scholarly journals VH CDR3-Focused Somatic Hypermutation in CLL IGHV-IGHD-IGHJ Gene Rearrangements with 100% IGHV Germline Identity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4277-4277 ◽  
Author(s):  
Katerina Gemenetzi ◽  
Andreas Agathangelidis ◽  
Fotis Psomopoulos ◽  
Kostas Pasentsis ◽  
Evdoxia Koravou ◽  
...  
Keyword(s):  
Research Funding ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Gene Rearrangements ◽  
Rt Pcr ◽  
Bioinformatics Pipeline ◽  
Sample Mean ◽  
Mean Frequency ◽  
High Data ◽  
Ighv Gene

Classification of patients with chronic lymphocytic leukemia (CLL) based on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the sequence of the rearranged IGHV gene excluding the VH CDR3. This is mostly due to the difficulty in discriminating actual SHM from random nucleotides added between the recombined IGHV, IGHD and IGHJ genes. Hence, this approach may underestimate the true impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions i.e. the VH CDR3. Relevant to mention in this respect, studies from our group in CLL with mutated IGHV genes (M-CLL), particularly subset #4, have revealed considerable intra-VH CDR3 diversity attributed to ongoing SHM. Prompted by these findings, here we investigated whether SHM may also be present in cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3), focusing on two well characterized stereotyped subsets i.e. subset #1 (IGHV clan I/IGHD6-19/IGHJ4) and subset #6 (IGHV1-69/IGHD3-16/IGHJ3). These subsets carry germline-encoded amino acid (aa) motifs within the VH CDR3, namely QWL and YDYVWGSY, originating from the IGHD6-19 and IGHD3-16 gene, respectively. However, in both subsets, cases exist with variations in these motifs that could potentially represent SHM. The present study included 12 subset #1 and 5 subset #6 patients with clonotypic IGHV genes lacking any SHM (100% germline identity). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified by subgroup-specific leader primers and a high-fidelity polymerase in order to ensure high data quality. RT-PCR products were subjected to paired-end NGS on the MiSeq platform. Sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis was undertaken using an in-house purpose-built bioinformatics pipeline. Rearrangements with the same IGHV gene and identical VH CDR3 aa sequences were defined as clonotypes. Overall, we obtained 1,570,668 productive reads with V-region identity 99-100%; of these, 1,232,958 (mean: 102,746, range: 20,796-242,519) concerned subset #1 while 337,710 (mean: 67,542, range: 50,403-79,683) concerned subset #6. On average, 64.4% (range: 1.7-77.5%) of subset #1 reads and 49.2% (range: 0.7-70%) of subset #6 reads corresponded to rearrangements with IGHV genes lacking any SHM (100% germline identity). Clonotype computation revealed 1,831 and 1,048 unique clonotypes for subset #1 and #6, respectively. Subset #1 displayed a mean of 157 distinct clonotypes per sample (range: 74-267), with the dominant clonotype having a mean frequency of 96.9% (range: 96-98.2%). Of note, 44 clonotypes were shared between different patients (albeit at varying frequencies), including the dominant clonotype of 11/12 cases, which was present in 2-6 additional subset #1 patients. Subset #6 cases carried a higher number of distinct clonotypes per sample (mean: 219, range: 189-243) while the dominant clonotype had a mean frequency of 95.6% (range: 94.5-96.5%). Shared clonotypes (n=30) were identified also in subset #6 and the dominant clonotype of each subset #6 case was present in 3-5 additional subset #6 patients. Focusing on the VH CDR3, in particular the IGHD-encoded part, the following observations were made: (1) in both subsets, extensive intra-VH CDR3 variation was detected at certain positions within the IGHD gene; (2) in most cases, the observed aa substitutions were conservative i.e. concerned aa sharing similar physicochemical properties. Particularly noteworthy in this respect were the observations in subset #6 that: (i) the valine residue (V) in the D-derived YDYVWGSY motif was very frequently mutated to another aliphatic residue (A, I, L); (ii) in cases were the predominant clonotype carried I (also in the Sanger-derived sequence), several minor clonotypes carried the germline-encoded V, compelling evidence that the observed substitution concerned true SHM. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, very likely attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. While the prognostic/predictive relevance of this observation is beyond the scope of the present work, our findings highlight the possible need to reappraise definitions ('semantics') regarding SHM status in CLL. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

Higher Order Restrictions of the Immunoglobulin Repertoire in CLL: The Illustrative Case of Stereotyped Subsets #2 and #169

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5453-5453
Author(s):  
Katerina Gemenetzi ◽  
Andreas Agathangelidis ◽  
Fotis Psomopoulos ◽  
Karla Plevova ◽  
Lesley-Ann Sutton ◽  
...  
Keyword(s):  
Research Funding ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Illustrative Case ◽  
Gene Rearrangements ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Mean Frequency ◽  
Cdr3 Sequence ◽  
Sample Range

Stereotyped subset #2 (IGHV3-21/IGLV3-21) is the largest subset in CLL (~3% of all patients). Membership in subset #2 is clinically relevant since these patients experience an aggressive disease irrespective of the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene. Low-throughput evidence suggests that stereotyped subset #169, a minor CLL subset (~0.2% of all CLL), resembles subset #2 at the immunogenetic level. More specifically: (i) the clonotypic heavy chain (HC) of subset #169 is encoded by the IGHV3-48 gene which is closely related to the IGHV3-21 gene; (ii) both subsets carry VH CDR3s comprising 9-amino acids (aa) with a conserved aspartic acid (D) at VH CDR3 position 3; (iii) both subsets bear light chains (LC) encoded by the IGLV3-21 gene with a restricted VL CDR3; and, (iv) both subsets have borderline SHM status. Here we comprehensively assessed the ontogenetic relationship between CLL subsets #2 and #169 by analyzing their immunogenetic signatures. Utilizing next-generation sequencing (NGS) we studied the HC and LC gene rearrangements of 6 subset #169 patients and 20 subset #2 cases. In brief, IGHV-IGHD-IGHJ and IGLV-IGLJ gene rearrangements were RT-PCR amplified using subgroup-specific leader primers as well as IGHJ and IGLC primers, respectively. Libraries were sequenced on the MiSeq Illumina instrument. IG sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis conducted using an in-house, validated bioinformatics pipeline. Rearrangements with identical CDR3 aa sequences were herein defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. For the HC analysis of subset #169, we obtained 894,849 productive sequences (mean: 127,836, range: 87,509-208,019). On average, each analyzed sample carried 54 clonotypes (range: 44-68); the dominant clonotype had a mean frequency of 99.1% (range: 98.8-99.2%) and displayed considerable intraclonal heterogeneity with a mean of 2,641 subclones/sample (range: 1,566-6,533). For the LCs of subset #169, we obtained 2,096,728 productive sequences (mean: 299,533, range: 186,637-389,258). LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean: 148, range: 110-205); the dominant clonotype had a mean frequency of 98.1% (range: 97.2-98.6%). Intraclonal heterogeneity was also observed in the LCs, with a mean of 6,325 subclones/sample (range: 4,651-11,444), hence more pronounced than in their partner HCs. Viewing each of the cumulative VH and VL CDR3 sequence datasets as a single entity branching through diversification enabled the identification of common sequences. In particular, 2 VH clonotypes were present in 3/6 cases, while a single VL clonotype was present in all 6 cases, albeit at varying frequencies; interestingly, this VL CDR3 sequence was also detected in all subset #2 cases, underscoring the molecular similarities between the two subsets. Focusing on SHM, the following observations were made: (i) the frequent 3-nucleotide (AGT) deletion evidenced in the VH CDR2 of subset #2 (leading to the deletion of one of 5 consecutive serine residues) was also detected in all subset #169 cases at subclonal level (average: 6% per sample, range: 0.1-10.8%); of note, the 5-serine stretch is also present in the germline VH CDR2 of the IGHV3-48 gene; (ii) the R-to-G substitution at the VL-CL linker, a ubiquitous SHM in subset #2 and previously reported as critical for IG self-association leading to cell autonomous signaling in this subset, was present in all subset #169 samples as a clonal event with a mean frequency of 98.3%; and, finally, (iii) the S-to-G substitution at position 6 of the VL CDR3, present in all subset #2 cases (mean : 44.2% ,range: 6.3-87%), was also found in all #169 samples, representing a clonal event in 1 case (97.2% of all clonotypes) and a subclonal event in the remaining 5 cases (mean: 0.6%, range: 0.4-1.1%). In conclusion, the present high-throughput sequencing data cements the immunogenetic relatedness of CLL stereotyped subsets #2 and #169, further highlighting the role of antigen selection throughout their natural history. These findings also argue for a similar pathophysiology for these subsets that could also be reflected in a similar clonal behavior, with implications for risk stratification. Disclosures Sutton: Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1524-1533 ◽  
Author(s):  
Fiona Murray ◽  
Nikos Darzentas ◽  
Anastasia Hadzidimitriou ◽  
Gerard Tobin ◽  
Myriam Boudjogra ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Germ Line ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Chain Gene ◽  
Distinctive Features ◽  
Ighv Gene ◽  
Gene Usage

Abstract Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, “stereotyped” amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

scholarly journals Immunoglobulin gene analysis in chronic lymphocytic leukemia in the era of next generation sequencing

Leukemia ◽  
2020 ◽  
Vol 34 (10) ◽  
pp. 2545-2551 ◽  
Author(s):  
Frédéric Davi ◽  
◽  
Anton W. Langerak ◽  
Anne Langlois de Septenville ◽  
P. Martijn Kolijn ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Somatic Hypermutation ◽  
Technological Development ◽  
Data Interpretation ◽  
Medical Diagnostics ◽  
Lymphocytic Leukemia ◽  
Immunoglobulin Gene ◽  
New Era ◽  
Ighv Gene ◽  
Therapeutic Decision Making

Abstract Twenty years after landmark publications, there is a consensus that the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is an important cornerstone for accurate risk stratification and therapeutic decision-making in patients with chronic lymphocytic leukemia (CLL). The IGHV SHM status has traditionally been determined by conventional Sanger sequencing. However, NGS has heralded a new era in medical diagnostics and immunogenetic analysis is following this trend. There is indeed a growing demand for shifting practice and using NGS for IGHV gene SHM assessment, although it is debatable whether it is always justifiable, at least taking into account financial considerations for laboratories with limited resources. Nevertheless, as this analysis impacts on treatment decisions, standardization of both technical aspects, and data interpretation becomes essential. Also, the need for establishing new recommendations and providing dedicated education and training on NGS-based immunogenetics is greater than ever before. Here we address potential and challenges of NGS-based immunogenetics in CLL. We are convinced that this perspective helps the hematological community to better understand the pros and cons of this new technological development for CLL patient management.

Lack of Prognostic Significance of the Conventional and Novel Prognostic Markers in Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4354-4354
Author(s):  
Valter Gattei ◽  
Riccardo Bomben ◽  
Michele Dal Bo ◽  
Antonella Zucchetto ◽  
Francesca Rossi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Research Funding ◽  
Prognostic Markers ◽  
Lymphocytic Leukemia ◽  
Cytogenetic Abnormality ◽  
Mutational Status ◽  
Ighv Gene ◽  
Trisomy 12 ◽  
Gene Status

Abstract Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases. Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL. Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 andSF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis. Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p<0.0001), and of UM IGHV cases (55% vs 25%, p<0.0001). Analysis of recurrent mutations highlighted a higher prevalence of NOTCH1 mutations (26% vs 8%, p<0.0001) and of BIRC3 mutations (21% vs 1%, p<0.0001) in tris12 vs control group CLL. Conversely, no differences were found in the fraction of cases with TP53 mutations (3% vs 4%, p=0.38) or SF3B1 mutations (7% vs 7%, p=0.89), and in cases expressing ZAP-70 (62% vs 52%, p=0.09). The impact of these features on OS was tested by univariate analysis: in tris12 CLL, only the UM IGHV gene status predicted shorter OS (HR=2.37, p=0.0063), while none of the other characteristics reaching statistical significance as OS predictors (CD49d HR=1.36, p=0.36; CD38 HR=0.42, p=0.052; ZAP-70 HR=3.12, p=0.07; TP53 HR=2.33, p=0.25; NOTCH1 HR=1.40, p=0.22; SF3B1 HR=2.05, p=0.17; BIRC3 HR=1.22, p=0.61). On the other hand, in the control cohort, a significantly higher HR was found for CD49d (HR 3.11, p<0.0001) and CD38 (HR 3.45, p<0.0001) expression, TP53 (HR 2.88, p=0.0026), NOTCH1 (HR 3.57, p<0.0001), and SF3B1 (HR 2.57, p=0.0038) mutations, as well as for the UM IGHV gene status (HR=2.81, p<0.0001), but not for ZAP-70 expression and BIRC3 mutations (HR=1.74 and HR=1.91, p=0.15 and p=0.37, respectively). Conclusions. Mutational status of IGHV genes was the sole prognostic factor able to stratify OS in tris12 CLL. Despite the high frequency of NOTCH1 and BIRC3 mutations, as well as of CD49d and CD38 overexpression, these markers failed to convey a prognostic risk in tris12 CLL. The lack of a significant clinical impact for TP53 and SF3B1 mutations might be partly explained by the low number of mutated cases combined with a relative short follow up in our tris12 cohort. These findings are in keeping with the hypothesis of a different patho-biological mechanism occurring in tris12 CLL, which however remains to be fully elucidated. Disclosures D'Arena: Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

scholarly journals Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 944-944
Author(s):  
Filippo Vit ◽  
Francesca Maria Rossi ◽  
Tiziana D'Agaro ◽  
Tamara Bittolo ◽  
Antonella Zucchetto ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Excision Repair ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Direct Role ◽  
Region Gene ◽  
Mutational Status ◽  
Ighv Gene

Abstract Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era. Aim. To apply a NGS approach to investigate ID in CLL. Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B). Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F). Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed. Disclosures Zaja: Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.

scholarly journals Chemoimmunotherapy for Patients with Chronic Lymphocytic Leukemia: MD Anderson Experience Beyond FCR300

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2047-2047 ◽  
Author(s):  
Dai Chihara ◽  
Philip A Thompson ◽  
Hagop M. Kantarjian ◽  
Susan M. O'Brien ◽  
Alessandra Ferrajoli ◽  
...  
Keyword(s):  
Cumulative Incidence ◽  
Research Funding ◽  
Risk Model ◽  
Lymphocytic Leukemia ◽  
Cox Proportional Hazards Model ◽  
High Dose ◽  
First Line ◽  
Ighv Gene ◽  
The Impact

Abstract Background: Novel, targeted therapies, such as ibrutinib, have transformed outcomes for patients with relapsed CLL and for older and unfit patients in the first-line setting. However, chemoimmunotherapy (CIT) remains the standard-of-care in fit patients. We reported that a subgroup of patients with IGHV mutated CLL experience prolonged PFS and potential cure after first-line CIT withfludarabine, cyclophosphamide and rituximab (FCR). However, FISH data was not available for this cohort of patients. Accurate knowledge of which patients are likely to experience prolonged PFS after FCR is essential to better select patients who may benefit from CIT in the era of novel therapies. Patients and Methods: We analyzed 492 patients who were treated on six clinical trials of first-line CIT between 2004 and 2015. Treatments were FCR, (n=277) FCR with high dose rituximab (n=65), FCR plusmitoxantrone (n=30), FCR plusalemtuzumab (n=60) and FCR with GM-CSF (n=60). Progression-free survival (PFS) and overall survival (OS) were calculated and pretreatment characteristics were evaluated for association with survival outcomes using a Cox Proportional Hazards model. Cumulative incidence was calculated by competing risk (death without event) regression analysis. Results: The median age of patients was 59 (range 28-84). Sixty-seven percent of the patients were male, 33% of the patients had mutated IGHV gene. Thirty percent of patients had del(13q), 19% had Trisomy12, 21% had del(11q), 8% had del(17p) and 21% were negative by FISH. Fifty-nine percent of patients received six cycles of CIT. With a median follow up duration of 6.2 years, the median PFS and OS were 6.3 years and not reached, respectively. Recently reported risk model by Rossi and colleagues using IGHV mutation status and FISH results (Blood 2015) discriminated PFS very well; 5-year PFS for low risk {mutated without del(11q)}, intermediate risk {unmutated or del(11q)} and high risk group {del(17p)} were 81%, 45% and 22%, respectively. Of note, there was a plateau in PFS after 8 years in patients with mutated IGHV gene, with 10-year PFS of 63% (Figure A). There was a significantly improved OS after relapse by the time. Three-year OS in patients who started salvage chemotherapy in 2004 to 2012 and 2012 to 2016 were 59% and 83%, respectively, suggesting the impact of improved salvage treatment options, particularly B cell signaling pathway inhibitors (Figure B). Five-year cumulative incidence of Richter transformation (RT) and AML/MDS was 4.8% and 4.2%, respectively (Figure C, D). There was a difference in onset for these two complications; 52% of RT occurred within 2 years, while 62% of AML/MDS occurred in 2-4 years after CIT. Overall, 110 patients (22.4%) died during the follow-up; the three major causes of death were CLL progression (4.9%), Richter transformation (3.7%) and AML/MDS (3.3%). Conclusion: Patients with mutated IGHV gene and who do not have del(11q) or del(17p) have favorable outcomes and demonstrate a plateau on the PFS curve, consistent with prior studies. Effective salvage therapy has improved outcomes at relapse, but the development of RT and AML/MDS remain major causes of mortality in CLL patients. Given favorable outcomes for patients with mutated IGHV gene treated with FCR, further studies are warranted to identify predictors of non-response among the mutated patients, risk factors for development of AML/MDS and RT and whether choice of first-line therapy can modulate this risk. Disclosures Thompson: Pharmacyclics: Consultancy, Honoraria. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria; Incyte: Research Funding; Celgene: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Novartis: Consultancy, Honoraria; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; BMS: Research Funding; Infinity: Research Funding. Wierda:Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding.

Analysis of Non-Expressed IGK Locus Rearrangements in Chronic Lymphocytic Leukemia Indicates a Role for Secondary Rearrangements in Shaping the Expressed Immunoglobulin Repertoire.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 972-972 ◽  
Author(s):  
Chrysoula Belessi ◽  
Kostas Stamatopoulos ◽  
Katerina Hatzi ◽  
Anastasia Hadzidimitriou ◽  
Fotini Marantidou ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Light Chain ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Receptor Editing ◽  
Rt Pcr ◽  
Stop Codons ◽  
Immunoglobulin Kappa ◽  
Immunoglobulin Repertoire ◽  
Dna Pcr

Abstract Immunoglobulin kappa (IGK) locus rearrangements were analyzed in 164 κ-light chain (LC) expressing chronic lymphocytic leukemia (CLL) cases and 95 λ-LC expressing CLL cases. In κ-CLL, 151 IGKV-J transcripts were successfully amplified by RT-PCR; 147/151 were in frame; four out-of-frame IGKV-J transcripts were heavily mutated and contained one or more stop codons, presumably introduced by somatic hypermutation; in all four cases the corresponding expressed transcript was also mutated. DNA-PCR identified 24/164 κ-CLL cases (15%) with double IGKV-J rearrangements; 9/24 non-expressed IGKV-J rearrangements were in-frame; 3/9 were mutated. The most frequently used IGKV genes in expressed IGKV-J rearrangements were: 3–20, 1-39/1D-39, 1–5 and 4-1; the IGKV4-1 gene was by far the most frequent in non-expressed IGKV-J rearrangements (9/24 cases, 33%). In λ-CLL, a total of 65 IGKV-J rearrangements were amplified in 59/95 cases (62.0%); six cases had two different rearrangements. IGVK4-1 was the most frequently used gene (14/65 sequences, 21.5%), followed by IGKV1-16 (8 cases,12.3%), 1-33/1D-33 and 2-30 (7 cases each, 10.8%). IGKV-J transcripts were detected by RT-PCR in 10/59 λ-CLL cases with IGKV-J rearrangements, of which four were in-frame and six out-of-frame. Seven IGKV-J rearrangements in λ-CLL had less than 100% homology to germline; 3/7 were in-frame; 6/7 patients had mutated IGHV and 5/7 had mutated IGLV genes. In κ-CLL, biased usage of the IGKJ1/IGKJ2 genes was observed both in expressed (69%) and non-expressed rearrangements (78%); in contrast, in λ-CLL, downstream IGKJ (IGKJ3-5) usage was observed in 32/59 sequences (53%). Nonproductive rearrangements involving the kappa deleting element (KDE) that render the IGK locus inactive were also analyzed. In κ-CLL, 22/147 cases (15%) carried IGKV to KDE rearrangements, while 24/147 cases (16.5%) carried IGKJ-C- INTRON (JKI) to KDE rearrangements. In λ-CLL, IGKV-KDE rearrangements were amplified in 55/94 cases (59%); JKI-KDE rearrangements were amplified in 52/94 cases (56%). IGKV1D-43 was the most frequent gene in IGKV-KDE joints in κ-CLL (4/22 cases); in contrast, the most frequent genes in IGKV-KDE joints in λ-CLL were IGKV3-20 and IGKV2-30 (9/55 and 7/55 cases, respectively). In conclusion, the present study confirms IGK locus rearrangements in the vast majority of λ-LC expressing CLL cases. Differential usage of IGKJ genes along with significant IGKV repertoire differences in both IGKV-J and IGKV-KDE rearrangements between κ- and λ-CLL allude to prolonged IGK locus recombination before CLL clonogenic cells became λ-producers. The inactivation of productive and potentially functional IGKV-J joints by secondary rearrangements indicates a role for receptor editing in shaping the expressed IG repertoire in CLL. Finally, the identification of mutated, non-expressed IGKV-J rearrangements both in κ- and λ-CLL might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in a proportion of cases.

Molecular Profiling of Cancer Testis Antigens in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4701-4701
Author(s):  
Jason A Dubovsky ◽  
Emmanuel Berchmans ◽  
John J. Powers ◽  
Lin Hui-Yi ◽  
Yang Gao ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Chi Square ◽  
Clinical Criteria ◽  
Cancer Testis Antigens ◽  
Mutational Status ◽  
Cancer Testis

Abstract Abstract 4701 Background Chronic Lymphocytic Leukemia (CLL) is characterized by the progressive accretion of long-lived mature B-lymphocytes. Although the classical Rai and Binet staging is still commonly used, a new molecular understanding has identified specific signatures which could help predict disease progression and survival. Given the recent success of immunotherapeutic strategies and immunomodulatory drugs in the treatment of CLL we sought to identify potentially immunologically relevant targets and their relation to well known disease criterion. Methods Our study characterized the mRNA expression of 29 known cancer-testis antigens (CTAs) in 66 patients with CLL at varying stages of disease using a RT-PCR based expression panel. Relevant clinical criterion such as RAI stage, B2m, ZAP-70, IGVH mutational status, CD38, cytogenetics by FISH analysis, WBC count, age, gender, and treatment among others were then taken into account. The binary RNA expression data associated with the clinical and demographic factors were evaluated using chi-square or Wilcoxon rank sum analysis. Results Of the cancer-testis antigens tested, the MAGE family of CTAs revealed statistically significant correlations with multiple clinical criteria. Our analysis reveals a correlation between previous chemo-immunotherapy treatment and MAGE-A1, B2, E1, MAD-CT-2, SPA-17, and PAGE-5 expression. Beyond treatment, total white blood cell count was shown to have a significant association with MAGE family members A1, A3, and B2 expression. In addition, MAD-CT-2 and MAGE-B2 were significantly correlated with the expression of FMC-7 and SSX-4 and LAGE-1 correlated with the presence of B-cell symptomatology. Conclusions Preliminary RT-PCR based CTA phenotyping has unveiled interesting correlations to clinical criteria, opening multiple avenues for future immunotherapeutic interventions as well as possible prognostic value in CLL. Further investigation to better understand the biological value of this information in warranted. Disclosures: Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

scholarly journals Remarkable Functional Constraints on the Antigen Receptors of CLL Stereotyped Subset #2: High-Throughput Immunogenetic Evidence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1839-1839 ◽  
Author(s):  
Katerina Gemenetzi ◽  
Andreas Agathangelidis ◽  
Lesley-Ann Sutton ◽  
Elisavet Vlachonikola ◽  
Chrysi Galigalidou ◽  
...  
Keyword(s):  
Research Funding ◽  
Sanger Sequencing ◽  
Lymphocytic Leukemia ◽  
Molecular Evidence ◽  
Variable Load ◽  
Illumina Platform ◽  
Antigen Binding Site ◽  
Mean Frequency ◽  
Sample Range ◽  
Self Association

Abstract Subset #2 is the largest subset carrying stereotyped B cell receptor immunoglobulin (BcR IG) in chronic lymphocytic leukemia (CLL). This particular BcR IG is composed of heavy (HC) and light (LC) chains encoded by the IGHV3-21 and the lambda IGLV3-21 gene, respectively. The clonotypic IGHV3-21 genes display a variable load of somatic hypermutation (SHM), being mostly classified as mutated (M-CLL) but also including unmutated (U-CLL) cases. Subset #2 cases, independently of the SHM status, have a particularly dismal clinical outcome similar to that of patients with TP53 aberrations, although lacking such aberrations. Subset #2 BcR IG display a series of distinctive features, including conservation at certain VH and VL CDR3 positions and recurrent SHMs; as well as a capacity for self-association leading to cell autonomous signaling that is critically dependent on a substitution of Arginine (R) for Glycine (G) introduced by SHM at the lambda VL-CL linker region. These features implicate antigen selection in CLL subset #2 ontogeny. However, the available molecular evidence derives from low throughput immunogenetic analysis, precluding comprehensive assessment of antigenic impact on (sub)clonal composition. Here, we sought to overcome this limitation by performing next-generation sequencing (NGS) of HC and LC gene rearrangements of 20 subset #2 patients. RT-PCR products amplified by the IGHV3-21/IGHJ6 and IGLV3-21/IGLC primer pairs, respectively, were subjected to NGS on the MiSeq Illumina Platform. NGS data was analyzed by a validated bioinformatics pipeline. Rearrangements with identical CDR3 amino acid (aa) sequences were defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. Starting with HCs, we obtained 3,340,508 (mean: 291,751, range: 101,231-186,055) productive reads. On average, each analyzed sample carried 92 distinct clonotypes (range: 71-152), with the dominant clonotype having a mean frequency of 96% (range: 67-99%): in all cases the dominant clonotype was identical to that determined by Sanger sequencing. The dominant clonotype displayed considerable intraclonal heterogeneity with a mean of 5,082 subclones/sample (range: 2,946-11,041). Turning to LCs, we obtained 5,094,045 (mean: 231,547, range: 38,036-507,949) productive reads. LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean 222, range: 156-306). The dominant clonotype had a mean frequency of 96% (range: 74-98%); similar to HCs, it was identical to that determined by Sanger sequencing. Intraclonal heterogeneity was observed in the LCs as well, with a mean of 7,382 subclones/sample (range: 1,946-11,866), hence more pronounced vs their partner HCs. Viewing the entire subset #2 VH or VL CDR3 dataset (i.e. the CDR3 aa sequences from all clonotypes of all cases) as a single entity branching through diversification enabled the identification of 2 distinct VH CDR3 sequences present at varying frequencies in 16 and 13 cases, respectively; and, 3 distinct VL CDR3 sequences present at varying frequencies in all 20 cases: these results allude to important constraints on the composition of the antigen binding site. Focusing on SHM, the following notable observations could be made. (i) The G-to-R substitution at the VL-CL linker was a clonal event in all cases with R being degenerately encoded by different nucleotide sequences; altogether, these findings underscore the seminal role of this recurrent SHM, likely due to mediating self-association. (ii) A recurrent 3-nucleotide deletion was detected in the VH CDR2 of all cases, strongly supporting functional pressure. This change, previously identified by Sanger sequencing as a recurrent SHM in subset #2 (albeit at a frequency of only 25%), was clonal in 4 cases and subclonal in the remainder, where it was present in an average of 105 subclones/sample (range: 1-369). (iii) Certain positions in both the VH and VL domain bore the same aa substitution, mostly at subclonal level: the prime example concerned the G for Serine (S) substitution within the VL CDR3, detected in all samples at a mean frequency of 44.2% (range: 6.3-87%). In conclusion, we provide compelling immunogenetic evidence for functional pressure in the ontogeny of CLL subset #2. On this evidence, subset #2 emerges as perhaps the most striking example of antigen-driven leukemogenesis reported thus far. Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.

Immunoglobulin Heavy Chain (IgH) Gene Somatic Hypermutation Status Using Homoduplex-Separated RT-PCR Products.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4813-4813
Author(s):  
Melissa A. Melan ◽  
Kenneth A. Foon ◽  
Stanley M. Marks ◽  
Jeffrey A. Kant
Keyword(s):  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Rt Pcr ◽  
Mutation Status ◽  
Percent Identity ◽  
Pcr Product ◽  
Pcr Products ◽  
Reverse Primer ◽  
Almost All ◽  
Blast Program

Abstract Assessment of IgH somatic hypermutation status has been shown to be a valuable indicator for judging the prognosis of patients with chronic lymphocytic leukemia (CLL). Our laboratory has developed a streamlined method to improve the rate of successful evaluation of IgH mutation status. Six individual PCR reactions are first performed using random hexamer-generated cDNA as template. These reactions have identical reaction parameters, use a common JH reverse primer and one of six VH class-specific forward primers within Framework 1. PCR products are separated on acrylamide or MetaPhor® agarose gels following formamide denaturation. In almost all cases, a single homoduplex band is resolved indicating a class-specific clonal product. The homoduplex band is excised from the gel, eluted and directly sequenced. Mutation analysis is performed using the NCBI Ig BLAST program with percent identity determined for the region from the beginning of CDR1 to the end of Framework 3. To date, less than 10% of cases analyzed have not yielded a clearcut clonal PCR product using this approach

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