|Year : 2022 | Volume
| Issue : 1 | Page : 52-57
Somatic hypermutation in CLL: From bench to bedside
Shrinidhi Nathany, Anurag Mehta, Himanshi Diwan, Dushyant Kumar, Sakshi Mattoo, Surender Dhanda, Manoj K Panigrahi, Manoj Kumar
Department of Pathology (Molecular Diagnostics), Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
|Date of Submission||07-Jun-2022|
|Date of Acceptance||16-Jun-2022|
|Date of Web Publication||02-Sep-2022|
Dr. Shrinidhi Nathany
Department of Pathology (Molecular Diagnostics), Rajiv Gandhi Cancer Institute and Research Centre, Sector 5 Rohini, Sir Chhotu Ram Marg, New Delhi 110085
Source of Support: None, Conflict of Interest: None
Chronic lymphocytic leukemia (CLL) is a molecularly heterogeneous disease with a clonal proliferation of B lymphocytes. Immunoglobulin (Ig) sequence analysis is widely employed for prognostic stratification, and European Recommendation in CLL (ERIC) has laid down recommendations for testing and analysis of the same. immunoglobulin heavy chain variable region (IGHV) gene somatic hypermutation has been established as one of the most sensitive prognostic markers in CLL risk stratification, irrespective of the clinical stage or presence of any other alterations. Therapeutic decisions are now based on whether the IGHV is mutated—mCLL (mutated CLL) or unmutated, i.e., uCLL (unmutated CLL). Despite clear-cut clinical observations and differences in both therapeutic response and prognosis of IGHV unmutated group, the exact reason behind this is still elusive. IGHV mutation status has made it to the frontline in both clinical decision-making and the diagnostic battery of CLL testing. This is a focused comprehensive review on the structure, molecular biology, testing recommendations, and prognostic impact of IGHV mutation testing in CLL.
Keywords: CLL, IGHV, NGS, somatic hypermutation
|How to cite this article:|
Nathany S, Mehta A, Diwan H, Kumar D, Mattoo S, Dhanda S, Panigrahi MK, Kumar M. Somatic hypermutation in CLL: From bench to bedside. J Curr Oncol 2022;5:52-7
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is a molecularly heterogeneous disease with a clonal proliferation of B lymphocytes. Immunoglobulin (Ig) sequence analysis is widely employed for prognostic stratification, and European Recommendation in CLL (ERIC) has laid down recommendations for testing and analysis of the same.
This is a comprehensive review focused on immunoglobulin heavy chain variable region (IGHV) mutation in CLL, with a detailed excerpt on Ig gene structure, rearrangement, testing methods, and prognostic implications.
| Ig Gene Rearrangement|| |
Ig receptors are expressed on the surface of B lymphocytes, which bind antigens via the B-cell receptors (BCR) in complex with CD79a/CD79b heterodimer. Ig molecules are heterodimers comprising two heavy (H) chains and two identical light (L) chains joined by disulfide bonds. The N terminal of both H and L chains contain a variable (V) region, whereas the C-terminal houses the constant (C) region. The constant regions are not engaged in antigen recognition, whereas the juxtaposed variable regions of H and L chains (VH and VL) form the antigen-binding site. The structure of VH and VL determines the specificity and affinity of Ig molecules for the antigens.
Ig molecules are encoded by numerous tandemly arranged gene segments. The IGH locus maps to chromosome 14q32.33, which consists of four segments V (variable), D (diversity), J (joining), and C (constant) oriented in 5′-3′ direction. There are 38–46 functional IGHV gene segments, subdivided into six to seven subgroups, 23 IGHD segments, six IGHJ segments, and nine IGHC segments [Figure 1].
|Figure 1: The region structure of variable region of IGH with areas marked for insertions/deletions|
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The variable region consists of four conserved framework regions (FR), namely FR1, FR2, FR3, and FR4, and three complementarity determining regions (CDR), namely CDR1, CDR2, and CDR3, which are hypervariables. The CDR region forms six loops, which create a surface for the direct antigen interaction [Figure 2]A. The V regions are assembled through a coordinated process of V(D)J recombination of VH, DH, and JH genes on the heavy chain and VL and JL genes on the light chain. Recombination activating gene (RAG1 and RAG2) recombinase causes double-stranded breaks at specific recombination signal sequences (RSSs) to initiate this recombination process.,
|Figure 2: (A) The domain structure of variable region of IGH with areas marked for and recombination of V(D)J. (B) Primer positions for IGHV sequencing|
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V(D)J recombination mechanism
The first step in this process involves the binding of RAG with high-mobility group proteins (HMGB1 and HMGB2). VH and JH segments are flanked by 23 RSS, and the DH is flanked by 12 RSS. Famously known as the 12/23 rule, the RAG complex either binds to a 12 RSS or a 23 RSS, resulting in a 12- or 23-signal complex. A nicking and hairpin formation occurs between the coding region and the RSS. Following this another type of RSS is taken to form a paired complex, which helps in the hairpin formation. Nonhomologous end-joining DNA repair pathway molecules are engaged at the ends of the coding segments, which open the ends and rejoin them forming imprecise coding joints containing extra nucleotides. Through this random adding and the removal of nucleotides during v-D and D-J ligation, two N regions, namely, N1 and N2, are created, which is the source of the extreme variability of VH CDR3, which lies at the VDJ junction.,
Numerous different processes can generate the IGH variable region diversity with respect to V(D)J recombination. Random assortment of different germline VH, DH, and JH can lead to somatic diversity. Combinatorial diversity that occurs due to various combinations of germline V, D, and J segments is further amplified by the diversification of D to J and V to D junctions during V(D)J recombination through the deletion of nucleotides. The process of V(D)J recombination has been schematically depicted in [Figure 2]A and B.
Productive and unproductive sequences
Although junctional diversity usually leads to the deletion/addition of extra nucleotides, the real number is random. Considering that the ATG start codon at 5′ of VH exon is fixed and considering that the splice junction between JH and an IGHC gene (C mu or C delta) C mu is also a fixed, only a section of VHDJH places the VH ATG in frame with the coding sequence of JH to create a mu protein termed as a “productive sequence.” Productive rearrangement of one locus in turn impedes the rearrangement of other IGH locus on the other chromosome (allelic exclusion), thus warranting mono specificity of B lymphocytes.
Out-of-frame rearrangements cannot encode a heavy chain protein and are thus called as “unproductive sequence.” If the rearrangement of one allele is unproductive, the second will undergo a second recombination, which if fails, cell will die of apoptosis. Similar rearrangements also occur at the light chain immunoglobulin kappa (IGK) and immunoglobulin lambda (IGL) loci.,
| B-cell Ontogeny in Relation to IGHV and Somatic Hypermutations|| |
The first stage of B-cell development is early pro-B cell in bone marrow defined by the beginning of IGHD-IGHJ recombination. The late pro-B marks the joining of IGHV to the IGHD-IGHJ rearrangement leading to the transcription and synthesis of mu heavy chain. Once completed, the expression of mu heavy chain marks pre–B-cell stage. This cytoplasmic mu in complex with CD79A/79B is transiently expressed on the surface of pre-BCR. Subsequently, rearrangements of the light chain loci occur, and there is an expression of surface IgM, which marks the immature B cell. At this stage, the self-reactive clones are eliminated by the receptor editing, and the cells migrate to spleen where they develop into the naive B cells. Alternative splicing of IGH occurs, leading to co-expression of IgM and IgD with same antigen specificity.,,,
On encounter with antigens in the secondary lymphoid organs, the naive B cells undergo signaling leading to B-cell proliferation of antigen-specific clones. In some germinal centers, the B cells undergo proliferation at high rates and undergo somatic hypermutation (SHM) and class switch recombination. Activation-induced cytidine deaminase mediates the SHM process, which introduces missense mutations into the rearranged Ig loci, localized in variable regions of heavy and light chains. Specific hotspot motifs targeted include RGYW and its inverse repeat WRCY. Clonal progeny of the activated B cells is thus generated with diversified Ig rearrangements, which are later clonally selected based on the encountered antigen. Finally, repeated rounds culminate in affinity maturation and class switch recombination, leading to the fusion of IGHV-IGHD-IGHJ rearrangement to a downstream constant segment. This results in the production of other isotypes (except IgM and IgD) but with the same antigen specificity.,
| BCR Stereotypy|| |
Approximately 30% of CLL patients express near-identical BCRs, so-called “stereotyped” receptors. BCR “stereotypy” refers to highly delimited and sometimes identical variable heavy complementarity determining region 3 (VH-CDR3) sequences among different CLL patients reported in ~30%–35% of cases., In a recent analysis of a series of 21,123 IGHV sequences from CLL patients, 23 “major” (i.e., most populated) subsets were identified and represented 12% of all CLLs. However, in another major analysis on 29,856 CLL patients, 41% could be assigned to specific subsets.,,,, Stereotypy encompasses common somatic mutations, similar genetic and epigenetic profile, similar functional responses through the BCR, as well as similar clinical outcomes., The tool recommended for assigning to subsets is known as ARResT/AssignSubsets. A detailed description of a few of the major CLL subsets is depicted in [Table 1].
| IGHV Testing Strategies and Recommendations|| |
IGHV gene SHM has been established as a sensitive prognostic marker in CLL risk stratification, irrespective of the clinical stage or presence of any other alterations. Therapeutic decisions are now based on whether the IGHV is mutated—mCLL (mutated CLL) or unmutated, i.e., uCLL (unmutated CLL). Hence an accurate detection of the same is warranted using robust technologies. Unmutated clonal IGH gene rearrangement is defined as very high nucleotide sequence identity homologous to the closest germline IGHV sequence; mCLL in contrast is defined by IGHV sequence distinct by some percentage relative to its germline sequence. By convention, the SHM status in CLL is reported on the dominant clonal population recognized at diagnosis and is defined as a deviation of >2% (mutated status) or ≤2% (unmutated status) from the closest germline IGHV reference sequence.,
What samples to test, what substrates can be used, and what are primers
The main target for testing is the lymphocytes, which are present both in peripheral blood/bone marrow and hence either can be used as a specimen for testing. According to the ERIC,, both DNA and RNA can serve as starting material; however, cDNA has an advantage of specially identifying the rearrangement. Primers that can be used include leader primers, which allow for the sequencing of the entire IGHV region, or specific FR primers can be used. In cases that yield borderline results, both leader primers and FR1 primers can be used to settle any discrepancy. The primer positions are depicted in [Figure 2]B.
Conventionally, Sanger sequencing using DNA or RNA as starting material has been the method for the detection of IGHV mutational status. Considered as the gold standard, this involves two steps: polymerase chain reaction (PCR) followed by capillary electrophoresis to detect clonality and followed by dye-terminator sequencing. After clonality detection, the clonal region is subjected to sequencing, and web-based tools such as IMGT (International ImMunoGeneTics Information System) and IgBlast are used for facilitating the analysis and characterizing which V, D, and J alleles are used. The ERIC guidelines have laid down recommendations for the final interpretation and analysis of IGHV mutation status based on the type of sequence obtained, productive/unproductive, etc. A flow chart depicting the analysis and interpretation is depicted in [Figure 3]. To assess the stereotypy, ARResT (antigen receptor research tool) is used, which assigns to subsets if applicable. However, Sanger sequencing suffers from limitations including labor intensive workflow, technical complexities, and limited scalability. Additionally, cases with more than one clonal IGH may be under-appreciated using this technology because of the inability to consistently quantify relative abundance of individual clones. There may be situations such as single unproductive sequence and discordant SHM status in double-productive sequences, which may lead to interpretative errors in 3%–4% of cases.,,,,
|Figure 3: Interpretation of sequences as per ERIC recommendations after Sanger sequencing, especially in problematic cases|
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Since its inception, next-generation sequencing (NGS)-based technologies have had widespread applications. The usage of NGS for the detection of SHM was first explored by McClure et al. Since then, many commercialized kits have been made available on different platforms. NGS provides a more detailed insight into the intraclonal diversity and subclonal architecture as well as circumvents the need of labor-intensive lab processes. The ERIC–EuroClonality–NGS Working Group has laid emphasis and focus on NGS-based Immunogenetics implemented in 2012. They have collaborated systematically to develop robust pipelines for the NGS-based determination of SHM, encompassing both in vitro and in silico aspects. Stamatopoulos et al. used the LymphoTrack IGH SHM Assay Kit (Invivoscribe, San Diego, CA, United States) on complementary DNA (cDNA) from purified CD19+CD5+ cells. They showed multiple clonal IGH rearrangements in 25% of samples tested for CLL. A depiction of NGS workflow and interpretation in the light of EuroClonality have been depicted in [Figure 4]. Additionally, minimal residual disease assessment in CLL can be done using NGS, typically tracking the same clone identified at diagnosis for SHM determination.
|Figure 4: The workflow process of NGS for IGHV sequencing along with the representative case of unmutated and mutated IGHV|
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| Prognostic Significance of IGHV Status|| |
Why is the IGHV mutation status a prognostic indicator in CLL? IGHV mutation status has been described widely to correlate with clinical outcomes of patients treated with chemo-immunotherapy (CIT). Two landmark articles published in 1999 depicting the importance of IGHV mutation status showed a marked improvement of progression-free survival and overall survival in mutated cases., Another meta-analysis also confirmed an improved progression free survival (PFS) in IGHV mutated cases (9.2–18.9 years in the mutated versus 1–5 years in the unmutated cases). Cytogenetically, mutated cases are usually associated with the favorable group of cytogenetic alterations, and unfavorable alterations track with the unmutated subgroup. The CLL international prognostic index also includes IGHV as a parameter, apart from other clinical, biochemical, and genomic features. It has been shown to predict time to first treatment and overall survival (OS) in CLL, with the relative risk of IGHV status being two times than those of clinical features such as age and stage and second only to the TP53 mutation status in the order of importance. The original FCR300 study demonstrated a PFS of 53.9% for mutated compared with 8.7% in unmutated patients after a follow-up of 12.8 years. In the CLL8 study, it was demonstrated that IGHV unmutated with the presence of del17p (TP53) was the strongest negative prognostic factor for both PFS and OS. Although the response duration to CIT in an unmutated group is shorter, there appears to be no difference in the duration of response when treated with BCR pathway kinases. Instead, the responses in the unmutated have been shown to be more rapid in the unmutated group when compared with the mutated group.
| Conclusions and Future Perspectives|| |
Despite clear-cut clinical observations and differences in both therapeutic response and prognosis of IGHV unmutated group, the exact reason behind this is still elusive. IGHV mutation status has made it to the frontline in both clinical decision-making and the diagnostic battery of CLL testing. Therefore, future studies to simplify testing are warranted, including tests with rapid turnaround time and less-intensive workflow processes.
| References|| |
Hallek M, Al-Sawaf O Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. Am J Hematol 2021;96:1679-705.
Sutton LA, Hadzidimitriou A, Baliakas P, Agathangelidis A, Langerak AW, Stilgenbauer S, et al
; European Research Initiative on CLL (ERIC). Immunoglobulin genes in chronic lymphocytic leukemia: Key to understanding the disease and improving risk stratification. Haematologica 2017;102:968-71.
Radaev S, Zou Z, Tolar P, Nguyen K, Nguyen A, Krueger PD, et al
. Structural and functional studies of igalphabeta and its assembly with the B cell antigen receptor. Structure 2010;18:934-43.
Scott GB, de Wynter EA, Cook GP Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system. Mob DNA 2010;1:9.
Melarkode Vattekatte A, Shinada NK, Narwani TJ, Noël F, Bertrand O, Meyniel JP, et al
. Discrete analysis of camelid variable domains: Sequences, structures, and in-silico structure prediction. PeerJ 2020;8:e8408.
Roth DB V(D)J recombination: Mechanism, errors, and fidelity. Microbiol Spectr 2014;2.
Bassing CH, Swat W, Alt FW The mechanism and regulation of chromosomal V(D)J recombination. Cell 2002;109:S45-55.
Jackson KJ, Kidd MJ, Wang Y, Collins AM The shape of the lymphocyte receptor repertoire: Lessons from the B cell receptor. Front Immunol 2013;4:263.
Tsakou E, Agathangelidis A, Agathagelidis A, Boudjoghra M, Raff T, Dagklis A, et al
. Partial versus productive immunoglobulin heavy locus rearrangements in chronic lymphocytic leukemia: Implications for B-cell receptor stereotypy. Mol Med 2012;18:138-45.
Rosenquist R, Ghia P, Hadzidimitriou A, Sutton LA, Agathangelidis A, Baliakas P, et al
. Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia: Updated ERIC recommendations. Leukemia 2017;31:1477-81.
Agathangelidis A, Chatzidimitriou A, Gemenetzi K, Giudicelli V, Karypidou M, Plevova K, et al
. Higher-order connections between stereotyped subsets: Implications for improved patient classification in CLL. Blood 2021;137:1365-76.
Jackson TR, Ling RE, Roy A The origin of B-cells: Human fetal B cell development and implications for the pathogenesis of childhood acute lymphoblastic leukemia. Front Immunol 2021;12:637975.
Oppezzo P Chronic Lymphocytic Leukemia. London: IntechOpen; 2012. p. 464. Available from: https://www.intechopen.com/books/647
. [Last accessed on 2022 May 1].
Ten Hacken E, Gounari M, Ghia P, Burger JA The importance of B cell receptor isotypes and stereotypes in chronic lymphocytic leukemia. Leukemia 2019;33:287-98.
Rossi D, Gaidano G Biological and clinical significance of stereotyped B-cell receptors in chronic lymphocytic leukemia. Haematologica 2010;95:1992-5.
Vlachonikola E, Sofou E, Chatzidimitriou A, Stamatopoulos K, Agathangelidis A The significance of B-cell receptor stereotypy in chronic lymphocytic leukemia: Biological and clinical implications. Hematol Oncol Clin North Am 2021;35:687-702.
Darzentas N, Stamatopoulos K Stereotyped B cell receptors in B cell leukemias and lymphomas. Methods Mol Biol 2013;971: 135-48.
Agathangelidis A, Vardi A, Baliakas P, Stamatopoulos K Stereotyped B-cell receptors in chronic lymphocytic leukemia. Leuk Lymphoma 2014;55:2252-61.
Agathangelidis A, Psomopoulos F, Stamatopoulos K Stereotyped B cell receptor immunoglobulins in B cell lymphomas. Methods Mol Biol 2019;1956:139-55.
Stamatopoulos K, Agathangelidis A, Rosenquist R, Ghia P Antigen receptor stereotypy in chronic lymphocytic leukemia. Leukemia 2017;31:282-91.
Bystry V, Agathangelidis A, Bikos V, Sutton LA, Baliakas P, Hadzidimitriou A, et al
; European Research Initiative on CLL. ARResT/AssignSubsets: A novel application for robust subclassification of chronic lymphocytic leukemia based on B cell receptor Ig stereotypy. Bioinformatics 2015;31:3844-6.
Davi F, Langerak AW, de Septenville AL, Kolijn PM, Hengeveld PJ, Chatzidimitriou A, et al
; ERIC, the European Research Initiative on CLL, and the EuroClonality-NGS Working Group. Immunoglobulin gene analysis in chronic lymphocytic leukemia in the era of next generation sequencing. Leukemia 2020;34:2545-51.
Agathangelidis A, Sutton LA, Hadzidimitriou A, Tresoldi C, Langerak AW, Belessi C, et al
. Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia: From patient material to sequence interpretation. J Vis Exp 2018.
McClure R, Mai M, McClure S High-throughput sequencing using the ion torrent personal genome machine for clinical evaluation of somatic hypermutation status in chronic lymphocytic leukemia. J Mol Diagn 2015;17:145-54.
Gupta SK, Viswanatha DS, Patel KP Evaluation of somatic hypermutation status in chronic lymphocytic leukemia (CLL) in the era of next generation sequencing. Front Cell Dev Biol 2020;8:357.
Molica S, Shanafelt TD, Giannarelli D, Gentile M, Mirabelli R, Cutrona G, et al
. The chronic lymphocytic leukemia international prognostic index predicts time to first treatment in early CLL: Independent validation in a prospective cohort of early stage patients. Am J Hematol 2016;91:1090-5.
Thompson PA, Tam CS, O’Brien SM, Wierda WG, Stingo F, Plunkett W, et al
. Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia. Blood 2016;127:303-9.
Fischer K, Bahlo J, Fink AM, Goede V, Herling CD, Cramer P, et al
. Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL: Updated results of the CLL8 trial. Blood 2016;127:208-15.
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