recently, NOTCH1 mutations have been linked to low CD20 levels in CLL and with a relative resistance to anti-CD20 immunotherapy in vitro. But to date, there is limited information on the correlation between cell surface marker expression and the presence of somatic mutations in CLL. The aim of this study was to evaluate potential associations between an extended phenotypic panel and the mutational status of 13 recurrently mutated genes in CLL detected by deep sequencing. Patients and Methods: To this end, we performed targeted NGS sequencing of blood samples, collected at diagnosis, from 131 CLL patients. Every patient underwent, at baseline, a flow cytometry characterization with a panel including (sIg)λ, (sIg)κ, CD19, CD5, CD11b, CD81, CD10, CD79b, CD29, CD38, FMC7, CD22, CD45, CD103, CD11c, CD25, ZAP70, CD11a, and CD24. We designed a TruSeq Custom Amplicon panel (Illumina, Inc. San Diego, CA, USA) containing 13 genes and covering 28.099 bases. The average amplicon size was 238 base pairs and ~ 99.1% of the regions were covered on both strands. Paired-end sequencing (2x250 bp) was performed with MiSeq v2.2 chemistry, and a mean depth of 998 reads/base within the regions of interest was obtained. Raw data were analyzed with IlluminaonJboard Real Time Analysis (RTA v.2.4.60.8) software and MiSeq Reporter. Results: With a median age of 68 y.o. (range, 33-95) and a slight male predominance, the median follow up time of our cohort was 43 months (24-104). We found that 47/131 (35%) patients harbored at least one mutation, with NOTCH1 (n = 13, 10%), ATM (n = 10, %), TP53 (n = 8, %), and SF3B1 (n = 8, 5.5%), as the most frequently mutated genes. Those patients with a NOTCH1 mutation showed a lower CD25 expression (25 mean fluorescence intensity units (MFIu)) than those without a mutation (45 MFIu), p=0.001. In addition, a higher expression of CD5 (265 vs. 219 MFIu, p= 0.02), of the monoclonal light chain (90.5 vs. 58.6 MFIu, p=0.03), and a higher percentage of CD38+ cells in the CD19+CD5+ compartment (37% vs. 19%, p=0.006) were significantly associated with the presence of, at least, one somatic mutation. We could not validate the recently reported association between the presence of NOTCH1 mutations and a low expression of CD20. In our cohort, the MFI expression in NOTCH1 mutated and non-mutated patients was 176 and 135 units, respectively (p=0.2) In the multivariate Cox analysis, the presence of a somatic variant in TP53 and a higher percentage of positive CD38 cells in the tumour population showed both a worse overall survival and shorter time to first treatment. The independence of these two variables was also supported by not finding a significative difference percentage of CD38 positive cells between TP53 mutated and non mutated cases (p=0.5). Conclusions: The associations described herein suggest potential pathogenic pathways in CLL, in particular the CD25-NOTCH1 axis, with a significative inferior expression of CD25 when activating NOTCH1 mutations are present. The relationship found between these two variables, with an inversed direction to that found in physiological conditions, has also been shown in the setting of NOTCH1-mutated acute lymphoblastic leukemia, emerging as a potential targetable pathway in this subset of CLL patients. Disclosures Maciejewski: Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

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